investigation of the effects of inhibiting n-glycosylation ... · investigation of the effects of...

Investigation of the Effects of Inhibiting

N-Glycosylation in Cancer

by

Reza Beheshti Zavareh, Hon. B. Sc.

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Department of Medical Biophysics

University of Toronto

© Copyright by Reza Beheshti Zavareh (2011)

ii

Investigation of the Effects of Inhibiting N-Glycosylation in Cancer

Reza Beheshti Zavareh

Doctor of Philosophy

Department of Medical Biophysics

University of Toronto

2011

Abstract

Glycosylation, the addition of sugar moieties to nascent proteins, is one of the

most common posttranslational modifications. Glycosylation regulates protein structure,

function and localization. Most cell surface proteins and secreted proteins are

glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-

glycosylation is a well-accepted feature of malignancy and is a potential prognostic

marker for some types of cancer. For example, increased expression of complex N-

glycans has been detected in cancers of breast, colon and has been correlated with

reduced survival of the patients. Therefore, understanding the role of N-glycosylation in

malignancy could be beneficial for developing novel therapeutic and prognostic

strategies.

To examine the role of N-glycosylation in malignancy, we applied chemical

biology and genetic approaches. First, we conducted a high throughput screen to identify

compounds that could block L-PHA-induced cell death. Our screen identified the cardiac

glycoside Na+/K

+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further

analysis of N-glycans consistently confirmed that inhibition of Na+/K

+-ATPase impairs

the N-glycosylation, as well as migration and invasion. Interestingly, other studies have

shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput

iii

screen identified Na+/K

+-ATPase inhibition as a novel strategy to target the N-

glycosylation pathway.

In addition, we used a genetic approach to investigate the role of N-

acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of

GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal

microscopy, knockdown of GlcNAc-TI decreased cell surface expression of 1 integrins

and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown

decreased the migration and invasion of malignant cells. Next, we investigated the effect

of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI

knockdown significantly decreased the lung colony formation of the highly metastatic

PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for

GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower

expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus,

GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI

inhibitors could have therapeutic benefits for cancer patients. Moreover, expression

levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.

iv

Acknowledgments

First of all, I would like to thank my supervisor, Dr. Aaron Schimmer, who is a

dedicated and inspired scientist, clinician and a great mentor. Aaron has been an

incredible teacher and my experience as a graduate student under his supervision has

been an exceptionally wonderful opportunity. I am forever indebted to him.

Secondly, I would also like to thank my supervisory committee members Dr. Jim

Dennis, Dr. David Rose and Dr. Harry Schachter. Jim and his lab members provided me

with continual support for multiple experiments and projects. David was always very

kind, supportive and inspiring throughout these years. Harry provided us with great

insight when we were facing with the most challenging questions. It was an honour for

me to learn the intricacies of N-glycosylation from scientists of such high caliber during

my training.

I would also like to thank the past and present members of the Schimmer lab,

especially, Marcela Gronda and Rose Hurren who have greatly contributed to my

education and success. I have had an wonderful opportunity to work with many

outstanding and kind people in the Schimmer lab including: Nazir Jamal, Craig Simpson,

Dr. Imtiaz Mawji, Dr. Clare Henderson, Dr. Yanina Eberhard, Amanda Wasylishen, Dr.

Mahadeo Sukhai, Dr. Wei Xu, Xiaoming Wang and others.

I have had the pleasure of collaborating with several amazing people over these

years. Dr. Pamela Cheung and Dr. Ken Lau from Jim’s lab taught me very important

instrumental assays. At the Robotic facility at SLRI, Dr. Alessandro Datti, Dr. Leigh

Rivers and Thomas Sun helped us tremendously with the high throughput screens.

v

I sincerely thank Dr. Maire Percy, my undergraduate supervisor who trusted me

and encouraged me to continue research. Maire’s moral support over these years has

been very helpful.

Finally, I would like to thank my parents and my family. I would like to honour

my father, Dr. Hossein Beheshti Zavareh who was a devoted pediatrician. His legacy of

excellence, strong morality and passion for helping others will guide me all my life. My

mother, Fatemeh Tehrani selflessly helped me in every step of my education and taught

me to dream big and achieve the best I can. I am blessed to have a caring sister, Zahra,

who always believes in me and is a source of energy and joy in my life. My brother-in-

law, Dr. Mehrdad Shamsi had a lot of constructive advice during my graduate studies. I

would not have been able to embark on the journey of coming to the University of

Toronto without the help and support of my uncle and aunt, Dr. Ali Tehrani and

Naghmeh Hejri. Sepasgozaram!

vi

List of Abbreviations

AMP Adenosine monophosphate

Asn Asparagine

BSA bovine serum albumin

Ca2+

Calcium 2+ ion

cDNA Complementary DNA

CD-MPR Cation-dependent Mannose-6-phosphaste receptor

CG Cardiac glycoside

CGs Cardiac glycosides

CI-MPR Cation-independent Mannose-6-phosphaste receptor

ConA Concanavalin A

DMEM Dulbecco’s modified Eagle’s medium

DNA Deoxyribonucleic acid

E9.5 Embryonic day 9.5 after fertilisation

ECM Extracellular matrix

EGF epidermal growth factor

EGFR epidermal growth factor receptor

EMT Epithelial-to-mesenchymal transition

ER Endoplasmic reticulum

Ets-1 Erythroblastosis virus E26 oncogene homolog

FGFR Fibroblast growth factor receptor

Fuc Fucose

Gal Galactose

GalNAc N-acetylgalactosamine

GlcNAc N-acetylglucsamine

GlcNAc-TI N-acetylglucosaminyl-transferase I

GlcNAc-TII N-acetylglucosaminyl-transferase II

GlcNAc-TIII N-acetylglucosaminyl-transferase III

GlcNAc-TIV N-acetylglucosaminyl-transferase IV

GlcNAc-TV N-acetylglucosaminyl-transferase V

GPI glycosylphosphatidylinositol

GT-A Glycosyltransferase type-A

her2/neu Human epidermal growth factor receptor 2/neural

IP3 inositol 1,4,5-trisphosphate

K+

Potassium + ion

L-PHA Lectin phytohemagglutinin

MALDI Matrix assisted laser desorption/ionizaion

Man mannose

MES 2-(N-morpholino)ethanesulfonic acid

Mgat1 mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-

acetylglucosaminyltransferase

Mgat5 mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-

glucosaminyltransferase

Mn2+

Manganese 2+ ion

MnCl2 Manganese chloride

vii

MS Mass spectrometry

Na+

Sodium + ion

Na+/K

+-

ATPase

sodium potassium adenosine triphosphatase pump

NH4HCO3 ammonium bicarbonate

N-glycan Nitrogen linked-glycan

OST oligosacaccharyltransferase

PBS Phosphate buffered saline

pH Potential of hydrogen

Ras Rat sarcoma oncogene

RER Rough endoplasmic reticulum

RNA Ribonucleic acid

RPMI Rosewell Park Memorial Institute

RTK Receptor tyrosine kinase

Sa Sialic acid

SCID severe combined immunodeficient

Ser Serine

SDS Sodium dodecyl sulfate

Src Rouse Sarcoma oncogene

SWA Swainsonine

Thr Threonine

TGF- Transforming growth factor

TOF Time of flight

VEGF Vascular endothelial growth factor

viii

Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgments ............................................................................................................. iv

List of Abbreviations ......................................................................................................... vi

Table of Contents ............................................................................................................. viii

List of Figures and Tables .................................................................................................. x

Chapter 1: Introduction: ...................................................................................................... 1

1.1 Glycosylation ....................................................................................................... 1

1.2 N-glycosylation .................................................................................................... 2

1.2.1 The synthesis of the dolichol-linked oligosaccharide ....................................... 3

1.2.2 The glycosylation step: Transfer of Glc3Man9GlcNAc2 to the nascent protein 5

1.2.3 The trimming and processing of the maturing N-glycan in ER and Golgi ....... 6

1.2.4 Elongation and capping of the terminal branches in the trans-Golgi ............... 7

1.3 The functional roles of N-glycans ...................................................................... 10

1.4 N-glycosylation and Cancer ............................................................................... 12

1.5 A chemical biology approach to identify novel strategies to inhibit N-

glycosylation ................................................................................................................. 16

1.6 A Chemical screen for novel N-glycosylation inhibitors ................................... 16

1.7 GlcNAc-TI: The gateway from oligomannose N-glycans to hybrid and complex

N-glycans ...................................................................................................................... 20

1.8 Rational and outline of the thesis ....................................................................... 24

Chapter 2 ........................................................................................................................... 26

Inhibition of the sodium/ potassium ATPase impairs N-glycan expression and function 26

2.1 Abstract ................................................................................................................... 27

2.2 Introduction ........................................................................................................... 28

2.3 Material and Methods ............................................................................................ 30

2.4 Results .................................................................................................................... 39

2.5 Discussion ............................................................................................................... 67

Chapter 3 ........................................................................................................................... 72

Suppression of N-glycan branching by N-acetylglucosaminyltransferase I knockdown

inhibits tumor cell migration, invasion and metastasis ..................................................... 72

3.1 Abstract .................................................................................................................. 73

3.2 Introduction: ............................................................................................................ 74

3.3 Materials and methods: ........................................................................................... 76

ix

3.4 Results: ................................................................................................................... 81

3.4 Discussion .............................................................................................................. 97

Chapter 4 : Discussion and future directions .................................................................. 101

4.1 Chemical biology approach to identify N-glycosylation inhibitors ................. 101

4.2 The genetic approach to inhibit N-glycan branching in cancer ........................ 105

References: ...................................................................................................................... 110

Appendix1 ....................................................................................................................... 119

The chemical screening of LoPAC and Prestwick library .............................................. 119

Appendix 2 ...................................................................................................................... 124

The spectra of MALDI-TOF data presented in chapter 2 ............................................... 124

x

List of Figures and Tables

Table 1.1. The types of sugar-amino acid linkages found in glycoproteins of mammalian

cells. .................................................................................................................................... 2 Figure 1.1 The synthesis of Glc3Man9GlcNAc2-P-P-dolichol , .......................................... 4 Figure 1.2. The Biosynthesis of N-glycans ......................................................................... 9 Figure 1.3. Na

+/K

+-ATPase maintains the proper [Na

+] and [K

+] across the cell

membrane. ......................................................................................................................... 18 Figure 1.4. The reaction and structure of N-acetylglucosaminyltransferase I. ................. 23 Figure 2.1 Scheme of the N-glycan biosynthesis pathway. .............................................. 30

Figure 2.2 A chemical screen identifies cardiac glycosides as inhibitors of L-PHA

induced cell death. ............................................................................................................ 40 Figure 2.3. Other cardiac glycosides inhibit L-PHA induced cell death. ......................... 42 Figure 2.4. Testing the effect of digoxin in murine MDAY lymphoma cells. ................ 43

Figure 2.5. Digoxin increases levels of ConA-binding glycoproteins through inhibition of

the Na+/K+-ATPase ......................................................................................................... 46

Figure 2.6 The effects of digoxin on cell surface ConA staining are due to an on target

effect of Na+/K

+ -ATPase inhibition ................................................................................. 48

Table 2.1. Digoxin and swainsonine alter the expression of oligosaccharides ................. 50

Figure 2.7 The graphic presentation of the MALDI-TOD data. ....................................... 52 Figure 2.8 The effect of digoxin on mRNA expression levels of key golgi

glycosyltransferases. ......................................................................................................... 54

Figure 2.9. The enzymatic activity of N-glycosylation enzymes in the presence of

increasing concentrations of digoxin. ............................................................................... 55 Figure 2.10 . Digoxin increases the intracellular levels of Na

+ and Ca

2+.......................... 56


increasing concentrations of Na+ and Ca

2+. ...................................................................... 57

Figure 2.12 Digoxin inhibits cell migration and invasion. .............................................. 61

Figure 2.13. Digoxin inhibits distant tumor formation in vivo ......................................... 65 Figure 3.1. GlcNAc-TI knockdown does not alter the proliferation of malignant cells. . 82 Figure 3.2 GlcNAc-TI knockdown decreases the migration and invasion of malignant

cells ................................................................................................................................... 85 Figure 3.4 Evaluation of the effects of GlcNAc-TI knockdown in an orthotopic model of

metastatic prostate cancer ................................................................................................. 91

Figure 3.5 GlcNAc-TI knockdown decreases distant tumor formation. ........................... 93 Figure 3.6 . Expression profiling of breast cancer patients............................................... 95 Appendix 1 Figure 1. The high throughput screen of LoPAC library ............................ 120 Appendix 1 Table 1 The statistically significant hits of LoPAC library ....................... 121

Appendix 1 Figure 2 The high throughput screen of Prestwick library ......................... 122 Appendix 1 Table 2 The top hits of the Prestwick library ............................................. 123

1

Chapter 1: Introduction:

1.1 Glycosylation

Glycosylation is the most common post translational modification and influences

protein function and structure. (1-3). In fact, Apweiler et al have estimated that more

than 50% of all eukaryotic proteins are glycoproteins(4). In mammalian cells,

glycosylation can have many forms including N-glycosylation (addition of a glycoside to

the amido group of the asparagine residue in the sequence Asn-X-Ser/Thr, where X can

be any amino acid except for proline), O-glycosylation (addition of GlcNAc, Gal, Man,

GalNAc, Glc or Fuc to the hydroxyl group of serine or threonine), C-glycosylation

(mannosylation of certain tryptophan residues in the sequence Trp-X-X-Trp) and

glypiation (addition of glycosylphosphaitylinositol anchor to the c-terminus of proteins

that localize to the cell membrane(5-7). Table 1 shows a summary of the various sugar-

amino acid linkages found in mammalian cells.

In this chapter, the N-glycosylation pathway is described first, followed by the

importance of the N-glycans in malignancy. Next, the rationale of developing N-

glycosylation inhibitors is explained. Finally, the outline and rationale of this thesis is

presented.

2

Table 1.1. The types of sugar-amino acid linkages found in glycoproteins of

mammalian cells.

1.2 N-glycosylation

Most cell surface proteins and secreted proteins are N-glycosylated. N-linked

glycans function as specific signals which can be recognized by a wide variety of

carbohydrate binding proteins (lectins) (8). N-glycosylation influences the localizing of

proteins and facilitates localization of proteins to the cell surface or secretion of proteins.

N-glycosylation involves a cascade of events and a series of enzymes. This cascade can

be divided into four successive steps which will be discussed in detail below(7, 9):

1. The synthesis of the activated precursor oligosaccharide (Glc3Man9GlcNAc2)

on dolichol pyrophosphate

2. Co-translational and post-translational transfer of Glc3Man9GlcNAc2 from

dolichol to the Asn residue of the target proteins in the lumen of the rough ER

3. The trimming and processing of the maturing glycoprotein in ER and Golgi

4. The elongation of the terminal branched in the trans-Golgi

Type of

glycosylation Subcellular

compartment Target sequence The glycan structure

N- ER/Golgi Asn-X-Ser/Thr Glc3Man9GlcNAc2

O- Cytoplasm, ER,

Golgi

Ser/Thr Glc/GlcNAc/Man

Gal/GalNAc/Fuc/xylose

C- ER Trp-X-X-Trp Man

Glypiation ER C-terminal GPI anchor

3

1.2.1 The synthesis of the dolichol-linked oligosaccharide

The N-glycosylation pathway begins by assembling a lipid linked 14-residue

oligosaccharide Glc3Man9GlcNAc2-P-P-dolichol. The assembly of this oligosaccharide

is initiated on the cytosolic side of the endoplasmic reticulum (ER) (Figure 1.1).

First, the GlcNAc-1phosphotransferase (GPT) transfers one GlcNAc-1phosphate

from UDP-GlcNAc to dolichol phosphate to form GlcNAc-P-P-dolichol.

GlcNAc-P-P-dolichol is further processed by addition of one more GlcNAc and three

mannoses to form Man3GlcNac2-P-P-dolichol by the action of cytoplasmic

glycosyltransferases to form the N-glycan “core” structure. Two more mannoses are

added to the core structure by mannosyltransferases from GDP-Man. This intermediate

structure is then translocated from the cytosolic side of the ER into the lumen of the ER.

The mechanism and effectors that promote the translocation of the intermediate structure

to the ER lumen are not well understood (10).

Once Man5GlcNAc2-P-P-dolichol is translocated into the ER lumen four more

mannoses and three glucoses are sequentially added to the growing oligosaccharide

structure by mannosyltransferases and glucosyltransfereases to form Glc3Man9GlcNAc2.

4

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5

1.2.2 The glycosylation step: Transfer of Glc3Man9GlcNAc2 to the

nascent protein

Once the synthesis of Glc3Man9GlcNAc2-P-P-dolichol is complete the

oligosaccharide is transferred en bloc to the asparagine residue of the nascent protein.

The oligosaccharide can be added to asparagine residues located in the consensus

sequence (sequon) Asn-X-Thr/Ser, where X can be any amino acid except for proline.

This reaction is catalyzed by a relatively large protein complex oligosaccharyltransferase

(OST) (11, 12) (Figure 1.1).

Of note, only 70% of asparagines with the appropriate consensus sequence are

glycosylated(4). Therefore, the presence of the sequon is essential but not sufficient for

the addition of the N-glycans. Studies have elucidated a number of factors that influence

whether asparagine residues with the appropriate consensus sequence will be

glycosylated. Asparagine residues are less likely to be glycosylated if the consensus

sequence is located toward the C-terminal end of the protein(13). In addition, increased

rates of protein folding decreases N-glycosylation as the asparagine site is buried in the

folded protein(14). Conversely, Asn residues are more likely to be glycosylated when

levels of Glc3Man9GlcNAc2-P-P-dolichol are increased or the expression of OST is

increased (1, 7).

6

1.2.3 The trimming and processing of the maturing N-glycan in ER and

Golgi

Once the Glc3Man9GlcNAc2 is transferred to the sequon, further processing of the

structures is initiated. First, the outermost glucose is removed by the action of

glucosidase I. Subsequently, glucosidase II removes the last two glucoses

(1,3glucose). Finally, mannosidase removes one more mannose from the

oligosaccharide to produce Man8GlcNAc2. The trimming and processing of N-glycans to

produce Man8GlcNAc2 facilitates protein folding and exit from the ER. Specifically,

protein folding is promoted by lectins calnexin/calreticulin binding intermediate glycan

structures and act as chaperons in the protein folding process. These molecular

chaperons prevent the export of the misfolded protein chains from ER. Calnexin and

calreticulin also facilitate the formation of disulfide binds during the protein folding.

In order for the glycoproteins to be released from calnexin and calreticulin, glucosidase II

removes the last 1,3glucose. At this stage, properly folded proteins can leave the ER.

However misfolded proteins can be recognized by UDP-Glc:glycoprotein

glucosyltransferase (GT) and reglucosylated. The reglucosylated protein can bind

calnexin/calreticulin again to promote proper folding. The deglucosylation-

reglucosylation cycle is repeated until the protein is properly folded and it can leave the

ER. Alternatively, the misfolded protein is targeted for degradation(15).

The glycoprotein with the Man8GlcNAc2 oligosaccharide is capable of

translocating from the ER lumen to the cis-Golgi. In the cis-Golgi additional enzymes

further trim and modify the N-glycans. The N-glycans can be processed according to one

of several pathways. Oligosaccharides can go through minimal processing in Golgi and

exit the N-glycosylation pathway as high mannose type structures. However, typically,

7

the N-glycans in mammals go through a much more elaborate processing in the Golgi.

Initially, mannosidases 1A, 1B and 1C remove three mannoses from the N-glycan to

produce Man5GlcNAc2. This heptasaccharide is an important intermediate in the

conversion of high mannose structures to hybrid and complex type N-glycans (Figure

1.2).

Man5GlcNAc2 is further modified by N-acetylglucosaminyltransferase I

(Mgat1/GlcNAc-T1) which adds a GlcNAc in 1,2 linkage to the trimannosyl core of the

Man5GlcNAc2. Following the addition of the GlcNAc branch by GlcNAc-TI, -

mannosidase II can remove the mannoses from the other arm of the trimannosyl core (the

arm) to produce GlcNAcMan3GlNAc2. GlcNAcMan3GlNAc2 then serves as a

substrate for GlcNAc-TII which adds the second 1,2 linked GlcNAc. The biantennary

product of this reaction is the first complex-type N-glycan synthesized. Subsequent

branching occurs by the sequential action of GlcNAc-T III, IV, V which add further

branches to produce tri, tetra antennary complex N-glycans. The declining efficiency of

GlcNAc-TI to GlcNAc-TV activities results in heterogeneity; a divergent array of N-

glycans can be added to proteins and allows detailed regulation of protein localization

and function (Figure 1.2).

1.2.4 Elongation and capping of the terminal branches in the trans-

Golgi

The final step in the N-glycosylation cascade involves processing of the hybrid

and complex N-glycans by a wide variety of enzymes in the trans-Golgi. These final

modifications on the N-glycans can be divided into two broad types: elongation and

capping(16).

8

The elongation of the terminal GlcNAc is generally occurs by a galactose in a 1-

4 or linkage resulting in a terminal Gal1-4GlcNAc (Type 2 N-acetyllactosamine

“LacNAc”) or GalGlcNAc (type 1 N-acetyllactosamine “LacNAc”). The terminal

1-4 LacNAc branch can in turn be lengthened by sequential addition of GlcNAc and Gal

to produce poly- N-acetyllactosamine chains. Interestingly, in early mammalian

development, galactose residues in poly- N-acetyllactosamine chain can be targeted by

the GlcNAc transferase (I-transferase) to produce the blood group I antigens

(17)(Figure 1.2).

The second type of trans-Golgi modifications is capping of the terminal Gal1-

4GlcNAc and GalGlcNAc. The terminal branches can be capped with a wide

variety of structures including -linked sialic acids, fucose, sulphation and addition of

ABH and Lewis antigenic N-glycans (e.g.: Lewisx, Lewis

y, Lewis

a, Lewis

b, Sialyl-

Lewisx, etc)(7) (Figure 1.2).

9

Figure 1.2. The Biosynthesis of N-glycans

The biosynthesis of N-glycans involves the following steps: (i) Sequential conversion of

the high mannose N-glycan Man9GlcNAc2 to Man5GlcNAc2; (ii) Addition of 2-linked

GlcNAc to the terminal 3-linked Man of Man5GlcNAc2 by GlcNAc-TI to form

GlcNAc1Man5GlcNAc2; (iii) Removal of two Man residues from

GlcNAc1Man5GlcNAc2 by -mannosidase II to form GlcNAc1Man3GlcNAc2; (iv)

Addition of a second 2-linked GlcNAc to the terminal 6-linked Man of

GlcNAc1Man3GlcNAc2 by GlcNAc-TII to form GlcNAc2Man3GlcNAc2; (v) Addition

of L-Fucose , Galactose and sialic acid residues. The abbreviations used are:

oligosaccharyltransferase, OT; the -glucosidases, GI, GII; the -N-

acetylglucosaminyltransferases, TI, TII,TIII, TIV, TV; the -1,2mannosidases, MI; -

1,3/6mannosidase, ManII; 1,2-galactosyltransferases, Gal-T; -sialyltransferases, Sa T;

UDP-N-acetylglucosamine, UDP-GlcNAc; UDP-galactose, UDP-Gal; CMP-sialic acid,

CMP-SA. The four types of N-glycans are shown: high-mannose-, hybrid-, bisected- and

complex-type N-glycans. Adapted from :(18). Copyright permission granted by publisher,

2011 ©

Biochim Biophys Acta. 1999 Dec 6;1473(1):21-34

10

1.3 The functional roles of N-glycans

N-glycosylation plays an important role in protein folding, function,

localization, stability and solubility. For example, as discussed previously,

calenxin/calreticulin bind monoglucosylated N-glycan Glc1Man9GlcNAc2 in the ER

lumen and serve as chaperons to promote protein folding. Calnexin and calreticulin also

facilitate the formation of disulfide bonds during the protein folding. These molecular

chaperons prevent the export of the misfolded protein chains from ER (15). One of the

other intracellular functions of N-glycans is targeting misfolded proteins to the ER-

associated degradation (ERAD).

N-glycosylation also promotes targeting of hyrolases to lysosomes. To target

hydrolases to lysosomes, GlcNAc-phosphate is added to selected mannose residues on

the hydrolases by N-acetylglucosamine-1-phosphotransferase. Subsequently, the

GlcNAc is removed by N-acetylglucosamine-1-phosphodiester -N-

acetylglucosaminidase so that the man-6-phosphate remains on the enzyme. The man-6-

phosphate on lysosomal hydrolases is then recognized by cation-dependent man-6-

phosphate receptor (CD-MPR) or cation-independent man-6-phosphate receptor (CI-

MPR) that promote their import to lysosomes. (19, 20). Therefore, N-glycans regulate

many important intracellular processes.

N-glycosylation also promotes protein secretion and modulates cell surface

localization and retention of proteins. Interesting examples of this function are the effects

of N-glycosylation on growth factors and growth factor receptors. The growth factor

VEGF requires N-glycosylation for secretion and chemical inhibitors of N-glycosylation

decrease the secretion of VEGF in tumor cells (21, 22). In addition, N-glycosylation

11

promotes the cell surface localization of EGFR and consequently its responsiveness to

cytokines(23). Chemical inhibition of N-glycosylation with tunicamycin or swainsonine

inhibits the N-glycosylation of EGFR and decreases cell surface expression of EGFR

(22). Similar findings have been reported for other growth factor receptors including

FGFR and TGF-R (24). Thus, N-glycans play an important role in regulating protein

secretion and localization and thereby modify cell growth and differentiation.

Another major role of N-glycans is the regulation of cell-cell and cell-ECM

adhesion. The binding of N-glycans with carbohydrate binding proteins, lectins, is the

key regulator of these interactions. For instance, E-, P-, and L-selectins bind sialylated

and fucosylated glycans (e.g. Sialyl-Lewisx) and facilitate the adhesion of leukocytes to

endothelium. As such, selectin promote the trafficking of leukocytes from the circulation

into lymphatic system (leukocyte extravasation) (25). Similarly, galectins, S-type lectins

which bind N-acetyllactosamine sequences on N-glycans have been implicated in cell-cell

adhesion, and cell-ECM interaction(26). Interestingly, most of the oligosaccharide

ligands of the selectins and galectins are the more mature complex N-glycans which are

synthesized in medial- and trans-Golgi. Additionally, N-glycosylation also regulates cell

migration. Integrins, a major family of cell-cell and cell-ECM adhesion receptors, are

heavily N-glycosylated proteins. These N-glycans modulate the interaction of integrins

with ECM. For example, the interaction of integrin with fibronectin is controlled

by N-glycans and these N-glycans regulate the dependent fibronectin fibrilogenesis

(27, 28). The N-glycan mediated integrin-fibronectin interaction has a direct role on cell

migration(29). For example, overexpression of GlcNAc-TV in lung epithelial cells

decreased adhesion to fibronectin-coated surface and increased cell migration (30).

12

Therefore, complex N-glycans add another dimension and complexity to the cellular

functions of N-glycosylation by regulating cell adhesion and migration (31).

Another mechanism by which N-glycosylation regulates cellular functions is by

modulating the galectin-glycoprotein lattice. This lattice plays an important role at the

level of plasma membrane to regulate the diffusion and retention of cell surface

glycoproteins. The lattice formation is promoted by the expression of complex N-

glycans. Galectins bind with tetraantennary N-glycans synthesized by GlcNAc-TV with

the highest affinity. Therefore, increased branching of N-glycans can increase the lattice

recruitment of glycoproteins and consequently increase cell membrane residency (32-34).

1.4 N-glycosylation and Cancer

Aberrant glycosylation is a well-accepted feature of malignant transformation.

One of the earliest observations of aberrant glycosylation in cancer was detection of large

N-glycans on the surface of SV-40-transformed cells(35). In addition, early work

demonstrated that lectin-resistant mutants of highly malignant cells had decreased

metastasis. Definite proof of the transforming properties of aberrant glycosylation can be

found in studies in which GlcNAc-TV was overexpressed in an immortalized non-

tumorigenic lung epithelial cell line. Overexpression of GlcNAc-TV produced cells

capable of metastasis(30, 36). In addition, activation of multiple oncogenes including ras

(37), Src(38), her2/neu (39), Ets-1(40) and polyoma middle T antigen(41), has been

shown to increase the expression of N-glycosylation enzymes and thus provides more

evidence for the direct role of oncogenic activity in aberrant N-glycosylation.

13

Multiple mechanisms have been proposed for how increased N-glycosylation

promotes malignant cell transformation. The increased expression of N-glycosylation

enzymes leads to modification of growth factor receptors and integrins with highly

branched complex N-glycan. Increased expression of complex N-glycan on growth factor

receptors is responsible for increased cell-surface retention and consequently more

responsiveness to cytokines. The same modifications in integrins have been associated

with increased tumor cell migration and invasion. Clinical observations also points

toward the same effects of N-glycosylation on tumor metastasis. Using L-PHA staining,

several studies have measured the abundance of complex N-glycans on the surface of

tumors. For example, malignant cells from breast and colon carcinoma show increased

L-PHA-staining compared to control non-malignant cells and the increased N-glycan

branching correlates with increased metastasis and reduced survival of these patients(42).

Therefore, the tumor cells with aberrant glycosylation have a selective advantage for

more autonomous growth and metastatic potential.

Aberrant N-glycosylation in tumor cells is not limited to overexpression of 1-

6GlcNAc branches. Sialyl-Lewisx and Sialyl-Lewis

a are tumor-associated N-glycans also

frequently overexpressed in malignant cells. It is proposed that the interaction of these

N-glycans with selectins on the surface of endothelium is involved in the attachment of

the circulating tumor cells with the secondary site of the tumor and the process of

extravasation. Sialyl-Lewisa

is likely involved in the adhesion of human colon, pancreas

and stomach cancers, and Sialyl-Lewisx is responsible for adhesion of lung, liver and

ovarian cancer cells (43). In fact, increased expression of Lewis antigens has been

correlated with worse outcome in colorectal carcinoma patients(44).

14

Given the role of N-glycosylation in cancer, several studies have evaluated the

effects of inhibiting N-glycosylation pathway in malignant cells. In malignant cells,

genetic and chemical inhibition of the N-glycosylation pathway has been shown to inhibit

tumor growth, decrease cell migration and invasion and delay tumor growth and

metastasis. For example, blocking the synthesis of Glc3Man9GlcNAc2-P-P- dolichol with

tunicamycin induces cell death and decreases metastasis. For example, B16 metastatic

melanoma cells were treated with 0.5 g/mL tunicamycin. Tunicamycin decreased cell

growth and lung tumor colony formation(45). Likewise, in a breast cancer xenograft

tunicamycin decreased growth of the breast tumors by 65% in these mice (46)

Similarly, inhibition of α-mannosidase II also has anticancer effects. α-

mannosidase II is a Golgi enzyme that is involved in the synthesis of complex N-glycans.

Swainsonine is a natural indolizidine alkaloid and a potent competitive inhibitor of the α-

mannosidase II enzyme. Cells treated with swainsonine have decreased expression of

complex-type N-glycans and therefore have very low levels of cell-surface L-PHA

staining. By blocking α-mannosidase II, hybrid-type N-glycans accumulate and ConA

binding increases. In cancer cells, treatment with swainsonine does not induce cell death.

Rather, swainsonine inhibits tumor migration and invasion (47-49). In xenograft mouse

models of colorectal carcinoma, melanoma and breast carcinoma, swainsonine inhibited

tumor growth (50-53). Given its promising clinical efficiency, swainsonine was

advanced into Phase I clinical trials for patients with refractory malignancy. In these

phase I trials, patients with advanced malignancies received swainsonine as a continuous

IV infusion or as an oral formula. Pharmacodynamic correlative studies demonstrated

that swainsonine inhibited α-mannosidase II in patients (54). For example, swainsonine

15

inhibited L-PHA staining of peripheral blood lymphocytes in patients receiving drug. A

few clinical responses were reported among the 19 patients who received swainsonine in

the various clinical trials. For example, one patient with head and neck cancer had more

than 50% tumor shrinkage (55). However, swainsonine did not progress beyond phase I

trials primarily because of hepatic and neurological toxicities. The cause of the

swainsonine toxicity may be related to inhibition of the related lysosomal -

mannosidases (Ki ~ 100 nM). By inhibiting the lysosomal -mannosidases swainsonine

promotes the accumulation of oligomannosides in body fluids and tissues.

Oligomannosides accumulation in brain could explain the neurological symptoms of

swainsonine treatment (56). Thus, targeting the N-glycosylation pathway at other sites

with more specific inhibitors might abrogate such untoward toxicities and produce

clinical benefit.

GlcNAc-TV is a key Golgi enzyme in the processing of multiantennary N-

glycans. This gene is overexpressed in some malignant cells(18). Studies have also

demonstrated the association of increased GlcNAc-TV activity with enhanced cell

invasiveness and metastatic potential (57). Finally, GlcNAc-TV expression is increased

in response to a number of oncogenes(56). Given these finding, inhibition of GlcNAc-

TV may be a novel anticancer strategy. Supporting this notion, Granovsky et al showed

that the Mgat5-deficient mice had suppressed tumor growth and metastasis in MMTV-

PyMT model of cancer. Similar observation were made in Pten+/- mice deficient in the

late branching enzyme GlcNAc-TV (18, 41, 42, 58). In addition, Knockdown of

GlcNAc-TV with siRNA in metastatic and invasive breast carcinoma cells decreased

EGF responsiveness and decreased cell motility and invasiveness(59).

16

1.5 A chemical biology approach to identify novel strategies to inhibit

N-glycosylation

In patients with solid tumors, metastatic disease is the cause of 90% of human

cancer deaths while the primary tumor site (save for central nervous system malignancy)

rarely causes death. Micrometastasis may be already present at the time when the primary

tumor is diagnosed. However, recent studies demonstrate that circulating tumor cells can

remain after completion of therapy. These circulating tumor cells can contribute to

relapse and metastasis (60). Thus, there is a need for novel anticancer agents that prevent

metastasis.(61).

An accumulating body of evidence shows a causative role for abnormal

glycoproteins in cancer progression and metastasis(18) . Therefore, modification of the

glycosylation pathway may be a novel strategy for the treatment of malignancy and

prevention of metastasis. Here, we used a chemical biology approach to target the N-

glycosylation pathway.

1.6 A Chemical screen for novel N-glycosylation inhibitors

L-PHA (phytohemagglutinin) binds and cross-links cell surface glycoproteins

with tetraantennary complex type N-glycans and thereby induces agglutination and cell

death. In a classic forward genetic screen, mutations in glycosylation enzymes were

identified by incubating cells with PHA after treatment with mutagenic agents (62-66).

We optimized and automated this screen to identify small molecule that blocked L-PHA

induced cell death. In this screen, Jurkat cells were treated with aliquots of two chemical

libraries (LoPAC and Prestwick). Twenty four hours after treatment L-PHA was added

17

and viability measured by an MTS assay. From screening ~2500 compounds, we

identified dihydroouabain that consistently blocked the L-PHA-induced cell death.

Dihydroouabain and cardiac glycosides (CGs) are approved for treatment of

cardiac arrhythmias and congestive heart failure.(67). The cardiotonic effect of CGs is

due to inhibition of Na+/K

+-ATPase. As a result of Na

+/K

+-ATPase inhibition,

cytoplasmic Ca2+

is increased which, in turn, increases the contractile force of the cardiac

myocytes(Figure 1.3) (68).

18

Figure 1.3. Na+/K

+-ATPase maintains the proper [Na

+] and [K

+] across the cell

membrane.

Under normal conditions, Na+/K

+-ATPase pumps Na

+ out of and K

+ into a cell. The

pump hydrolyzes one molecule of ATP inside the cell in order to pump three Na+ out and

two K+ in. The electrochemical gradient generated by Na

+/K

+-ATPase drives the Na

+-

Ca2+

Exchanger to move Ca2+

out of the cell.

Binding of cardiac glycosides to the extracellular domain of the subunit of Na+/K

+-

ATPase results in an increased intracellular [Na+]. Consequently the Na

+-Ca

2+

Exchanger which relies on the Na+ transmembrane concentration gradient cannot expel

Ca2+

from the cytosol. Thus, the net result of Na+/K

+-ATPase inhibition is an increased

cytosolic Na+

and Ca2+

(69).

[Na+] gradient [K+] gradient3Na+/1ATP

2K+/1ATP

Na+

Ca2+

[Ca2+] gradient

Na+-Ca2+ Exchanger

Na+/K+ -ATPase

Cytosol

Extracellular space

19

The Na+/K

+-ATPase is ubiquitous in all eukaryotic cells. This pump consists of

an catalytic subunit which contains the CG biding site, a regulatory subunit and a or

FXYD modulating subunit. There are four isoforms, 1-4 (transcribed by ATP1A1-4

genes); three isoforms, 1-3 (transcribed by ATP1B1-3) and seven FXYD isoforms,

FXYD1-7 (transcribed by genes of the same name)(69). The and FXYD exhibit a

tissue-specific pattern of expression. In addition, the pattern of isoform expression is

influenced by the developmental stage, hormonal regulations and pathological conditions.

The 12 possible complex combinations show a differential ouabain-sensitivity and the

FXYD subunit plays an additional role in modulating this sensitivity(70).

The cytoplasmic domain of the Na+/K

+-ATPase interacts with many important

signaling proteins including EGFR, SRC kinase and inositol 1,4,5-triphosphate (IP3)

receptor. The Na+/K

+-ATPase assembles these signaling protein complexes in coated

pits. Binding of CGs to Na+/K

+-ATPase can activate the signaling cascades downstream

of these proteins(71).

Several anticancer effects have been reported for CGs in the past 4 decades (67).

For example, in vitro digoxin induces cell death of malignant cells through mechanism

related to activation of Cdk5 (72), Src kinase (73) and p21 (74). However, the

concentration of digoxin required for these effects does not appear to be clinically

achievable.

Some data suggest that systemic administration of CGs to patients may have anti-

cancer effects. For example, Stenkvist et al reported the outcome of 207 patients with

breast carcinomas of which 32 were on cardiac glycoside treatment for cardiac

indications (75, 76). Remarkably, five years after mastectomy, recurrence rate of patients

20

on CGs was close to 10 times less than patients not taking CGs(77). In another recently

published study, a large prospective cohort of men (47,881) were followed from 1986 till

2006. Among these men, 936 were taking digoxin for arrhythmia or congestive heart

failure. In this study, men who used digoxin had a 25% lower risk of prostate cancer

compared to men not taking digoxin(78). Thus, these epidemiological studies support a

potential anti-cancer role for cardiac glycosides at clinically relevant concentrations.

In our study, we showed that Na+/K

+-ATPase inhibitors blocked the N-glycan

branching at a point downstream of the α-mannosidase II inhibitor swainsonine. We

showed that Na+/K

+-ATPase inhibitors also blocked tumor cell migration and invasion as

well as distant tumor formation in two different mouse models. Thus, our results may

help explain previously reported anticancer effects of CGs. Moreover, targeting Na+/K

+-

ATPase could be a new strategy for the development of anticancer therapies.

1.7 GlcNAc-TI: The gateway from oligomannose N-glycans to hybrid

and complex N-glycans

GlcNAc-TI is the obligatory glycosyltransferase for the synthesis of hybrid- and

complex-type N-glycans. In its absence, none of the other Golgi glycosyltransferases can

function and therefore oligomannose N-glycans accumulate indicating that there are no

compensatory pathways for this transferase(79). Stanley’s group showed mice lacking

GlcNAc-TI dies at mid-gestation approximately by day E9.5(80). The catalytic domain

of GlcNAc-TI has one Rossman fold in the first 120 amino acids, which is the

characteristic of all (glycosyltransferase type-A) GT-A glycosyltransferases(16).

GlcNAc-TI has a DXD domain indicating that this enzyme is a metal ion dependent

glycosyltransferase. Based on the stereochemistry of its reaction, GlcNAc-TI is

21

considered an inverting enzyme because the -linked GlcNAc in the UDP--GlcNAc

donor substrate is transferred to Man5GlcNAc2 acceptor via a -linkage. Rini’s group

solved the structure of rabbit GlcNAc-TI and provided a molecular explanation for the

previously explained “sequential Bi-Bi mechanism” of GlcNAc-TI (81, 82). In this

reaction UDP-GlcNAc/Mn2+

must bind first to form a 13 residue loop which creates the

acceptor binding site. Once the reaction is complete, the acceptor is released first

followed by UDP (Figure 1.4).

Given the critical role of GlcNAc-TI in N-glycosylation, we wished to explore the

effects of GlcNAc-TI inhibition in cancer cells. To date, the effects of inhibiting

GlcNAc-TI in malignant cells are unknown. Potentially, inhibiting GlcNAc-TI may

block metastasis and decrease cell invasion by decreasing the synthesis of complex N-

glycan structures (41). The converse is also possible, as high levels of GlcNAc-TI

activity in epithelial cells can deprive GlcNAc-TIV and V of their common substrate,

UDP-GlcNAc, because of the ultrasensitivity of branching pathway to this substrate (83).

Therefore, potentially inhibiting GlcNAc-TI could increase the levels of UDP-GlcNAc in

Golgi and make it more available for the downstream N-acetylglucosaminyltransferases,

including GlcNAc-TV. As such, inhibiting GlcNAc-TI could result in the counterintuitive

effect of increased metastasis and invasion, dependent on the residual state of the

branching pathway and the supply of substances. Therefore, we investigated the effects

of GlcNAc-TI knockdown in tumor cells and studied the effect on cell migration,

invasion and metastasis.

Our investigations showed that partial knockdown of GlcNAc-TI by shRNA in

malignant cells that suppressed complex N-glycans by 50-70%, did not alter proliferation

22

or viability. Rather, GlcNAc-TI inhibition blocked tumor cell migration and invasion.

Moreover, GlcNAc-TI knockdown decreased metastasis of human prostate cancer cells in

a xenograft orthotopic model of prostate metastasis. Finally, we demonstrated that lower

levels of GlcNAc-TI were correlated with a decreased risk of future metastasis. As such,

GlcNAc-TI may be a target for anti-cancer therapies and levels of GlcNAc-TI may be

prognostic in cancer patients.

23

Figure 1.4. The reaction and structure of N-acetylglucosaminyltransferase I.

A. GlcNAc-TI attaches the first GlcNAc to the arm of the trimannosyl core in a

linkage. B. GlcNAc-TI ribbon diagram. The colour coding is as follows: UDP-GlcNAc

and Mn2+

are shown in yellow, the 13 amino acid residue loop structured after UDP-

GlcNAc binding is shown in red, domains 1 and 2 are shown in cyan and brown

respectively and the linker connecting domain 1 and 2 is shown in green. The ribbon

diagram adopted from ref:(82). Copyright permission granted by the publisher, 2011 ©

UDP /Mn2+

GlcNAc-TI

Asn

Man GlcNAc

Asn

1,2

N

N

UDP /Mn2+

A

B

24

1.8 Rational and outline of the thesis

N-glycosylation regulates the localization and function of cell surface

glycoproteins and is required for normal cellular growth, proliferation, differentiation and

homeostasis. Aberrant activity of this pathway is commonly seen in malignant cells and

promotes tumor cell invasion and metastasis. Here, I used a chemical biology approach

and a genetic approach to identify novel strategies to inhibit N-glycosylation in

malignancy.

First, as a chemical biology approach to identify small molecule inhibitors of N-

glycan biosynthesis, I developed a chemical screen based on the ability of the L-PHA to

bind and cross-link surface glycoproteins with 1,6GlcNAc-branched complex type N-

glycans and thereby induce agglutination and cell death. The most potent inhibitor of L-

PHA-induced cell death in this screen was the cardiac glycoside (CG) dihydroouabain.

In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced

agglutination and cell death. All of the CGs tested inhibited L-PHA- induced death and

the most potent CG tested was digoxin. Demonstrating the functional importance of CGs

on N-glycan mediated processes associated with metastasis, CGs inhibited tumor cell

migration in invasion. Furthermore, CGs prevented distant tumor formation in two

mouse models of metastatic prostate cancer. The effects of CGs on N-glycosylation were

due to their inhibition of the Na+/K

+-ATPase and resultant alterations of intracellular

sodium and calcium. These observations could help explain previously reported

anticancer effects of these compounds and highlight new strategies for the development

of novel anticancer therapies (Chapter2).

25

Then, I investigated the mechanism by which inhibition of Na+/K

+-ATPase

blocked the N-glycosylation. Specifically, I sought to investigate the sensitivity of the N-

glycosylation enzymes to changes in ion concentration induced by blocking the Na+/K

+-

ATPase. I demonstrated that GlcNAc-TV was more sensitive to changes in Na+ and Ca

2+

ion concentration than GlcNAc-TI and Golgi -mannosidase II was not affected by

altering these ions. Thus, inhibitors of the Na+/K

+-ATPase may impair the Golgi-residing

N-glycosylation enzymes by altering intracellular Na+

and Ca2+

(Chapter 2).

Finally, I investigated the effect of GlcNAc-TI knockdown in N-glycan regulated

processes of migration, invasion and metastasis. I established tumor cell lines with

GlcNAc-TI knockdown. The knockdown was confirmed by measuring mRNA

expression, enzymatic activity and cell-surface L-PHA staining. When compared with

the cells infected by anti-RFP shRNA, HeLa cells with GlcNAc-TI knockdown had

significantly decreased migration and invasion. Furthermore, GlcNAc-TI knockdown in

PC3N7 cells prevented distant tumor formation in an orthotopic mouse model of

metastatic prostate cancer. These observations suggest a role for GlcNAc-TI in cancer

cell metastases. Therefore, we examined the association between Mgat1 mRNA

expression in primary tumors and the incidence of subsequent metastasis. We found for

the first time that patients with the lowest levels of Mgat1 mRNA in their primary tumors

were less likely to develop metastatic disease after therapy. Thus, inhibitors of GlcNAc-

TI enzyme could have therapeutic use as anti-cancer agents and GlcNAc-TI expression

levels might confer prognostic information in patients with cancer (Chapter 3).

26

Chapter 2

Inhibition of the sodium/ potassium ATPase impairs N-glycan

expression and function

Reza Beheshti Zavareh, Ken S. Lau, Rose Hurren, Alessandro Datti, David J. Ashline,

Marcela Gronda, Pam Cheung, Craig D. Simpson, Wei Liu, Amanda R. Wasylishen, Paul

C. Boutros, Hui Shi, Amudha Vengopal, Igor Jurisica, Linda Z. Penn, Vern N. Reinhold ,

Shereen Ezzat, Jeff Wrana, David R. Rose, Harry Schachter, James W. Dennis, Aaron D.

Schimmer

A similar report was published as follows:

Cancer Res. 2008 Aug 15;68(16):6688-97.

Attribution of the data:

Data is generated and analyzed by primary author with the exception of:

Reza Beheshti Zavareh and Hui Shi : Fig 2.5.A

Reza Beheshi Zavareh and Ken Lau Fig 2.5.B

Reza Beheshti Zavareh and

David Ashline : Table 2.1, Fig 2.7

Reza Beheshti Zavareh and Ken Lau: Fig 2.9, 2.11

Reza Beheshti Zavareh and David Hedley: Fig 2.10


Meenakashi Venkatesan: Fig 2.11

Reza Beheshti Zavareh and Wei Liu: Fig 2.12


Amudha Vengopal: Fig 2.13

27

2.1 Abstract

Aberrant N-linked glycans promote the malignant potential of cells by enhancing the

epithelial-to-mesenchymal transition and the invasive phenotype. To identify small

molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on

the ability of the tetravalent plant lectin L-PHA (phytohemagglutinin) to bind and cross-

link surface glycoproteins with 1,6GlcNAc-branched complex type N-glycans and

thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a

library of off-patent chemicals (n=1280) to identify molecules that blocked L-PHA-

induced death. The most potent hit from this screen was the cardiac glycoside (CG)

dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-

PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-

induced death in Jurkat cells and the most potent CG tested was digoxin with an EC50 of

60 ± 20 nM. Digoxin also increased the fraction of some ConA (concavalinA)-binding

N-glycans. Using MALDI-TOF mass spectrometry, digoxin specifically increased

Gn1M3Gn2F1 and Gn2M3Gn2F1 oligosaccharides demonstrating a block downstream of

the enzyme N-acetylglucosaminyltransferase II. Consistent with this effect on the N-

glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell

migration and invasion. Furthermore, digoxin prevented distant tumor formation in two

mouse models of metastatic prostate cancer. Thus, taken together, our high throughput

screen identified CGs as inhibitors of the N-glycan pathway. These molecules can be

used as tools to better understand the role of N-glycans in normal and malignant cells.

Moreover, these results may partly explain the anti-cancer effect of CGs in

cardiovascular patients.

28

2.2 Introduction

Malignant cells display characteristic changes in N-glycan structures on surface

glycoproteins including receptors and transporters that contribute to cancer progression

and metastasis (reviewed in (18)). Oncogene activation stimulates increased expression

of the Golgi enzymes that generate 1,6GlcNAc-branched tetra-antennary N-glycans.

These N-glycans are found on proteins including growth factor receptors and integrins,

and have been shown to enhance growth signaling in motile tumor cells (28, 83, 84).

Moreover, the expression of tetra antennary N-glycans is necessary for epithelial-to-

mesenchymal transition (EMT)(85). Chemical or genetic disruption of the N-

glycosylation pathway in cancer cells decreases these malignant features(56, 86).

Therefore, targeting defective glycosylation pathways may be a novel approach for the

treatment of malignancy and a strategy to prevent metastasis.

The N-glycosylation pathway begins in the lumen of the rough endoplasmic

reticulum and remodeling in the Golgi apparatus generates structural diversity. To

initiate the pathway, the oligosaccharide precursor Glc3Man9GlcNAc2 is transferred en

bloc from dolichol-pyrophosphate onto asparagine (Asn) residues in the sequence Asn-X-

Ser/Thr (where X can be any amino acid except for proline) to form an asparagine-linked

glycan (N-glycan). Once attached to the protein, this precursor is modified through a

well-defined pathway leading to the sequential removal of the three glucoses and one

mannose by the actions of rough endoplasmic reticulum glycosidases to form

Man8GlcNAc2-Asn. In the Golgi, Man8GlcNAc2-Asn is further modified by the removal

of mannoses via Golgi mannosidases and by the addition of GlcNAc via GlcNAc

transferases, leading to the generation of hybrid and complex N-glycans. Finally other

29

sugars such as fucose, galactose and sialic acid are added to the N-glycans to increase

their diversity (Figure 2.1) (reviewed in (3, 87)).

To better understand the N-glycosylation pathway and identify strategies to target

the aberrant N-glycosylation in neoplastic cells, we designed, automated and conducted a

chemical screen of off-patent drugs and chemicals. This high-throughput screen

identified cardiac glycoside (CG) Na+/K

+-ATPase inhibitors that blocked N-

glycosylation. Subsequent evaluation demonstrated that inhibition of the Na+/K

+-

ATPase increased the fraction of ConA-binding N-glycans by blocking the N-

glycosylation pathway downstream of N-acetylglucosaminyltransferase II (GlcNAc-TII).

Moreover, the effects of CG on N-glycosylation appeared functionally important as these

compounds inhibited the glycosylation mediated processes of cell migration and invasion

as well as decreased distant tumor formation in vivo.

30

2.3 Material and Methods

Figure 1

Figure 2.1 Scheme of the N-glycan biosynthesis pathway.

The N-glycan biosynthesis pathway is shown. The biosynthesis of N-glycans involves

the following steps: (i) Sequential conversion of the high mannose N-glycan M9Gn2 to

M8Gn2, M7Gn2, M6Gn2 and M5Gn2 (oligosaccharides before -mannosidase II in

Table 1); (ii) Addition of 2-linked GlcNAc to the terminal 3-linked Man of M5Gn2

by GlcNAc-TI to form Gn1M5Gn2 (oligosaccharides before -mannosidase II in Table

1); (iii) Removal of two Man residues from Gn1M5Gn2 by -mannosidase II to form

Gn1M3Gn2 (Oligosaccharides after -mannosidase II - Hybrid structures in Table 1);

(iv) Addition of a second 2-linked GlcNAc to the terminal 6-linked Man of

Gn1M3Gn2 by GlcNAc-TII to form Gn2M3Gn2 (Oligosaccharides after -mannosidase

II - Complex structures in Table 1); (v) Addition of L-Fucose (F) and Galactose (Hx)

residues. Swainsonine (SW) is a known inhibitor of the -mannosidase II enzyme. The

abbreviations used are: oligosaccharyltransferase, OT; the -glucosidases, GI, GII; the

-N-acetylglucosaminyltransferases, TI, TII, TIV, TV; the -1,2mannosidases, MI; -

1,3/6mannosidases, MII, MIII; 1,2-galactosyltransferases, Gal-T; -sialyltransferases,

Sa T; UDP-N-acetylglucosamine, UDP-GlcNAc; UDP-galactose, UDP-Gal; CMP-sialic

acid, CMP-SA. The Golgi apparatus subdomains (Cis, Medial and Trans) are shown by

separate boxes. The N-glycan structures that bind ConA and L-PHA are shown by gray

highlights.

31

Reagents.

The LOPAC chemical library, L-PHA, digoxin, digitoxin, dihydrooaubain and

fibronectin were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Cell culture.

Jurkat human leukemia, WRO human thyroid carcinoma, PPC-1 human prostate cancer

and Colo320 colorectal adenocarcinoma cells were maintained in RPMI 1640. HT1080

human fibrosarcoma, 5637 human bladder carcinoma and HeLa human cervical cancer

cells were maintained in Dulbecco’s modified Eagle Medium (DMEM). All cells were

supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and antibiotics.

WRO cells were also supplemented with 1 mM sodium pyruvate. All cell lines were

cultured in a standard humidified incubator at 37°C in a 5% CO2 atmosphere.

High-throughput screen for inhibitors of L-PHA induced cell death.

Liquid handling was performed by a Biomek FX Laboratory Automated Workstation

(Beckman Coulter, Mississauga, ON). Jurkat cells (5,000 cells/well) were seeded in 96

well plates followed by the addition of aliquots from the LOPAC library of 1280 off

patent drugs and chemicals with a final DMSO concentration of 0.05%. Jurkat cells were

selected for this assay as their growth in suspension conditions facilitated the automated

nature of this screen. Twenty four hours after addition of the compound library, L-PHA

was added at a final concentration of 20µg/mL(88). Forty eight hours after the addition

of L-PHA, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) reduction

assay according to the manufacture’s protocols (Promega, Madison, WI) and as

previously described (89). Cell viability was calculated relative to vehicle treated (0.1%

32

DMSO) control cells on each plate. To identify statistically significant hits, we calculated

the Z-score for each hit relative to the negative control. These Z-scores were transformed

into p-values based on the standard-normal distribution. To control for multiple-testing,

we employed a false discovery rate (FDR) correction to generate a q-value for each

compound (90). Statistically significant hits were selected as those with a q-value of less

than 5%, at which level four compounds were identified.

Measurement of ConA-binding glycopeptides.

Quantification of ConA-binding glycans after swainsonine and digoxin treatment was

performed as follows. Briefly, Jurkat cells (1 x 107) were treated with 2 µM swainsonine,

100 nM digoxin, or buffer control for 24 hours. After treatment, cells were harvested,

washed and sonicated. The homogenate was centrifuged and the pellet was solubilized in

6 M guanidine-HCl in 0.1 M Tris buffer (pH 8.0) containing 20 mM dithiothreitol to

reduce disulfide bonds. Solid iodoacetamide was then added to a final concentration of

60 mM and the solution was incubated for one hour to block free sulfhydryl groups. The

solution was then centrifuged to remove any remaining insoluble residue. The

solubilized, reduced and alkylated proteins were precipitated with 10 volumes of cold

absolute ethanol-glacial acetic acid.

The pellet was collected, dried and suspended in 50 mM NH4HCO3. Trypsin (Promega)

at 1:100 (w:w) relative to total protein, was added (half at the beginning of the digestion

and the other half 3 hours later) and after an overnight digestion, the enzyme was

denatured by boiling. After proteolysis and centrifugation, the supernatant was dried and

redissolved in PBS. The peptide mixture was loaded onto a ConA-Sepharose column

equilibrated with PBS buffer. After washing the column using PBS, the ConA-bound

33

glycopeptides were eluted with 15% methyl--D-mannoside in PBS. A SepPak-C18

cartridge was then used to remove the methyl--D-mannoside. The glycopeptides were

eluted from SepPak-C18 with 50% acetonitrile in 0.1% TFA and the solution was

lyophilized. Equal amounts of material were dissolved in H2O and the amount of hexose

in the sample was determined by the phenol-sulfuric acid method (91).

Con-A binding to cell surface glycans.

Cells (500 cells/well) were seeded in 96-well plates. After adhering overnight, cells were

treated with increasing concentrations of digoxin for 48 hours. After treatment, cells

were fixed with 3.7% formaldehyde and washed. Surface N-glycans were stained with

ConcanavalinA (ConA - 20 µg/mL) conjugated to tetramethylrhodamine B

isothiocyanate (TRITC) (EY Laboratories, San Mateo, CA) and nuclei were stained with

Hoechst 33342. The total intensity of ConA staining on each cell was quantified using

Cellomics ArrayScan II (Cellomics, Pittsburgh, PA) (83).

siRNA transfections.

One x105 cells were seeded in six-well plates and transfected the next day using

Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and double-stranded siRNAs targeting

either the human Na+/K

+-ATPase, or Non-Targering siRNA (siControl) (Smartpool,

Dharmacon, Lafayette, CO, USA). Cells were harvested 72 hours post-transfection and

then assayed for Con-A binding to cell surface glycans.

Reverse-transcriptase real-time PCR.

First-strand cDNA was synthesized from 1 g of DNase-treated total cellular RNA using

random primers and SuperScript II reverse transcriptase (Invitrogen) according to the

manufacturer’s protocols. Real-time PCR assays were performed in triplicate with 5 ng

34

of RNA equivalent cDNA, SYBR Green PCR Master mix (Applied Biosystems, Foster

City, CA, USA), and 400 nM of gene-specific primers. Reactions were processed and

analyzed on an ABI 7900 Sequence Detection System (Applied Biosystems).

Forward/reverse PCR primer pairs for human cDNAs were as follows: murine Na+/K

+-

ATPase: Forward 5’- TGT GAT TCT GGC TGA GAA CG-3’, Reverse 5’- TCT TGC

AGA TGA CCA AGT CG-3’, 18S: Forward 5’- AGG AAT TGA CGG AAG GGC

AC-3’, Reverse 5’-GGA CAT CTA AGG GCA TCA CA-3’.Relative mRNA expression

was determined using the CT method as described (92).

DNA constructs and generation of stable cell lines.

Stable cell lines expressing the alpha subunit of the murine Na+/K

+-ATPase were

engineered by transfecting PPC1 human prostate cancer cells with cDNA corresponding

to the murine Na+/K

+-ATPase in pcDNA3 vector using Lipofectamine 2000 (Invitrogen,

Burlington, ON, Canada) according to the manufacturer’s instructions. Cells stably

expressing murine Na+/K

+-ATPase were selected with 800 µg/mL G418 (Invitrogen).

MALDI-TOF mass spectrometry to semi quantify N-glycan expression.

Jurkat cells (1 x 108) were treated with 100 nM digoxin, 2 µM Swainsonine or buffer

control. After incubation, cells were lysed in 35 mM Tris, 8M urea, 4% CHAPS, 65 mM

DTT, pH 8.0 followed by sonication and freezing. Equal amounts of protein were

dialyzed with cassettes (Slide-A-Lyzer, molecular weight cutoff of 7,000, Pierce,

Rockford, IL) in 10 mM NH4HCO3/0.02% SDS. Following dialysis, samples were

concentrated via vacuum centrifugation. Samples were deglycosylated at 37ºC for 48

hours with PNGase F (New England Biolabs, Ipswich, MA). Oligosaccharides were

purified via C18 and porous graphitized carbon solid phase extraction. The composition

35

of N-glycans was semi-quantified using mass spectrometry via MALDI-TOF, using

DHB as matrix, on a MALDI-CFR (Shimadzu Biotech, Columbia, MD) as previously

described (93, 94). The intensities of the peaks were arbitrarily normalized to the level of

(Hexose)2(HexNAc)2(Deoxyhexose)1.

Glycosyltransferase assays.

To measure the effects of Na+ and Ca

2+ on the activity of GlcNAc-TI and GlcNAcT-V,

recombinant GlcNAc-TI and GlcNAc-TV with a truncated transmembrane domain were

incubated with their acceptors Man(1,3)Man(1,6)βGlc-O(CH2)7CH3 and GlcNAc1-

2Mana1-2Man1octylCH3 (Toronto Research Chemicals), respectively in 0.5 mM UDP-

[63H]GlcNAc (44400 dpm/nmol) in 125 mM MES pH 6.5, 50 mM GlcNAc, 1 mM UDP-

GlcNAc, 0.8 mM AMP and 10 mM MnCl2 (for GNTI only) with increasing

concentrations of CaCl2 and NaCl. Transfer of [63H]GlcNAc to the acceptor was

quantified by liquid scintillation counting(83).

Mannosidase assay.

To measure the effects of Na+ and Ca

2+ on the enzymatic activity of Golgi Mannosidase

II(GMII), recombinant drosophila GMII(dGMII) was incubated with its substrate para-

nitrophenyl -D-mannopyranoside (Sigma) in 40 mM MES pH 5.75 and increasing

concentration of Na+ and Ca

2+. Conversion of the substrate to para nitrophenol was

measured with a previously described colorimetric assay (95).

Measurement of intracellular Ca2+

and Na+.

Changes in cytosolic calcium were measured by staining cells with indo-1 AM

(Invitrogen Canada, Burlington, Canada) (final concentration 3 μM) and obtaining an

electronic ratio of indo-1 emission at 405 over 525 nm at an excitation of

36

360 nm as previously described(96). After incubation, cells were stained with Na Green

(Molecular probes) and the intensity of staining was measured using the Axiovert

fluorescent microscope (40X magnification). In order to determine alterations in

intracellular Na+, cells were stained with Na Green (Molecular probes) and the intensity

of staining was measured using the Axiovert fluorescent microscope (40X

magnification).

Scratch wound healing assay.

Scratch wound healing assays were performed as previously described(30, 97). Briefly,

HT1080 (1 x 106) cells were treated with digoxin or buffer control. After 24 hours, cells

were harvested and seeded (1x 105) on fibronectin-coated four-well chamber slides.

After adhering overnight, the monolayer was scratched with a plastic pipette tip.

Migration of the cells over six hours was captured with a digital camera (Nikon,

Mississauga, ON) mounted on an inverted microscope (Nikon, Mississauga, ON).

Cellular migration was measured in relative units (pixels).

Migration and invasion assays.

Invasion and migration assays were performed as previously described(97). Briefly WRO

cells (2 x 105) were treated with digoxin or buffer control for 24 hours. After treatment

cells were harvested and seeded in uncoated invasion chambers for migration assay or

BioCoat Matrigel Invasion Chambers (BD Biosciences, Mississauga, ON) in serum-free

RPMI 1640 medium containing 0.2% BSA were used for invasion assays. Growth

medium containing 5% FBS was used as a chemoattractant in the bottom well.

Following 24 hours of incubation, cells that had migrated or invaded the lower surface of

the membrane were stained with Diff-Quik Stain (BD Biosciences). The number of

37

migrating or invading cells were imaged and counted using the Aperio ScanScope CS

whole slide Scanner (Aperio Technologies, Vista, CA) and Image-Pro Plus Software

(version 4.5; Media Cybernetics Inc., Silversprings, MD).

In vivo studies.

The effects of digoxin on distant tumor formation in vivo were evaluated as

previously described (89). Briefly, dsRED-PPC-1 cells that stably express dsRed2

fluorescent protein were treated in culture with digoxin (100nM) or buffer control for 20

hours. After treatment, 3.5x106 viable cells (as determined by trypan blue exclusion

assay) were either injected via the tail vein or subcutaneously into the hind limbs into

sublethally irradiated (3.5 Gy) male severe combined immunodeficient (SCID) mice

between 5 and 7 weeks of age. The number of cells injected was at least 3-fold above the

minimum threshold required for distal tumor formation. Mice injected with tumor cells

subcutaneously were maintained for 2 weeks and then sacrificed via carbon dioxide

inhalation. Tumors were excised and weighed. Mice injected with tumor cells

intravenously were maintained for 4 weeks after injection or until moribund, at which

time, the animals were sacrificed via carbon dioxide inhalation for complete examination.

In a separate model, DsRed labeled PPC-1 cells (3x106) were injected

intravenously into sublethally irradiated SCID mice. Mice were then treated with digoxin

or buffer control daily for 2 weeks. Four weeks after injection of the cells, the mice were

sacrificed via carbon dioxide inhalation and dissected.

Red fluorescent tumors were detected via whole body imaging and whole organ

imaging using a Leica MZ FLIII fluorescent stereomicroscope with a 100 W mercury

lamp, a 560/40 excitation filter, and a 610 long-pass emission filter. Images were

38

acquired using a Olympus DP70 digital camera at 0.8 X magnification and analyzed

using Image Pro Plus 6.0 (MediaCybernetics). A single common threshold was applied

to identify and measure fluorescence in each organ (98). The number of fluorescent spots

was recorded for each lung lobe. All quantification was performed on unmanipulated

images.

Mice were obtained from an in-house breeding program and housed in laminar-

flow cage racks under standardized environmental conditions with ad libitum access to

food and water. All experiments were performed according to the regulations of the

Canadian Council on Animal Care.

39

2.4 Results

Inhibitors of L-PHA induced-cell death identified via a high-throughput screen.

Phytohemagglutinin is a tetravalent plant lectin that binds and cross-links complex N-

glycoproteins on cell surfaces and induces agglutination and cell death(99). To identify

compounds that inhibit N-glycan remodeling in malignant cells, we developed,

automated, and conducted a chemical screen for inhibitors of L-PHA-induced cell death

in Jurkat cells. In the optimized assay, Jurkat cells were seeded in 96-well plates treated

with aliquots of the LOPAC (n=1280) library of off-patent drugs and chemicals (final

concentration ~ 5 µM and 0.05% DMSO). Twenty four hours after incubation, cells were

treated with L-PHA (final concentration 20 µg/mL), and forty-eight hours later cell

viability was measured by the MTS assay (Figure 2.2). As controls, cells received L-

PHA or buffer alone. To identify molecules that inhibited L-PHA induced cell death, we

employed a statistically robust methodology. For each compound in the LOPAC library,

we calculated a Z-score using the extensively replicated L-PHA control wells. These Z-

scores were converted into p-values using the standard normal distribution. As we

screened 1280 distinct compounds, we then applied a false discovery rate (FDR)

correction for multiple-hypothesis testing to the vector of p-values (90). Statistically

significant hits were defined as compounds with a q-value (adjusted p-value) less than

0.05, indicating a false-positive rate of at most 5%. Four statistically significant hits were

identified in this manner. Of the four statistically significant hits, secondary screening

validated one compound: the Na+/K

+-ATPase inhibitor dihydroouabain. This compound

restored viability of L-PHA-treated cells to 70% or over of untreated cells.

40

0

10

20

30

40

50

60

70

80

90

0 200 400 600 800 1000 1200 1400

Chemicals

Via

bilit

y (

% c

on

tro

ls)

Figure 2.2

Dihydroouabain

A

Figure 2.2 A chemical screen identifies cardiac glycosides as inhibitors of L-PHA induced

cell death.

Jurkat cells (5000) were seeded in 96 well plates and treated with aliquots of the LOPAC

chemical library (final concentration ~5 µM). Twenty four hours after treatment, L-PHA (final

concentration 20 µg/mL) was added. Forty eight hours after L-PHA addition, cell viability was

measured by an MTS assay. Viability was expressed as a percentage of buffer treated control

cells. Statistically significant hits were selected as those with a q-value of less than 5%. Four

statistically significant hits were identified in this manner which restored viability of L-PHA-

treated cells to 70% or over of untreated cells (dashed line).

41

Other cardiac glycosides inhibit L-PHA induced cell death.

Dihydroouabain belongs to the cardiac glycoside (CG) family of Na+/K

+- ATPase

inhibitors which are used clinically to treat patients with heart failure and atrial

arrhythmias (100). Moreover, CGs have potential anti-tumor properties in patients (75,

77), but the mechanism by which they exert this effect is unknown. To determine

whether the effects on N-glycan remodeling were specific to dihydroouabain or a class-

effect of CGs, we evaluated a panel of CGs for inhibition of L-PHA-induced cell death.

Jurkat cells were treated with L-PHA along with increasing concentrations of digoxin,

digitoxigenin, digitoxin and dihydroouabain and cell viability was measured by the MTS

assay (Figure 2.3). All CGs tested blocked L-PHA-induced cell death. The most potent

CG tested was digoxin that inhibited L-PHA-induced cell death with an EC50 of 60 ± 20

nM. This result is in keeping with digoxin being a more potent inhibitor of the Na+/K

+-

ATPase than dihydroouabain and digitoxigenin(101) . In addition to inhibition of L-

PHA-induced cell death, CGs also blocked L-PHA mediated agglutination of the Jurkat

cells. Of note, at higher concentrations of the CGs or longer periods of incubation, the

CGs directly induced cell death, consistent with previous reports (74, 102). Compared to

the human isoform, CGs bind the 1 subunit of murine Na+/K

+-ATPase with less

affinity, thereby rendering murine cells resistant to any effects of CGs that are mediated

through this ATPase(68, 103). Consistent with this prediction, the tested CGs did not

block L-PHA-induced cell death nor agglutination of MDAY-D2 murine leukemia cells,

suggesting that CGs inhibition of L-PHA toxicity to Jurkat cells requires its known

function as inhibitors of human Na+/K

+-ATPase (Figure 2.4).

42

Dihydroouabain

50

60

70

80

90

100

110

0.01 0.1 1 10 100

µM

Via

bil

ity(%

Co

ntr

ol)

Digitoxin

50

60

70

80

90

100

110

0.01 0.1 1 10 100

µMV

iab

ilit

y (

% C

on

tro

l)

Digitoxigenin

50

60

70

80

90

100

110

0.01 0.1 1 10 100

µM

Via

bil

ity (

% C

on

tro

l)

Digoxin

0

20

40

60

80

100

0 50 100 150

(nM)

Via

bilit

y (

% C

on

tro

l)

Figure 2.3

Figure 2.3. Other cardiac glycosides inhibit L-PHA induced cell death.

Jurkat cells (5000) were seeded in 96 well plates and treated with increasing concentrations

of dihydroouabain, digitoxigenin, digitoxin and digoxin. Twenty four hours after treatment,

L-PHA (final concentration 20 µg/mL) was added. Forty eight hours after L-PHA addition,

cell viability was measured by an MTS assay. Viability was expressed as a percentage of

buffer treated control cells. Data represents the mean ± SD percent viable cells.

43

0

20

40

60

80

100

0.01 0.1 1 10 100

Via

bili

ty (

% C

ontr

ol)

M

0

20

40

60

80

100

0.01 0.1 1 10 100

Via

bili

ty (

% C

ontr

ol)

M

Figure 2.4. Testing the effect of digoxin in murine MDAY lymphoma cells.

A. MDAY cells (5000 cells/mL) were seeded in 96 well plates and treated with

increasing concentrations of digoxin. 72 hours after treatment, cell viability was

measured by an MTS assay. Viability was expressed as a percentage of buffer treated

control cells. Data represents the mean.

B. MDAY cells (5000 cells/mL) were seeded in 96 well plates and treated with

increasing concentrations of digoxin. Twenty four hours after treatment, L-PHA (final

concentration 20 µg/mL) was added. Forty eight hours after L-PHA addition, cell

viability was measured by an MTS assay. Viability was expressed as a percentage of

buffer treated control cells. Data represents the mean.

A

B

44

Digoxin increases the levels of total and cell surface ConA-binding glycoproteins

To explore the effect of inhibiting the Na+/K

+-ATPase on the N-glycan profile of cancer

cells, we measured the effects of digoxin on levels of ConA-binding high mannose and

hybrid N-glycans. Jurkat cells were treated with digoxin (100nM), the known Golgi -

mannosidase II inhibitor, swainsonine (2 µM) (50, 99), or buffer control for 24 hours.

After treatment, the abundance of ConA-binding glycopeptides was measured by the

phenol-sulfuric acid method, as described in the Methods section. Digoxin and

swainsonine increased the abundance of ConA-binding glycopeptides 2.3 and 1.8 fold,

respectively, compared to controls (Figure 2.5.A).

To further explore the effects of Na+/K

+-ATPase inhibition on ConA-binding

glycans and to evaluate the generalizability of this finding, we measured the binding of

ConA to the surface of a panel of solid tumor cell lines. Colo320, 5637, Hela, WRO,

PPC-1, and HT1080 cells were treated with increasing concentrations of digoxin for 48

hours. After treatment, cells were fixed and stained with TRITC-labeled ConA. The

binding of fluorescently-labeled ConA was quantified by the Cellomics Array scan

(Figure 2.5.B). Consistent with a block in N-glycan pathway, digoxin increased the

binding of ConA to the surfaces of all the cell lines studied.

To determine whether the effects of digoxin on the surface expression of ConA

binding proteins were due to inhibition of its known target Na+/K

+-ATPase, we over-

expressed in PPC1 human prostate cancer cells the 1 subunit of murine Na+/K

+ -ATPase

that is less sensitive to cardiac glycosides. Increased expression of the Na+/K

+-ATPase

was confirmed by Q-RTPCR. Over-expression of the 1 subunit of murine Na+/K

+-

ATPase blocked the effects of digoxin and prevented the increase in surface ConA

45

staining (Figure 2.6.A). Likewise, we transfected PPC-1 cells with siRNA against the

1 subunit of human Na+/K

+ -ATPase (ATP1A1) and knockdown of the ATP1A1 mRNA

was confirmed by Q-RTPCR. Compared to cells treated with control siRNA, PPC-1 cells

transfected with siRNA against the Na+/K

+ -ATPase displayed increased ConA binding

proteins on the cell surface (Figure 2.6.B). Thus, taken together, these results

demonstrate that inhibition of the Na+/K

+ -ATPase blocks the N-glycan pathway.

46

Figure 2.5

0

10

20

30

40

50

60

70

80

Control Swainsonine DigoxinGly

co

pe

ptid

e C

on

ce

ntr

atio

n (

µg

/ µ

L)

A

P = 0.0008

***

P<0.0001

***

Figure 2.5. Digoxin increases levels of ConA-binding glycoproteins through

inhibition of the Na+/K+-ATPase

A) Jurkat cells (1 x 107) were treated with, 100 nM digoxin, 2 µM

Swainsonine or buffer control for 24 hours. After treatment, cells were

harvested and the levels of ConA-binds glycopeptides were measured by the

colorimetric technique as described in the materials and methods. Data

represents the mean + SD concentration of ConA-binding glycopeptides.

B) Colo320, 5637, PPC-1, Hela, WRO and HT1080 cells (1 x 104 cells /mL)

were seeded in 96-well plates and treated with increasing concentrations of

digoxin for 48 hours. After incubation, cells were stained with TRITC-labeled

ConA and Hoechst 33342. The intensity of ConA staining was measured using

the automated Arrayscan microscope as described in the materials and methods.

Data represents the mean ± SD fluorescent intensity(Next page).

47

0 50 100 15015000

20000

25000

30000

35000

5637

Digoxin (nM)

Co

nA

In

ten

sit

y

0 50 100 15015000

20000

25000

30000

35000

Colo320

Digoxin (nM)

Co

nA

In

ten

sit

y

0 50 100 150

2500

4500

6500

8500

10500

WRO

Digoxin (nM)

Co

nA

In

ten

sit

y

0 50 100 1505000

7500

10000

12500

15000

Hela

Digoxin (nM)

Co

nA

In

ten

sit

y

0 50 100 150

5000

10000

15000

HT1080

Digoxin (nM)

Co

nA

In

ten

sit

y

B

Figure 2.5

PPC1

0 50 100 15010000

20000

30000

40000

50000

Digoxin (nM)

Co

nA

In

ten

sit

y

48

Figure 2.6

AP=0.026

*

P=0.7NS

Vector mATP1A10

50

100

150 Control

Digoxin

Co

nA

In

ten

sit

y(%

Co

ntr

ol)

B

Control ATP1A1 siRNA0

20000

40000

60000

Co

n A

In

ten

sit

y

P=0.0003

***

Figure 2.6 The effects of digoxin on cell surface ConA staining are due to an on

target effect of Na+/K

+ -ATPase inhibition

A) PPC-1 cells stably transfected with cDNA corresponding to the 1 subunit of

murine Na+/K

+-ATPase or vector control were treated with digoxin (50 nM) for 48

hours. After incubation, cells were stained with TRITC-labeled ConA and Hoechst

33342 and the intensity of ConA staining was measured using the automated

Arrayscan microscope as described in the materials and methods. Data represents the

mean + SD fluorescent intensity.

B) PPC-1 cells were transfected with siRNA corresponding to the human Na+/K

+ -

ATPase or a control siRNA. Three days after transfection, cells were harvested and

stained with TRITC-labeled ConA and Hoechst 33342 and the intensity of ConA

staining was measured using the automated Arrayscan microscope as described in the

materials and methods. Data represents the mean + SD fluorescent intensity.

49

MALDI-TOF mass spectrometry shows a block down stream of -mannosidase II.

In order to identify the site in the N-glycan pathway blocked by Na+/K

+-ATPase

inhibitors, Jurkat cells were treated with digoxin, the known Golgi -mannosidase II

inhibitor swainsonine, or buffer control. After treatment, the expression profile of

intracellular oligosaccharides was measured by MALDI-TOF mass spectrometry (Table

2.1 and Figure 2.7). Swainsonine increased the abundance of M9Gn2, M8Gn2, M7Gn2,

M6Gn2 and M5Gn2, which is consistent with a block at Golgi -mannosidase II and

accumulation of these precursors. In contrast treatment with digoxin decreased the

abundance of these oligosaccharides, but increased the levels of Gn1M3Gn2F1 hybrid

and Gn2M3Gn2F1 bi-antennary complex N-glycans. Slight increases in Gn3M3Gn2F1

tri-antennary and Gn4M3Gn2F1 tetra- antennary complex N-glycans were also seen.

These results indicate that inhibition of the Na+/K

+-ATPase by digoxin blocks the N-

glycan pathway downstream of the N-acetylglucosaminyltransferase II (GlcNAc-TII)

enzyme thereby promoting the accumulation of hybrid and bi-antennary complex N-

glycans. Digoxin therefore inhibits the N-glycan remodeling through a mechanism

distinct from swainsonine.

50

Table 2.1. Digoxin and swainsonine alter the expression of oligosaccharides

Jurkat cells (1 x 107) were treated with 100 nM digoxin, 2 µM Swainsonine (SW) or

buffer control for 24 hours. After incubation, oligosaccharides were isolated and analyzed

by MALDI-TOF mass spectrometry at Glycomics facility of Dr. Reinhold. The ability of

glycans to bind to Con-A is based on previous studies using standard N-glycans and Con

A-Sepharose columns (104, 105). Previous work from several groups (e.g. (106)) with

mass spectrometry of N-glycans has shown that the m/z values in Table 1 correspond to

structures in which Hexose is D-Mannose (M) or unidentified (Hx, probably D-

Galactose), HexNAc is N-Acetyl-D-glucosamine (Gn), and Deoxyhexose is L-Fucose

(F).

51

m/z Structure Ctrl Digoxin SW ConA

Binding

1257 M5Gn2 37.39 28.88 67.63 ++

1403 M5Gn2F1 1.35 0 49.71 ++

1419 M6Gn2 25.22 15.06 36.41 ++

1581 M7Gn2 16.21 11.11 26.01 ++

1606 Gn1M5Gn2F1 1.35 0.987 21.96 ++

1622 Hx1Gn1M5Gn2 1.8 1.4 4.62 ++

1743 M8Gn2 20.72 10.86 26.01 ++

1905 M9Gn2 13.96 8.64 16.76 ++


Binding

1136 GnM3Gn2 2.25 4.19 2.31 +

1282 GnM3Gn2F1 8.11 17.78 6.94 +


Binding

1485 Gn2M3Gn2F1 4.5 29.87 5 .20 ++

1645 Hx1Gn2M3Gn2

F1

0 0 0 ++

1689 Gn3M3Gn2F1 1.8 5.43 2.89 --

1810 Hx2Gn2M3Gn2

F1

1.8 1.48 2.89 --

1892 Gn4M3Gn2F1 0 2.47 2.31 --

Oligosaccharides before -mannosidase II

Oligosaccharides after -mannosidase II- Hybrid structures

Oligosaccharides after -mannosidase II- Complex structures

Table 2.1

52

Oligosaccharides before -mannosidase II

M5

Gn

2

M5

Gn

2F

1

M6

Gn

2

M7

Gn

2

Gn

1M

5G

n2

F1

Hx

1G

n1

M5

Gn

2

M8

Gn

2

M9

Gn

2

M5

Gn

2

M5

Gn

2F

1

M6

Gn

2

M7

Gn

2

Gn

1M

5G

n2

F1

Hx

1G

n1

M5

Gn

2

M8

Gn

2

M9

Gn

2

M5

Gn

2

M5

Gn

2F

1

M6

Gn

2

M7

Gn

2

Gn

1M

5G

n2

F1

Hx

1G

n1

M5

Gn

2

M8

Gn

2

M9

Gn

2

0

20

40

60

80

Ctrl Digoxin SW

Rela

tive a

bu

nd

an

ce

Oligosaccharides after -mannosidase II

(Hybrid Structures)

Gn

M3

Gn

2G

nM

3G

n2

F1

Gn

M3

Gn

2G

nM

3G

n2

F1

Gn

M3

Gn

2G

nM

3G

n2

F1

0

5

10

15

20

Rela

tive a

bu

nd

an

ce

Ctrl

Digoxin

SW

Oligosaccharides after -mannosidase II(Complex structures)

Gn

2M

3G

n2

F1

Hx

1G

n2

M3

Gn

2F

1

Gn

3M

3G

n2

F1

Hx

2G

n2

M3

Gn

2F

1

Gn

4M

3G

n2

F1

Gn

2M

3G

n2

F1

Hx

1G

n2

M3

Gn

2F

1

Gn

3M

3G

n2

F1

Hx

2G

n2

M3

Gn

2F

1

Gn

4M

3G

n2

F1

Gn

2M

3G

n2

F1

Hx

1G

n2

M3

Gn

2F

1

Gn

3M

3G

n2

F1

Hx

2G

n2

M3

Gn

2F

1

Gn

4M

3G

n2

F1

0

10

20

30

Rela

tive a

bu

nd

an

ce

Ctrl

Digoxin

SW

Figure 2.7 The graphic presentation of the MALDI-TOD data.

The data from Table 1 are summarized here. Data represent the normalized intensity of

each oligosaccharide relative to (Hexose)2(HexNAc)2(Deoxyhexose)1

53

Increased Ca2+

and Na+ concentrations inhibit the enzymatic activity of GlcNAc-TV

As inhibitors of the Na+/K+ ATP-ase blocked the N-glycosylation pathway downstream

of -mannosidase II, we examined the effects of digoxin on GlcNAc-TV expression and

function. To determine whether Na+/K+ ATP-ase inhibition alters GlcNAc-TV

expression, Jurkat cells were treated with increasing concentration of digoxin and levels

of Mgat5 mRNA were measured by Q-RTPCR. No change in Mgat5 mRNA was

detected after digoxin treatment (Figure 2.8). However, Mgat1 mRNA was decreased

with an increasing concentration of digoxin.

To determine whether digoxin directly inhibits the function of GlcNAc-TV,

recombinant GlcNAc-TV was treated in a cell-free assay with increasing concentrations

of digoxin. No change in GlcNAc-TV activity was observed at concentration of digoxin

up to 100 nM (Figure 2.9), indicating that digoxin is not a direct inhibitor of GlcNAc-

TV.

Inhibition of Na+/

K+ ATP-ase increases intracellular Na

+, and intracelluar Ca

2+

also increases due to increased activity of the Na+/Ca

2+ contraport (69). Potentially,

digoxin alters the intracellular ions could reversibly impair the function of GlcNAc-TV. .

In Support of this hypothesis, concentration of digoxin that impaired N-glycans

expression increased intracellular Na+ and Ca

2+ in malignant cell lines as measured by

staining cells with Na Green (Molecular Probes) and Indo-1-Am (Molecular Probes),

respectively (Figure 2.10). Therefore, we examined the effects of altering Na+ and Ca

2+

concentrations on the function of GlcNAc-TV. Recombinant GlcNAc-TV activity was

assessed in a cell-free assay in the presence of increasing concentration of Na+

and Ca2+

.

Increasing the Na+ or Ca

2+ concentration impaired the function of GlcNAc-TV(Figure

54

2.11). In contrast, no significant change in GlcNAc-TI or dGMII activity was observed

at the same Na+ and Ca

2+ concentrations. Thus, the GlcNAc-TV enzymes are sensitive to

alteration in Na+ and Ca

2+ and therefore blocking the Na

+/K

+ ATP-ase could reversibly

inhibit the function of GlcNAc-TV by altering intracellular Na+ and Ca

++ concentrations.

Rela

tive m

RN

A e

xp

ressio

n

0. 25 50 75 0. 25 50 75 0. 25 50 75 0. 25 50 75 0. 25 50 750

1

2

3

Mgat1

Mgat4B

Mgat4A

Mgat3

Mgat5

Digoxin (nM)

Figure 2.8 The effect of digoxin on mRNA expression levels of key golgi

glycosyltransferases.

Jurkat cells were treated with digoxin (100 nM) for 24 hours. Cells were

harvested and mRNA extracted. Levels of Mgat1,Mgat3,Mgat4A and Mgat5

mRNA were measured by QRTPCR. Data represent the mean + SD relative

expression of mRNA relative to control sequence (n = 3 independent

experiments performed in triplicate)

55

GlcNAc-TI assay

0 25 50 75 100 1250

500

1000

1500- GlcNAc-TI acceptor

+ GlcNAc-TI acceptor

Digoxin (nM)

nm

ole

3H

-UD

P-G

lcN

Ac/g

lysate

GlcNAcTV assay

0 25 50 75 100 1250

25

50

75

100- GlcNAc-TV acceptor

+ GlcNAc-TV acceptor

Digoxin (nM)

nm

ole

3H

-UD

P-G

lcN

Ac/g

lysate


increasing concentrations of digoxin.

recombinant GlcNAc-TI and GlcNAc-TV with a truncated transmembrane domain were

incubated with their acceptors Man(1,3)Man(1,6)βGlc-O(CH2)7CH3 and GlcNAc1-

2Mana1-2Man1octylCH3 (Toronto Research Chemicals), respectively (+) and without the

acceptors (-) in 0.5 mM UDP-[63H]GlcNAc (44400 dpm/nmol) in 125 mM MES pH 6.5, 50

mM GlcNAc, 1 mM UDP-GlcNAc, 0.8 mM AMP and 10 mM MnCl2 (for GNTI only) with

increasing concentrations of digoxin. Transfer of [63H]GlcNAc to the acceptor was

quantified by liquid scintillation counting.

56

Figure 2.10 . Digoxin increases the intracellular levels of Na+ and Ca

2+.

A. HT1080 cells were treated with digoxin for 24 hours. After incubation, cells were

stained with Na Green (Molecular probes) and the intensity of staining was measured

using the Axiovert fluorescent microscope (40X magnification). B. Jurkat cells were

treated with 50 nM Digoxin and incubated over night. After incubation cells were stained

with Indo-1-AM and an electronic ratio of indo-1 emission at 405 over 525 nm at an

excitation of 360 nm was obtained as described previously (96).

Control

Figure 3.3

Control Digoxin0

1000

2000

3000

4000

5000

6000

RF

U

A

B

% o

f M

ax

imu

m C

ell

Nu

mb

er

Ratio: Calcium 405/525

Digoxin

57


increasing concentrations of Na+ and Ca

2+.

A. Recombinant GlcNAc-TI and GlcNAc-TV enzymes were incubated in the presence of

their respective acceptors and UDP-[63H]GlcNAc in buffers with increasing

concentrations of Na+. The enzymatic activities of the GlcNAc-TI and GlcNAc-TV were

measured as described previously (83). Data represent the mean ± SD fold change in

disintegration per minute (DPM) compared to incubation in the presence of 15.6 mM

Na+. B. Recombinant dGMII was incubated with its substrate p-nitrophenyl--D-

mannopyranoside (pNP-Man) in 40 mM MES pH 5.75, with increasing concentration of

Na+. The reaction was stopped by the addition of 50 mL of 0.5 M Na2CO3. The

conversion of pNP-Man to para nitrophenol was measured at 405 nm using a plate reader

(SoftmaxPro, Molecular Devices Inc). Data represents mean + SD of relative activity

compared to 0 mM Na+. C. Recombinant GlcNAC-TI and GlcNAc-TV were incubated

with their respective acceptors with UDP-[63H]-GlcNAc as described above with

increasing concentration of Ca2+

. Data represents mean + SD fold change in DPM

compared to incubation in presence of 3 mM Ca2+

. D. Recombinant dGMII was

incubated with its substrate pNP-Man as described above with increasing concentration

of Ca2+

. Data represents mean + SD of relative activity compared to 0 mM Ca2+

.

58

Figure 2.11

Figure 3.1

A

B

0 100 200 3000.0

0.4

0.8

1.2

1.6

Mgat1

Mgat5

Sodium (mM)

En

zym

e a

cti

vit

y (

Fo

ld D

PM

Ch

an

ge)

0 100 200 3000.0

0.4

0.8

1.2

1.6

Sodium (mM)

Rela

tive

-man

no

sid

ase

acti

vit

y

dGMII

0 15 30 45 600.0

0.4

0.8

1.2

Mgat1

Mgat5

Calcium (mM)

En

zym

e a

cti

vit

y (

Fo

ld D

PM

Ch

an

ge)

0 5 10 150.0

0.4

0.8

1.2dGMII

Calcium (mM)

Rela

tive

-man

no

sid

ase

acti

vit

y

C

D

59

Digoxin decreases migration and invasion of malignant cells

Increased GlcNAc-branching of N-glycans on the cell surface promotes the malignant

and metastatic potential of cells by altering cell migration and invasion (18 , 107) . As

treatment with digoxin blocked the N-glycan remodeling, we evaluated the effects of this

compound on the N-glycosylation-mediated processes of cell migration and invasion. To

determine the effects on cell migration, we evaluated the effect of digoxin in a scratch-

wound healing assay. HT1080 human fibrosarcoma cells were treated with digoxin or

buffer control and seeded in fibronectin-coated four-well chamber slides. After adhering

overnight, the cell monolayer was scratched to create a wound. Migration of cells to heal

the wound was measured over time. Treatment with digoxin impaired cell migration and

delayed wound healing (Figure 2.12.A). Of note, at the concentrations tested in these

assays, digoxin-treated cells were more than 90% viable as measured by the MTS assay.

Furthermore, the treated cells migrated sufficiently to completely heal the wound by 24

hours. Thus, the effects of digoxin on cell migration cannot be attributed to simple

reductions in cell viability.

To further evaluate the effects of digoxin on cell migration, WRO human thyroid

carcinoma cells were treated with digoxin and migration through uncoated invasion

chambers with an 8 µm pore size was measured (Figure 2.12.B). Again, digoxin

inhibited cell migration but did not reduce cell viability as measured by MTS assay.

Finally, we assessed the effects of digoxin on cell invasion (Figure 2.12.C).

WRO cells were treated with digoxin and seeded into matrigel-containing invasion

chambers in serum free media. Media with 5% FBS was placed in the lower chamber as

a chemoattractant. Twenty four hours after seeding, cell invasion through the martigel

60

was measured. Digoxin treatment decreased cell invasion, but did not reduce cell

viability as measured by the MTS assay.

Thus taken together, concentrations of digoxin that blocked N-glycan remodeling

also inhibited the N-glycosylation mediated processes dependent on GlcNAc-branched N-

glycans; cell migration and invasion. These results suggest that the effects of digoxin on

N-glycan expression are functionally important.

61

Figure 2.12 Digoxin inhibits cell migration and invasion.

A) HT1080 cells (1 x 106) were seeded in 10 cm tissue culture plates. The next day

cells were treated with Digoxin 100 nM (squares) or buffer control (Ctrl) (circles) for

24 hours and then seeded (1 x 105) on fibronectin coated four-well chamber slides.

After adhering overnight, the monolayer was scratched with a plastic pipette tip.

Migration was captured overtime and expressed as the mean ± SD distance of migrated

cells in relative units (RU). * p = 0.03 by the Student’s t-test.

B) WRO cells (1 x 106) were treated with digoxin (50 nM) for 24 hours and then

plated into invasion chambers with 8µm pores. 5% FBS was used as chemo-attractant.

After 24 hours, cells that had migrated through the pores were fixed and stained. The

number of cells that migrated through the pores was counted automatically as

described in the material and methods. Data represent the mean + SD of migrating

cells compared to buffer treated cells.

C) WRO cells (1 x 106) were treated with digoxin (50 nM) for 24 hours and then

seeded into invasion chambers with matrigel. 5% FBS was used as chemo-attractant at

the bottom of the well. After 24 hours, the cells that had invaded through the pores

were fixed and stained. The number of cells that migrated through the pores was

counted automatically as described in the material and methods. Data represent the

mean + SD of migrating cells compared to buffer treated cells.

62

0

10

20

30

40

50

0 2 4 6 8

Time (hours)

Wo

un

d h

eali

ng

(R

U)

Figure 2.7

B

0

20

40

60

80

100

120

Control Digoxin

Mig

rati

on

(%

of

Co

ntr

ol)

0

20

40

60

80

100

120

Control Digoxin

Invasio

n

(% o

f C

on

tro

l)

C

A

Ctrl

Digoxin

*

P=0.0026

**P<0.0001

***

Figure 2.12

63

Digoxin decreases distant tumor formation in vivo

As aberrant GlcNAc-branching of N-glycans on the cell surface promotes

metastases, we tested the effects of digoxin on distant tumor formation in mouse models

of metastatic prostate cancer. In the first model, dsRed-labelled PPC-1 cells were treated

with digoxin (100 nM), or buffer control in culture. After 20 hours of treatment, cells

were injected intravenously into sublethally irradiated SCID mice. Three weeks after

injection, mice were sacrificed and distant tumor formation in the organs was imaged

with fluorescent microscopy.

Invasion of the prostate cancer cells was detected in the lung, bone, and liver. In

particular, metastasis of dsRed-PPC-1 cells to the lung was readily quantifiable using

image-based analysis(89, 98, 108, 109). Compared to buffer control, mice injected with

digoxin treated cells had decreased mean tumor number (142 71 vs 20 19 tumors, p =

0.0004 by Student’s t-test) within the lung (Figure 2.13.A). Median tumor number was

also significantly attenuated by drug treatment. It is important to note that both treated

and control cells were greater than 90% viable at the time of injection. To determine

whether the decreased distant tumor formation was simply due to decreased proliferation,

DsRed-PPC-1 cells were treated in culture with digoxin or buffer control and injected

subcutaneously into mice. In contrast to its effects on distant tumor formation, digoxin

did not significantly alter the growth of dsRedPPC-1 cells injected subcutaneously

compared to cells treated with buffer control (227 +50 mg vs 331 + 81 mg, p = 0.24 by

Student’s t-test) (Figure 2.13.B). Therefore, the reduction in tumor burden after

digoxin treatment is not solely due to reductions in cellular proliferation.

64

To further evaluate the effects of digoxin on distant tumor formation, sublethaly

irradiated SCID mice were injected intravenously with dsRed -PPC-1 cells and then

treated intraperitoneally with digoxin (1.35mg/kg and 0.675mg/kg) or buffer control daily

for 14 days. Four weeks after the injection of the cells, mice were sacrificed and

analyzed as above. Compared to buffer control, both concentrations of digoxin decreased

the mean number of tumors (48 32 (0.65mg/kg) vs 28 19 (1.35 mg/kg) vs 171 83

(control)) in the lungs (Figure 2.13.C). Similar reductions in median tumor number were

also observed. Thus, taken together, digoxin inhibits distant tumor formation in vivo.

65

Figure 2.13. Digoxin inhibits distant tumor formation in vivo

A) Fluorescent dsRed-PPC-1 cells were treated with digoxin (100 nM) (n = 8) or buffer

control (n = 7). Twenty hours after treatment cells were harvested and 3.5x106 viable cells

were injected into the tail veins of sublethally irradiated SCID mice. Three weeks after

injection, mice were sacrificed and their organs were imaged using a fluorescent microscope.

The numbers of distant tumors from all five lobes of the lungs were quantified using image

analysis software. Data represent the mean + SD number of tumors. P-values are based on

the Student’s t-test.

B) Fluorescent dsRed-PPC-1 cells were treated with digoxin (100 nM) (n = 6) or buffer

control (n = 5). Twenty hours after treatment cells were harvested and injected

subcutaneously into sublethally irradiated SCID mice. Two weeks after injection, mice were

sacrificed, and the tumors were excised and weighed. Data points represent the mean + SD

tumor weight. P-values are based on the Student’s t-test.

C) Fluorescent dsRed-PPC-1 cells (3.5x106 viable cells) were injected into the tail veins of

sublethally irradiated SCID mice. Daily for 14 days, the mice were treated with

intraperitoneal injection of digoxin (0.675 mg/kg/day (n = 9) or 1.35 mg/k/day (n = 9)) or

buffer control (n = 5). Four weeks after injection, mice were sacrificed and their organs were

imaged using a fluorescent microscope. The number of distant tumors fromed in all five lobes

of the lungs were quantified using image analysis software. Data represent the mean + SD

number or area of tumors. P-values are based on the Student’s t-test.

66

**

Figure 2.8

A

Control 100 nM0

50

100

150

200

Tu

mo

r n

um

be

r

P=0.0004

***

Control 100 nM 0

100

200

300

400

Su

bc

uta

ne

ou

s t

um

or

weig

ht

(mg

)

BP=0.22

NS

Control 0.675 1.35 0

50

100

150

200

250

Tu

mo

r n

um

be

r

CP<0.0015

P<0.0003***

Figure 2.13

67

2.5 Discussion

To better understand the impact of modulating N-glycosylation in malignant cells, we

used a chemical biology screen for inhibitors of L-PHA-induced cell death to identify

novel regulators of glycosylation. From this screen we identified the CG dihydroouabain

and determined that structurally related CGs also blocked L-PHA induced cell death. Of

the tested CGs, digoxin was the most potent inhibitor. These results, coupled with the

lack of effect on murine cells suggest that CGs block N-glycosylation by inhibiting their

known target, the Na+/K

+-ATPase. Further supporting a mechanism linked to the known

binding target, over-expression of the murine Na+/K

+-ATPase that is less sensitive to CG

inhibition abrogated the effects of digoxin on surface ConA binding N-glycoproteins.

Moreover, knockdown of the Na+/K

+-ATPase using siRNA recapitulated the effects of

digoxin. Thus, taken together, we conclude that inhibiting the Na+/K

+-ATPase blocks the

N-glycosylation pathway.

In this report we used four independent approaches to demonstrate the ability of

CGs to inhibit the N-glycosylation pathway. First, L-PHA induces cell death by binding

primarily to cell-surface N-glycans that occur downstream of the GnTII in the N-glycan

synthesis pathway, and CGs blocked L-PHA-induced cell death. Second, CGs increased

total cellular ConA-binding glycoproteins. Third, CGs increased cell surface ConA

binding N-glycoproteins. Finally, mass spectrometry revealed that digoxin increased the

Con-A-binding GnM3Gn2F1 hybrid and Gn2M3Gn2F1 biantennary N-glycans.

The profile of oligosaccharides after digoxin treatment indicates that digoxin

blocks the N-glycosylation pathway downstream of GlcNAc-TII. These results also

indicate that digoxin blocks the pathway through a mechanism distinct from the α-

68

mannosidase II inhibitor swainsonine. The inhibition of L-PHA induced cell death in

Jurkat cells could also be a consequence of inhibition of galactosyltransferases. In order

to further investigate the effect of digoxin on level of activity of galactosyltransferases

fluorescently-conjugated ricin lectins can be used to measure the cell-surface expression

of these N-glycans.

Inhibitors of the Na+/K

+-ATPase may impair the Golgi-residing N-glycosylation

enzymes by altering intracellular Na+ and Ca

2+. Currently, however, it is unknown

whether the ion concentrations needed to disrupt the function of N-glycosylation enzymes

can be achieved in cells after treatment with cardiac glycosides. The lumens of the ER

and Golgi have a [Ca2+

] of 0.1mM-1mM, and Ca2+

transporters actively pump calcium

into the lumens(110). Ca2+

channels regulate many other aspects of membrane signaling,

and changes in the mM concentration range had a pronounced effect, decreasing

GlcNAc-TI and GlcNAc-TV activities, which could alter glycoprotein binding to

galectins thereby relative proportions at the cell surface (83). Thus, the cation sensitivity

of GlcNAc-branching enzymes may be a mechanism of transducing ion channel signaling

to adapt the cell surface by changing the profile of glycoprotein receptors.

Interestingly, digoxin treatment decreased Mgat1 mRNA expression in a dose

dependent manner. Additional experiments are required to investigate whether the effect

on Mgat1 mRNA levels can explain the observed phenotypic changes. For example,

cells overexpressing Mgat1 can be treated with digoxin in order to determine if the

increased Mgat1 mRNA can compensate for the effect of digoxin and rescue the

phenotypes. Therefore, it is unknown whether the digoxin-induced inhibition of N-

69

glycosylation is a direct consequence of disturbance of intracellular ion levels or an

indirect effect through decreasing Mgat1 mRNA levels.

In this report, we demonstrated that digoxin inhibited cellular migration and

invasion which are known functional consequence of blocking the N-glycan branching.

For example, Mgat5-/-

mice have reduced cancer growth and metastasis(41). Likewise,

siRNA knock-down of GlcNAc-TV in malignant cells impairs cell migration and

invasion (107). While digoxin’s inhibition of cell migration and invasion are consistent

with its effects on the N-glycan remodeling, we cannot exclude that these effects are

related to other pathways impacted by the molecule.

CGs can induce cell death in malignant cells (74, 102) through multiple

mechanisms including activation of Cdk5 (72), Src kinase (73) and p21 (73). However,

the effects of CG on N-glycan pathway and cellular processes of migration and invasion

were not artifacts of cell death, as the concentrations of digoxin and times of incubation

required to inhibit N-glycan remodeling were lower than those required to induce cell

death. Furthermore, as the concentrations of CG required to inhibit the N-glycan

pathway are lower than the concentrations associated with activation of these other

pathways, we suspect that the effects of CG on N-glycosylation are not related to

activation of these other pathways.

Serum concentrations of 2 nM digoxin can be achieved in humans without

significant toxicity(111). While the concentration of digoxin required to inhibit N-

glycosylation exceeded 2 nM, potentially, chronic daily dosing of digoxin could

sufficiently reduce N-glycan branching to affect the function of down-stream effectors.

Interestingly, patients with breast carcinoma who were coincidently receiving CGs for

70

cardiac dysfunction had a lower rate of relapse and metastasis than patients not receiving

CGs (75, 77). Our results suggest that CGs could have an anti-tumor effect in patients by

altering the N-glycosylation profile in malignant cells. To assess the effects of digoxin

on distant tumor formation and mimic some of the processes of metastasis, we tested

digoxin in two mouse models. In both of these models, digoxin decreased distant tumor

formation. Thus, the decreased metastasis supports a mechanism of action linked to the

inhibition of glycosylation. Thus, digoxin could be a lead for a novel therapeutic agent

for the treatment of malignancy and an adjunct to prevent metastases. A limitation to the

xenograft studies, however, is that we cannot be certain that digoxin prevented metastases

through a mechanism related to ability to inhibit glycosylation. Potentially inhibition of

the Na+/K

+-ATPase may have anti-tumor effects through mechanisms distinct from

impairing glycosylation.

Interestingly, other glycosylation inhibitors have entered clinical trials for the

treatment of malignancy. Swainsonine, a small molecule inhibitor of Golgi -

mannosidase II, has anti-cancer activity in preclinical models (50, 51). Given these

results, swainsonine was advanced into a phase I clinical trial for patients with refractory

malignancy. In the context of this trial, tumor regression was noted in one patient with

head and neck cancer (54, 55). Thus, inhibiting -mannosidase II may be a clinically

effective anti-cancer strategy and inhibiting targets downstream of α-mannosidase II

might also produce anti-tumor effects without untoward toxicity.

In summary, we used a chemical biology approach to investigate the Golgi N-

glycan pathway. Our high-throughput screen identified Na+/K

+-ATPase inhibitors that

blocked the N-glycan branching at a point distinct from the α-mannosidase II inhibitor

71

and downstream of GlcNAc-TII. Na+/K

+-ATPase inhibitors also blocked the N-glycan

dependent processes of cell migration and invasion of cancer cells as well as distant

tumor formation in mouse models. Thus, our results help explain previously reported

anticancer effects of these compounds and highlight new strategies for the development

of anticancer therapies.

72

Chapter 3

Suppression of N-glycan branching by N-

acetylglucosaminyltransferase I knockdown inhibits

tumor cell migration, invasion and metastasis

Reza Beheshti Zavareh, Mahadeo A. Sukhai, Rose Hurren, Marcela Gronda, Xiaoming

Wang, Craig D. Simpson, Neil Maclean, Troy Ketela, Jason Moffat, David R. Rose,

Harry Schachter, James W. Dennis, Aaron D. Schimmer

Attribution of the data:

Data is generated and analyzed by the primary author with the exception of:

Reza Beheshti Zavareh and Rose Hurren 3.1.A

Reza Beheshti Zavareh and Xiaoming Wang 3.4.D and 3.5

Reza Beheshti Zavareh and Mahadeo Sukhai 3.6

73

3.1 Abstract

N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) is the enzyme that initiates the

biosynthesis of hybrid and complex N-linked glycans in medial Golgi. Aberrant activity

of the N-glycosylation pathway is seen frequently in malignant cells and contributes to

their metastatic potential. In order to evaluate the effects of GlcNAc-TI inhibition in

malignant cells, we knocked down GlcNAc-TI with shRNA. Target knockdown and

inhibition was confirmed by demonstrating mRNA knockdown, decreased cell-surface L-

PHA staining, and inhibition of enzymatic activity. Knockdown of GlcNAc-TI did not

induce cell death or inhibit proliferation of malignant cells. Rather, GlcNAc-TI inhibited

migration and invasion of the malignant cells. Knockdown of GlcNAc-TI decreased cell

surface expression of β1 integrin as investigated by confocal microscopy. Furthermore,

in an orthotopic prostate cancer xenograft mouse model, knockdown of GlcNAc-TI

decreased the number of tumors that had metastasized to the lung more than three-fold

compared to control. Finally, we examined the association between Mgat1 mRNA

expression in primary tumors and the incidence of subsequent metastasis. By analyzing

publically available gene expression array data sets from patients with breast cancer, we

determined that patients with the lowest levels of Mgat1 mRNA in their primary tumors

were least likely to develop metastatic disease after therapy.

In summary, this work highlights the role of the GlcNAc-TI in cancer cell metastasis.

Thus, GlcNAc-TI may be a novel target for anti-cancer therapies and levels of GlcNAc-

TI in primary tumors may help predict the risk of developing metastatic disease.

74

3.2 Introduction:

Asparagine-linked glycosylation is a post-translational modification that alters the

localization and function of cell surface proteins and is required for normal cellular

growth, proliferation and differentiation (3, 18, 83). Aberrant activity of this pathway is

seen frequently in malignant cells and contributes to their metastatic potential by

promoting cell invasion and migration (18, 112). UDP-N-acetylglucosamine:α-3-D-

mannoside β-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC 2.4.1.101, encoded

by the Mgat1 gene) controls the synthesis of complex N-glycans(81). Here, we have

explored the effects of inhibiting GlcNAc-TI in malignant cells.

N-glycosylation is initiated in the lumen of the rough endoplasmic reticulum

where an oligosaccharyltransferase transfers the oligosaccharide precursor

Glc3Man9GlcNAc2 en bloc from dolichol-pyrophosphate onto asparagine (N) residues in

the sequence NXS/T (where X can be any amino acid except proline) to form an N-

glycan (Figure 1.2) (Reviewed in Ref (9)). Once attached to the amide group of the

asparagine, this precursor is further modified by the sequential removal of the three

glucoses and one mannose by the actions of rough endoplasmic reticulum glycosidases to

form Man8GlcNAc2-N. In the Golgi, three more mannoses are removed from the latter

structure via Golgi mannosidases to produce Man5GlcNAc2-N. All the N-glycan

structures that are formed to this point are termed high mannose structures (9). GlcNAc-

TI transfers N-acetylglucosamine (GlcNAc) to the α-3-D-mannoside moiety of

Man5GlcNAc2-Asn (81). The product of GlcNAc-TI is required as a substrate for

GlcNAc-TII and in turn subsequent branching by GlcNAc-TIV and GlcNAc-TV to

produce branched typed N-glycans ((9),Figure 1.2). GlcNAc-TV catalyzes the addition

75

of 1,6GlcNAc branch which is directly linked to cancer progression. Increased

oncogene activity stimulates overexpression of these Golgi enzymes which in turn

induces tumor cell motility and invasion (30, 38, 113-115).

GlcNAc-TI is required for mouse development beyond day E9.5 (80), although

cell lines lacking the GlcNAc-TI activity grow at normal rates (79), suggesting GlcNAc-

TI and complex N-glycans function in cell-cell/substratum interactions. However, to

date, the effects of inhibiting GlcNAc-TI in malignant cells are unknown. Potentially,

inhibiting GlcNAc-TI may block metastasis and decrease cell invasion by decreasing the

synthesis of complex N-glycan structures (41). The converse is also possible, as high

levels of GlcNAc-TI activity in epithelial cells can deprive GlcNAc-TIV and V of their

common substrate, UDP-GlcNAc, because of the ultrasensitivity of branching pathway to

this substrate (83).

In this paper, we have shown that partial knockdown of GlcNAc-TI by shRNA in

malignant cells that suppressed complex N-glycans by 50-70%, did not alter proliferation

or viability. Rather, it inhibited tumor cell migration and invasion. Moreover,

knockdown of GlcNAc-TI decreased metastasis of human prostate cancer cells in a

xenograft orthotopic model of prostate metastasis. Finally, we demonstrated that, in

patients with breast cancer, lower levels of GlcNAc-TI in the tumor were correlated with

a decreased risk of future metastasis. As such, this work highlights the role of GlcNAc-

TI and branched N-glycans in cancer cell metastasis. Furthermore, GlcNAc-TI may be a

target for anti-cancer therapies and levels of GlcNAc-TI may be prognostic in cancer

patients.

76

3.3 Materials and methods:

Reagents.

Alexa Fluor 488-conjugated L-PHA, Alexa Fluor 647-conjugated L-PHA and 4’,6-

diamidino-2-phenylindole, dilactate (DAPI, dilactate) were purchased from Molecular

Probes (Eugene, OR, US). Fibronectin was purchased from Sigma-Aldrich (Oakville,

ON, Canada).

Cell culture.

HeLa human cervical cancer cells were maintained in Dulbecco’s modified Eagle

Medium (DMEM). PC3N7 human prostate cancer cells stably expressing red fluorescent

protein (RFP) (gift from G. Glinsky (Ordway Research Institute)) were maintained in

RPMI 1640(116). All cells were supplemented with 10% fetal bovine serum (FBS)

(Hyclone, Logan, UT) and antibiotics. All cell lines were cultured in a standard

humidified incubator at 37°C in a 5% CO2 atmosphere.

L-PHA binding to cell surface glycans.

Cells (500 cells/well) were seeded in 96-well plates. After adhering overnight, cells were

fixed with 3.7% formaldehyde and washed. Surface N-glycans were stained with L-PHA

( 20 µg/mL) conjugated to Alexa Fluor 488 or Alexa Fluor 647 and nuclei were stained

with DAPI ( 5 µg/ml). The total intensity of L-PHA staining on each cell was quantified

using Cellomics ArrayScan II (Cellomics, Pittsburgh, PA) (83).

MGAT1 silencing by Lentiviral-Delivered RNA Interference.

Construction of hairpin-pLKO.1 vectors (carrying a puromycin antibiotic resistance gene)

containing short hairpin RNA (shRNA) sequences and production of shRNA viruses have

been described in detail (117). The shRNAs targeting the GlcNAc-TI coding sequences

77

are as follows: Mgat1-sh1 (NM_002406), 5′-CCCTGAGATCTCAAGAACGAT-3′; and

Mgat1-sh2 (NM_002406), 5′-GCACCTCAAGTTTATCAAGCT-3′. The control shRNA

coding sequences are as follows: RFP, 5′-CTACAAGACCGACATCAAGCT-3′ and

LacZ, 5′-CCGTCATAGCGATAACGAGTT-3′. Lentiviral infections were done

essentially as described previously(117). Briefly, adherent cells were treated with 0.5 mL

of the virus followed by overnight incubation (37°C, 5% CO2) without removing the

virus. The next day, viral medium was replaced with fresh medium containing puromycin

(1 μg/mL) to select a population of resistant cells.

Reverse-transcriptase real-time PCR.

First-strand cDNA was synthesized from 1 g of DNase-treated total cellular RNA using

random primers and SuperScript II reverse transcriptase (Invitrogen) according to the

manufacturer’s protocol. Real-time PCR assays were performed in triplicate with 5 ng of

RNA equivalent cDNA, SYBR Green PCR Master mix (Applied Biosystems, Foster

City, CA, USA), and 400 nM of gene-specific primers. Reactions were processed and

analyzed on an ABI 7900 Sequence Detection System (Applied Biosystems).

Forward/reverse PCR primer pairs for human cDNAs were as follows: human GlcNAc-

TI: Forward 5’- CGG AGC AGG CCA AGT TC-3’, Reverse 5’- CCT TGC CCG CAG

TCC TA -3’, 18S: Forward 5’- AGG AAT TGA CGG AAG GGC AC-3’, Reverse 5’-

GGA CAT CTA AGG GCA TCA CA-3’. Relative mRNA expression was determined

using the CT method as described (92).

GlcNAc-transferase activities.

The enzyme activity of GlcNAc-TI was measured using a synthetic receptor as

previously described (83). Briefly, cell lysates were incubated with GlcNAc-TI acceptor

78

Manα(1,3) [Manα(1,6)]Glcβ1-O-(CH2)7CH3 (Toronto Research Chemicals, Toronto,

ON), in a solution containing 0.5 mM UDP-[63H]GlcNAc (44400 dpm/nmol), 125 mM

MES (pH 6.5), 50 mM GlcNAc, 1 mM UDP-GlcNAc, 0.8 mM AMP and 10 mM MnCl2

. After incubation, the reaction was stopped by adding ice-cold H2O. Transfer of

[63H]GlcNAc to the acceptor was quantified by liquid scintillation counting.

Migration and invasion assays.

Invasion and migration assays were performed as previously described (97, 118). Briefly

HeLa cells (2 x 105) were harvested and seeded in uncoated invasion chambers for

migration assay or in BioCoat Matrigel Invasion Chambers (BD Biosciences,

Mississauga, ON) for invasion assays. Growth medium containing 10% FBS was used as

a chemoattractant in the bottom well. Following 48 hours of incubation, cells that had

migrated or invaded the lower surface of the membrane were stained with Diff-Quik

Stain (BD Biosciences). The number of migrating or invading cells were imaged and

counted using the Aperio ScanScope CS whole slide Scanner (Aperio Technologies,

Vista, CA) and Image-Pro Plus Software (version 4.5; Media Cybernetics Inc.,

Silversprings, MD). In order to test the effect of simultaneous GlcNAc-TI inhibition and

swainsonine treatment, HeLa cells were treated with 2 M swainsonine for 72 hours prior

to the migration and invasion assay as described above.

Immunocytochemistry of 1 integrin.

Cells were seeded on fibronectin coated cover slips. After adhering overnight, cells were

fixed with 2% paraformaldehyde for 10 minutes, followed by permeabilization using 0.2

% Triton-X-100. After blocking by 5% BSA for 1 hour, cells were incubated overnight at

4oC with mouse anti-integrin (1:200, Millipore). Cover slips were washed with PBS

79

and then incubated with donkey anti-mouse IgG-Cy3 conjugated secondary antibody

(1:200, Millipore). Cover slips were mounted on slides using fluorescent mounting

medium and imaged using the 40X lens of the Zeiss LSM700 confocal microscope (Carl

Zeiss MicroImaging GmbH, Jena, Germany).

In vivo studies.

The effects of GlcNAc-TI knockdown on distant tumor formation was evaluated in vivo

as previously described (89). Briefly, PC3N7 cells stably expressing RFP with and

without GlcNAc-T1 knockdown, were injected orthotopically into the prostates of

sublethally irradiated (3.5 Gy) SCID mice. Mice injected with tumor cells were

maintained for 4 weeks after injection, at which time, the animals were sacrificed via

carbon cervical dislocation for complete examination. Red fluorescent tumors were

detected via whole body imaging and whole organ imaging using a Leica MZ FLIII

fluorescent stereomicroscope with a 100 W mercury lamp, a 560/40 excitation filter, and

a 610 long-pass emission filter. Images were acquired using a Olympus DP70 digital

camera at 0.8X magnification and analyzed using Image Pro Plus 6.0

(MediaCybernetics). A single common threshold was applied to identify and measure

fluorescence in each organ. The number of fluorescent spots was recorded for each lung

lobe. All quantification was performed on unmanipulated images. Mice were obtained

from an in-house breeding program and housed in laminar-flow cage racks under

standardized environmental conditions with ad libitum access to food and water. All

experiments were performed according to the regulations of the Canadian Council on

Animal Care.

80

Expression profiling of breast cancer patients.

Microarray data from a previously published breast cancer study was obtained from Gene

Expression Omnibus (GEO) data base (http://www.ncbi.nlm.nih.gov/gds, GDS806)

(119). The expression of GlcNAc-TI was compared in patients with and without cancer

recurrence after tamoxifen treatment (recurrence was defined as evidence of distal

metastasis within 5 years post-treatment (119)). Patients were classified by quartile of

GlcNAc-TI expression, and recurrence status in the highest and lowest quartiles was

assessed using the publicly available data. Statistical significance was calculated using

the chi-square test; P<0.05 was considered significant.

http://www.ncbi.nlm.nih.gov/gds

81

3.4 Results:

Genetic knockdown of GlcNAc-TI does not inhibit cell proliferation or reduce cell

viability

We began by evaluating the effects of inhibiting GlcNAc-TI inhibition on cell

proliferation. HeLa human cervical cancer cells were infected with lentivirus containing

shRNA targeting GlcNAc-TI or control sequences and stable clones were selected.

Target knockdown using two independent shRNA sequences was confirmed by Q-

RTPCR (Figure 3.1.A). Moreover, we demonstrated that the shRNA targeting GlcNAc-

TI but not control sequences reduced the enzymatic activity of GlcNAc-TI (Figure 3.1.B)

and decreased L-PHA cell-surface staining (Figure 3.1.C). GlcNAc-TI knockdown did

not alter cell viability and proliferation as determined by trypan blue staining, cell counts,

and an MTS assay (Figure 3.1.D).

82

Figure 3.1. GlcNAc-TI knockdown does not alter the proliferation of malignant cells.

HeLa cells were infected with shRNA in lentiviral vectors targeting GlcNAc-TI shRNA

(GlNAC-T1 sh1 and GlNAC-T1 sh2) or the control shRNA sequences. Stable clones were

selected by the addition of puromycin (1 g/mL).

A) Cells were harvested and mRNA extracted. Levels of GlcNAc TI mRNA were

measured by QRTPCR. Data represent the mean + SD relative expression of mRNA relative

to control sequence (n = 3 independent experiments performed in triplicate)

B) Cells were harvested and cell lysates were prepared using PBS 1% Triton X-100 at 0oC,

and GlcNAc-TI enzyme activity was measured. Data represent the mean + SD DPM/g

protein from a representative experiment.

C) HeLa cells (10,000 cells/mL) were seeded in a 96-well plate and incubated overnight.

The next day, cells were fixed and stained by AlexaFluor488-conjugated L-PHA and imaged

using the automated Arrayscan microscope. Data represents mean + SD cytoplasmic

fluorescent intensity (n = 3 independent experiments performed in triplicate)

D) HeLa cells (0.05 X 10 6) were seeded in a 6 well plate. For the following four days, cells

were harvested from each well. Cell number and viability were measured by trypan blue

exclusion. All the counts were normalized to the number of cells counted on day one. Data

represent the mean + SD relative cell growth compared to the number of cells counted on day

one. (n = 3 independent experiments performed in triplicate).

83

Control Mgat1-sh1 Mgat1-sh20

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Mgat1-Sh2

Time (Hours)

Cell

gro

wth

D

BP=0.0056

**

P<0.0001*** P=0.0003

***

P<0.0001***

P=0.0240*

P=0.0022**

Control Mgat1-Sh1 Mgat1-sh20.00

0.25

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Rela

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84

Genetic knockdown of GlcNAc-TI inhibits tumor cell migration and invasion

Increased GlcNAc-branching of N-glycans in mammary and colon cancers correlates

with metastasis and reduced survival, while mice deficient in the late branching enzyme

GlcNAc-TV delay progression in MMTV-PyMT and Pten+/- models of cancer (18, 41,

42, 58). GlcNAc-TI knockdown is an earlier step in the pathway and therefore, we

evaluated the effects of GlcNAc-TI knockdown on the migration and invasion of

malignant cells. To assess the effect of GlcNAc-TI inhibition on cell migration, HeLa

cells with GlcNAc-TI knockdown were seeded into chambers in serum-free medium.

Medium with 10% FBS was placed in the lower chamber as a chemoattractant. Forty-

eight hours after seeding, cell migration into the bottom chamber was measured. Similar

studies were conducted using Matrigel-coated filters to measure cell invasion. Compared

to cells infected with control sequences, GlcNAc-TI knockdown with two independent

shRNA sequences decreased both cell migration and cell invasion (Figure 3.2.A, 3.2.B).

We also tested the effects of simultaneous knockdown of GlcNAc-TI and inhibition of

mannosidase II by swainsonine treatment on migration and invasion. As previously

reported, swainsonine treatment alone inhibited migration and invasion (56). Of note, the

treatment of HeLa cells with swainsonine after knockdown of GlcNAc-TI did not result

in any further decrease in cell migration and invasion compared to knockdown of

GlcNAc-TI alone (Figure 3.2.C, 3.2.D). This result is consistent with -mannosidase II

acting downstream of GlcNAc-TI, and no additional or parallel effects

85

Figure 3.2 GlcNAc-TI knockdown decreases the migration and invasion of malignant

cells

A) HeLa cells were plated into chambers with 8-μm pores and 10% FBS was used as a

chemoattractant. After 48h, cells that had migrated through the pores were fixed and

stained. The number of cells that migrated through the pores was counted automatically.

Data represents mean + SD number of migrated cells (n = 3 independent experiments

performed in triplicate)

B) HeLa cells were plated into invasion chambers with Matrigel. After 48h, cells that had

invaded through the pores were fixed and stained. The number of cells that invaded through

the pores was counted automatically. Data represents mean + SD number of invaded cells

(n = 3 independent experiments performed in triplicate)

HeLa cells with GlcNAc-TI knockdown and cells with control shRNA were treated with or

without 2 swainsonine (SWA) for 72 hours prior. After treatment, cells were seeded

into chambers and migration (C) and invasion (D) were measured as above.

86

Control Control Mgat1-Sh2 Mgat1-Sh20

25000

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of

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rate

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Figure 3.2

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of

Invad

ed

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s

C

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SWA: - + - +

SWA: - + - +

P<0.0001***

P<0.0001***

P=0.005**

P=0.0002***

P=0.0002***

NS

P=0.0036**

NS

87

Inhibition of GlcNAc-TI reduces 1 integrin accumulation at the cell membrane

The cell-surface fibronectin receptor 51 integrin influences cell adhesion, and

promotes cell migration and invasion (120). The formation of the functional heterodimer

of 51 integrin requires N-glycosylation of both the 5 and 1 subunits (121).

Mutation of 1 integrin at NXS/T blocks N-glycosylation and prevents cell surface

localization of this integrin and inhibits cellular spreading and migration (122, 123).

Moreover, addition of 1,6GlcNAc branches to 51 integrin by GlcNAc-TV is required

for interactions with galectins at the cell surface which in turn promotes 51 integrin

recruitment and activation (28). Therefore, we examined changes in the localization of

the 1 integrin after GlcNAc-TI knockdown. Control or GlcNAc-T1 knockdown HeLa

cells were stained with mouse-anti 1 integrin and imaged with confocal microscopy.

Knockdown of GlcNAc-TI led to increased accumulation of β1 around the nucleus and

prevented 1 integrin accumulation at the cell membrane (Figure 3.3).

88

Figure 3.3 GlcNAc-TI knockdown alters the pattern of integrin localization.

Hela cells with GlcNAc-TI knockdown or control shRNA were seeded (0.25× 105 /well) on

fibronectin-coated cover slips. After adhering overnight, cells were fixed with 2%

paraformaldehyde for 10 min, followed by permeabilization by 0.2% Tween for 30 min. After

blocking by 5% BSA for 1 hour, cells were incubated over night at 4C with integrin

antibody (1:200). Cells were washed with PBS and then incubated with Cy3 conjugated

secondary antibody. After washing with PBS, cover slips were mounted on slides using

mounting medium. The slides were imaged using the 40X lens of Zeiss LSM700 confocal

microscope.

A) One single layer of representative cells is shown. Top image, HeLa cell with control

shRNA and bottom image GlcNAc-TI knockdown.

B) The three dimensional composition of the entire cells are shown. Top image, HeLa cell

with control shRNA and bottom image GlcNAc-TI knockdown.

89

A B

Co

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Figure 3.3

90

GlcNAc-TI knockdown decreased distant tumor formation in an orthotopic model

of prostate cancer metastasis

Next, we evaluated the effects of GlcNAc-TI knockdown on distant tumor formation in

an orthotopic mouse model of metastatic prostate cancer. PC3N7 prostate cancer cells

stably over-expressing RFP were infected with lentivirus containing shRNA targeting

GlcNAc-TI or control sequences and stable clones were selected. Target knockdown was

confirmed by Q-RTPCR, inhibition of enzymatic activity, and decreased cell surface L-

PHA staining (Figure 3.4.A, 3.4.B, 3.4.C). GlcNAc-TI knockdown or control RFP-

labeled PC3N7 cells were injected orthotopically into the prostate glands of sublethally

irradiated SCID mice (n = 15 per group). Four weeks after injection, mice were

sacrificed and distant tumor formation in the organs was imaged with fluorescent

microscopy (Figure 3.4.D). Tumors developed in the prostate both in mice injected with

GlcNAc-TI knockdown or control RFP-labeled PC3N7 cells. In mice injected with

control cells, tumor formation was detected in the prostate as well as clinically relevant

sites of metastases in prostate cancer, including lung, lymph nodes and liver. Qualitative

reductions in distant tumor formation were observed in mice injected with GlcNAc-TI

knockdown cells. Given the resolution of images of RFP-PC3N7 tumors in the lung, we

were also able to quantify the number and area of distant lung tumors in mice injected

with GlcNAc-TI knockdown or control cells. Compared to controls, knockdown of

GlcNAc-TI significantly decreased the number and total area of metastatic RFP-PC3N7

to the lung (Figure 3.5.A). Moreover, GlcNAc-TI knockdown increased the number of

mice without any detectable lung metastases. Thus, taken together, GlcNAc-TI

knockdown inhibits distant tumor formation in vivo.

91

Figure 3.4 Evaluation of the effects of GlcNAc-TI knockdown in an orthotopic model of

metastatic prostate cancer

RFP labeled PC3N7 cells were infected with shRNA in lentiviral vectors targeting GlcNAc-

TI shRNA (Mgat1-sh2) or the control shRNA sequences. Stable cell populations were


A) Cells were harvested and mRNA extracted. Levels of GlcNAc TI mRNA were measured

by QRTPCR. Data represent the mean + SD mRNA expression compared to control

sequence (n = 3 independent experiments performed in triplicate)

B) Cells were harvested and cell lysates were prepared using PBS 1%-Triton X-100 at 0oC,

and GlcNAc-TI enzyme activity was measured. Data represents the mean (+ SD) enzyme

activity (DPM/g protein) in control and knockdown cell from representative experiments.

C) PC3N7 cells (10,000 cells/mL) were seeded in a 96-well plate and incubated overnight.

The next day, cells were fixed and stained by AlexaFluor647-conjugated L-PHA and imaged

using the automated Arrayscan microscope. Data represents the mean (+ SD) cytoplasmic

fluorescent intensity (n = 3 independent experiments performed in triplicate)

D) RFP-labeled PC3N7 cells with GlcNAc-TI knockdown or control shRNA were harvested

and 0.5 × 106 viable cells were injected orthotopically into the prostate of the sublethally

irradiated SCID mice. Four weeks after injection, mice were sacrificed and their organs were

imaged using a fluorescent microscope. Representative images of the primary site of tumor

and tumor cell retention and growth in the lymph nodes and lungs of one mouse from each

group (control and GlcNAc-TI knockdown) are shown here

92

Control Mgat1-sh20

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Figure 3.5

Figure 3.5 GlcNAc-TI knockdown decreases distant tumor formation.

RFP labeled PC3N7 cells were infected with shRNA in lentiviral vectors targeting GlcNAc-

TI shRNA (Mgat1-sh2) or the control shRNA sequences. Stable cell populations were


A) The numbers of distant tumors from all five lobes of the lungs from mice injected either

with PC3N7 cells expressing active GlcNAc-TI (Control) or GlcNAc-TI shRNA were

quantified using image analysis software. Each point represents one mouse.

94

Expression profiling of breast cancer patients

Our preclinical studies suggest a role for GlcNAc-TI in cancer cell metastases.

Therefore, we examined the association between Mgat1 mRNA expression in primary

tumors and the incidence of subsequent metastasis. Microarray data on 60 patients with

breast cancer enrolled in a previous study were obtained ((119), GDS806). Mgat1

expression levels were extracted from the data set. Of the 60 patients in the data set, 28

developed distant metastases with a median time to recurrence of four years and 32

remained disease-free with median follow up of 10 years.(119). We ranked patients

based on Mgat1 expression (Figure 3.6.A). As shown in Figure 3.6.B, 12 of 15 patients

in the highest quartile of GlcNAc-TI expression developed metastatic disease. In

contrast, only three of 15 patients in the lowest quartile of Mgat1 expression developed

metastasis. Therefore, patients with the lowest levels of Mgat1 mRNA in their primary

tumors were least likely to develop metastatic disease after therapy.

95

Figure 3.6 . Expression profiling of breast cancer patients.

Microarray data from the patients with breast cancer in the GDS806 study were obtained

from Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/gds).

A) Mgat1 expression was obtained from the data set. Black circles: Patients who

developed metastasis. White circles: Patients who did not develop metastasis. Ma XJ et

al calculated the relative gene expression ratios as follows: Raw cy5/cy3 ratios per assay

were first normalized using non-linear local regression then between-array normalization

were performed by subtracting the median log2 ratios gene-wise across all arrays(119).

B) Mgat1 expression was divided into quartiles. The percent of patients who developed

metastasis is shown. (P=0.02 by chi-square test when the distribution of recurrence was

compared to a random distribution and P<0.000001 by chi-square test when the

distribution of recurrence was compared with the distribution of non-recurrent patients).

96

Figure 3.6

Figure 6

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97

3.4 Discussion

Increased N-glycan branching in malignant cells contributes to their metastatic

potential although experimental blockade of enzymes in the pathway remains

incomplete(18). Here, we have knocked down GlcNAc-TI in malignant cells to

investigate the effects on proliferation, migration, invasion and metastasis. In malignant

cells, knockdown of GlcNAc-TI did not alter their growth, viability or proliferation.

Rather, inhibition of Glc-NAc-T1 inhibited cell migration and invasion. Moreover,

GlcNAc-TI knockdown decreased metastasis in an orthotopic prostate cancer xenograft.

Mechanistically, GlcNAc-TI knockdown inhibited the localization of 1 integrin to the

cell membrane. Thus, taken together, GlcNAc-TI plays an important role in regulating

cell invasion and metastasis.

Knockdown of GlcNAc-TI did not reduce cell growth or viability. However,

inhibiting the steps in the N-glycosylation pathway upstream of GlcNAc-TI induces cell

death in both normal and malignant cells(16). For example, tunicamycin, a natural

nucleoside antibiotic, blocks the transfer of GlcNAc-1-phosphate from UDP –GlcNAc to

dolichol-P and thereby decreases the formation of dolichol-PP-GlcNAc (the scaffold for

synthesis of Glc3Man9GlcNAc2). Inhibition of N-glycosylation by tunicamycin induces

apoptosis in various cells (16, 124-128). Likewise, mutations in other enzymes upstream

of GlcNAc-TI such as mannosylphosphoryldolichol synthase, glucosyl transferase, and

hexokinase resulted in cell death in Chinese Hamster Ovarian cells upon stress with

increased temperature (129).

Prior to this study, the effects of inhibiting GlcNAc-TI in malignant cells were

unknown. Theoretically, based on modeling of branching pathway, inhibiting GlcNAc-

98

TI may have either promoted or inhibited tumor metastasis. GlcNAc1Man5GlcNAc2, the

product of GlcNAc-TI, is the substrate for -mannosidase II, then GlcNAc-TII, GlcNAc-

TIV and GlcNAc-TV an ordered pathway to tetra-antennary structures ((3). Thus,

inhibiting GlcNAc-TI would be expected to inhibit tumor metastasis by reducing levels

of tetra-antennary structures (18). However, the branching pathway is ultrasensitive to

UDP-GlcNAc which could result in the counter intuitive effect that partial knockdown of

GlcNAc-TI could increase UDP-GlcNAc to GlcNAc-TII, GlcNAc-TIV and GlcNAc-TV

and increase the expression of tetra-antennary structures(83). Based on our observation

that GlcNAc-TI knockdown decreased the cell-surface binding of L-PHA to 1,6GlcNAc

branched structures, the limiting factor of N-glycosylation in these malignant cell lines

must be the synthesis of GlcNAc1Man5GlcNAc2 by GlcNAc-TI. Thus, GlcNAc-TI

knockdown has a tumor-suppressive phenotype suggesting that suppressing levels of the

oncogenic tetra-antennary N-glycans is consistent with branching as a driver of

metastasis.

Migratory behavior of cells are mediated by adhesion receptors such as integrins

which link the extracellular-matrix proteins to the cytoskeleton(130). Galectins regulate

the integrin-mediated adhesion to extracellular matrix by binding to N-acetyllactosamine

sequences on integrin N-glycans and forming the galectin-glycoprotein lattice(131) (32).

For example, Galectin-3 has been shown to interact with GlcNAc-branched N-

glycans on integrin on tumor cells and promote cell spreading and motility.

Therefore, the level of N-glycan branching can influence the cell surface retention

of this protein (28). We demonstrated that GlcNAc-TI knockdown decreases the cell

99

surface localization of integrin. However, additional studies will be required to

delineate the molecular mechanism of observed phenotypes.

Inhibition of Golgi -mannosidase II with the alkaloid swainsonine blocks tumor

migration and invasion and inhibits metastasis in mouse models and was advanced into

clinical trials (54, 55). However, the drug did not progress beyond phase I trials due to

hepatic and neurological toxicity. Of note, swainsonine also strongly inhibits lysosomal

-mannosidases (Ki ~ 100 nM) which causes accumulation of oligomannosides in body

fluids and tissues. Oligomannosides accumulation in brain could explain the neurological

symptoms of swainsonine treatment (56). Thus, targeting the N-glycosylation pathway at

GlcNAc-TI or more downstream sites with more specific inhibitors might abrogate such

untoward toxicities and produce clinical benefit.

A potential advantage to developing GlcNAc-TI inhibitors as anti-tumor agents is

that this enzyme acts at the gateway to convert high mannose structures to hybrid and

complex N-glycans. Moreover, unlike other enzymes in the N-glycan pathway, no

alternate pathways have been discovered to bypass the action of GlcNAc-TI and produce

hybrid and complex N-glycans from high mannose structures (132). Thus, inhibitors of

GlcNAc-TI would be potentially useful anti-cancer agents.

Glyco-biomarkers are being extensively investigated for predicting response and

outcome in patients with cancer (133, 134). For example, higher levels of L-PHA

staining branched N-glycans have been associated with more advanced stages of colon

and breast cancer(42). The branched N-glycans are preferred substrates for extension by

polylactosamine and Lewis antigens. Sialyl Lewisa is used as a tumor marker in colon

100

and pancreatic cancer(43) and increased serum levels of sialyl Lewisx was recently shown

to be associated with circulating tumor cells in metastatic breast cancer(135). Consistent

with our in vitro and in vivo data, we demonstrated that patients with breast cancer and

the lowest levels of GlcNAc-TI in their tumors had the lowest risk of disease relapse.

Although an intriguing result, this association was derived from analysis of published

microarray data. Further studies need to be conducted prospectively to validate GlcNAc-

TI as a prognostic marker in this and other disease sites.

Thus, in summary, partial knockdown of GlcNAc-TI inhibits tumor migration and

invasion and decreases metastasis in vivo. Inhibitors of this enzyme could therefore have

therapeutic use as anti-cancer agents and GlcNAc-TI levels might confer important

prognostic information in patients with cancer.

101

Chapter 4 : Discussion and future directions

Aberrant N-glycosylation contributes to the metastatic potential of the cancer

cells. As such, inhibitors of N-glycosylation may be a novel anti-cancer strategy and an

adjuvant to standard chemotherapy. Here, we used a chemical biology approach to

identify novel inhibitors of the pathway. We also used a genetic approach to study the

effects of inhibiting GlcNAc-TI, one of the key enzymes in the synthesis of complex-type

N-glycans.

4.1 Chemical biology approach to identify N-glycosylation inhibitors

As a chemical biology approach to better understand the N-glycosylation

pathway, we conducted a high-throughput screen to find novel N-glycosylation inhibitors.

In this screen, we searched for compounds that inhibited L-PHA-mediated cell death in

Jurkat cells. From screening ~2500 off-patent chemicals and drugs, we identified the

cardiac glycoside Na+/K

+-ATPase inhibitors as having an inhibitory effect on the N-

glycosylation pathway. Among all the cardiac glycosides tested, digoxin was the most

potent inhibitor of L-PHA mediated cell death. Digoxin also inhibited the N-

glycosylation-regulated processes of tumor cell migration and invasion. Moreover,

digoxin blocked distant tumor formation in two mouse models of metastatic prostate

cancer. We used MALDI-TOF mass spectrometry to analyze the N-glycan profile of the

cells treated with digoxin. Our results indicate that Na+/K

+-ATPase inhibition blocks the

N-glycosylation pathway downstream of GlcNAc-TII.

The glycoside moiety of digoxin has a digitoxose trisaccharide and could

potentially compete with acceptor to bind the glycosyltransferases. Therefore, we tested

102

to see whether digoxin inhibited GlcNAc-TI or GlcNAc-TV directly. Digoxin did not

inhibit the enzymatic reaction of GlcNAc-TI or GlcNAc-TV per se. Next, we tested

whether the downstream effects of Na+/K

+-ATPase inhibition namely increased [Na

+]

and [Ca2+

] could inhibit these Golgi enzymes. Interestingly, GlcNAc-TV was much more

sensitive to increased [Na+] and [Ca

2+] than GlcNAc-TI. Therefore, our results suggest

that the cardiac glycoside-induced changes in N-glycan remodeling is a consequence of

on target effect of Na+/K

+-ATPase inhibition.

Our results also suggest an anti-metastatic effect for cardiac glycosides.

Interestingly, other groups have also reported anticancer effects of cardiac

glycosides(67). For example, Stenkvist et al studied 207 patients with breast carcinomas

including 32 women who were coincidentally also receiving cardiac glycoside.

Remarkably, five years after surgery, women who received CGs had dramatically lower

risk of breast cancer metastasis than women not receiving CGs(77). Thus the effects of

CGs on N-glycosylation could have influenced the rates of breast cancer metastasis. In

fact, based on this and other results, digoxin is in phase II clinical trial in conjunction

with multiagent chemotherapy and immunotherapy in patients with malignant melanoma

(136).

Other studies, however, have demonstrated that CGs are directly toxic to

malignant cells. The direct cytotoxicity of CGs on cancer cells is unlikely to be related to

effects on N-glycosylation. Rather the direct toxicity of CG is likely mediated by other

factors including increasing osmotic stress with resultant changes in cell signaling

pathways(137). It is noteworthy, however, that the concentration of CG required to

103

inhibit N-glycosylation are lower than the concentration required to induce cell death in

other studies.

Although a very low dose of digoxin (50 nM) was used to inhibit N-glycosylation

in our study, this concentration exceeds the serum concentration achievable in human.

Digoxin has a narrow therapeutic range (1.0-2.5 nM or 0.8-2.0 g/L) and higher

concentrations can cause life-threatening toxicity including cardiac rhythm disturbance,

hyperkalemia, nausea, vomiting and anorexia (138). While the concentration of digoxin

we used exceeded the therapeutic range, it is important to note that our experiments were

based on short term (24 hours) treatment of cells in vitro. Potentially, longer daily dosing

of digoxin could target the N-glycosylation pathway in tumor cells of cancer patients.

This possibility can be investigated further in vitro and in vivo. In order to test whether

clinically relevant concentrations of digoxin can target N-glycosylation, cells can be

treated with 1.0-2.5 nM of digoxin over a long period. L-PHA and ConA staining can be

performed daily to find the first time point when low dose digoxin treatment has any

effect on the N-glycan profile of the cells. When the effect on N-glycosylation is

confirmed the same regiment can be used to treat cells in migration and invasion assays.

Unfortunately, murine and rodent animal models are unsuitable for testing the

effects of cardiac glycosides. As such, in vivo testing of anticancer efficiency of CGs is

limited. The murine and rodent Na+/K

+-ATPase is highly resistant to inhibition by

cardiac glycosides. Given the interest in understanding the anticancer effects of cardiac

glycosides and the great benefit of mouse models in studying metastasis, it would be

worthwhile to generate a transgenic mouse which carries the human subunit of

Na+/K

+-ATPase and therefore is sensitive to cardiac glycosides. The human oubain-

104

sensitive and murine oubain insensitive forms of the subunit of Na+/K

+-ATPase differ

slightly and conversion is achievable by a single point mutations(139).

Given the anticancer effects of CGs and given the very narrow therapeutic

window, a medicinal chemistry approach could be used to generate novel compounds

with reduced cardiotoxicity while retaining their anticancer effects(69). Indeed, by

applying a novel neoglycorandomization technique Langenhan et al synthesized

analogues of CGs that induced cell death but had less effects on the Na+/K

+-ATPase-

(140). Therefore, through chemical modification, novel compounds which are not

cardioactive but have cytotoxic effects can be developed. However, our results

demonstrate that inhibition of N-glycosylation by CGs is due to inhibition of the Na+/K

+-

ATPase. Therefore, we do not think the anti-metastatic effects of CGs can be dissociated

from the effects on the Na+/K

+-ATPase.

Our results suggest that cardiac glycoside-mediated inhibition of N-glycosylation

could be a consequence of increased intracellular [Ca2+

] as a compensation to inhibition

of the Na+/K

+-ATPase (Chapter 2). ER and Golgi are agonist-sensitive Ca

2+ stores and

both of these organelles have Ca2+

-transport ATPase which actively maintains this

concentration gradient(141). Interestingly, the Golgi PMR1/SPCA Ca2+

pump is also

responsible for pumping Mn2+

into the Golgi (142). Hypothetically, when the

intracellular [Ca2+

] increases, the ratio of [Mn2+

/ Ca2+

] decreases and less Mn2+

is pumped

into the Golgi. Mn2+

is required for the enzymatic reaction of many glycosyltransferases

including GlcNAc-TI inside the Golgi (16) and decreased [Mn2+

] could inhibit the N-

glycosylation pathway. Thus, the effects of CG on N-glycosylation could be due to

decreased levels of Mn2+

in the Golgi as an ultimate compensatory mechanism to

105

inhibition of Na+/K

+-ATPase. In support of this hypothesis, patients with Hailey-Hailey

disease may have defects in N-glycosylation. Hailey-Hailey disease is an autosomal

dominant disorder caused by mutation in PMR1/SPCA (143). As a result of this

mutation the PMR1 pump is dysfunctional resulting in decreased Mn2+

in the Golgi.

Patients with this disease have skin lesions which are pruritic vesicles or bulla on an

erythematous base. In addition they have defects in keratinocyte adhesion(144). I

hypothesize that the defects in keratinocyte adhesion are linked to the effects of abnormal

ion concentration on the enzymatic activity of the N-glycosylation enzymes. To test this

hypothesis, one could obtain peripheral blood leukocytes from these patients and measure

N-glycosylation activity via L-PHA and ConA staining. I also hypothesize that

fibroblasts from Hailey-Hailey disease will have abnormal N-glycosylation that can be

reversed by addition of Mn2+

. Likewise, I anticipate that effects in N-glycosylation can

be induced in normal cells by knocking down PMR1 with siRNA. If correct, PMR1

inhibitors could be a novel anticancer strategy. The anticancer effects could be achieved

without the cardiotonic side effect of CGs that inhibit the Na+/K

+-ATPase.

4.2 The genetic approach to inhibit N-glycan branching in cancer

N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) is at the gateway of high

mannose N-glycans to hybrid and complex N-glycans. These complex N-glycans provide

a sticky surface on the cell membrane via interactions with selectins and galectins. Thus,

malignant cells which express increased complex N-glycans on their surface have a

selective advantage for invading the surrounding tissues and completing the metastatic

cascade. As such, we hypothesized that inhibiting GlcNAc-TI would decrease cell

106

surface complex N-glycans and decrease invasion, migration and metastasis. However

another scenario resulting from GlcNAc-TI knockdown could have increased metastasis.

Knocking down GlcNAc-TI will leave more UDP-GlcNAc in Golgi to be added by

GlcNAc-T II-TV to the maturing N-glycan. As a result there would be an increase in the

tetraantennary structure and the cells would become more invasive. Our results support

the first model. Knockdown of GlcNAc-TI decreased cell surface complex N-glycans

and inhibited cell migration, invasion and metastasis.

The main limitation of our study is that we conducted all the in vitro experiments

using the HeLa human cervical cancer cell line but for the in vivo experiment we used the

PC3N7 human prostate cancer cell line. In order to keep the experiments consistent in

terms of the cancer type and tissue of origin, other techniques of migration and invasion

can be applied so that the same measurements could be repeated in our prostate cancer

cell line. Alternatively, HeLa cells can be labeled by RFP so they can be used in a

xenograft model. Testing the effect of GlcNAc-TI inhibition in other cancer disease sites

such as breast, colon and pancreas could also improve the strength of the study.

Another limitation of our study is that we used a retrospective breast cancer data

set and there were many base line parameters we could not control. Accessibility to the

primary tumors is another issue in this case as the gene expression microarray data need

to be confirmed by Q-RT-PCR for further validation.

GlcNAc-TI knockdown altered the integrin cellular localization from the cell

membrane to area around the nucleus. Although this is an interesting observation, it is

uncertain whether alteration of the integrin is sufficient to explain the phenotype of

the GlcNAc-TI knockdown. To test the functional significance we could express a

107

integrin that localizes to the cell surface without the need for N-glycosylation and

determine whether this overexpression rescues the phenotype produced by GlcNAc-TI

knockdown.

Developing specific chemical GlcNAc-TI inhibitors could be beneficial for

understanding the N-glycosylation pathway. In addition, such inhibitors could be novel

anticancer agents. To date, no chemical GlcNAc-TI inhibitors have been reported. To

aid in developing such compounds, the X-ray crystal structure of rabbit GlcNAc-TI has

been resolved (82). Given the high degree of homology between the rabbit and human

GlcNAc-TI a model of human form can be built. Then a virtual screen could be

performed to dock a library of compounds in silico into the active site of GlcNAc-TI and

thereby find potential inhibitors(145). As an alternative to identifying GlcNAc-TI

inhibitors, a fluorescent-based or FRET-based GlcNAc-TI assay could be developed for

high throughput screening of chemical libraries(134). The GlcNAc-TI radiochemical

enzymatic assay that we used in our study is laborious and cannot be automated for high-

throughput screening. Therefore, given the significance of GlcNAc-TI in the N-

glycosylation pathway and potential antimetastatic benefits, drug discovery projects

could be carried out for this enzyme.

Another extension of the work in my thesis is to find genetic targets that can

modify the N-glycosylation pathway. Here, an siRNA screen can be done in adherent

cells to identify sequences which modify lectin staining or lectin induced cell death.

Multiple fluorescently labeled lectins such as ConA, SBA, MAA, L-PHA, SNA, and

WGA can be used and high throughput confocal microscopy can be employed to

108

visualize lectin binding. Potential hits from these screens could help us better understand

the glycosylation pathway and could provide us with novel targets for drug discovery.

Even though our expression profiling of patients had its limitations, our results

support a future project to investigate the prognostic importance of Mgat1 and other

genes in the pathway (other N-acetylglucosaminyltransferases, galactosyltransferases,

fucosyltransferases and silalytransferases) in patients with cancer. This study will require

a prospective cohort of patients treated with the same regimen. Several different assays

can be used to measure the abundance of the components of the N-glycosylation pathway

in the primary tumor. As one approach, mRNA could be extracted from primary tumor

specimens and levels of Mgat1 mRNA could be measured by Q-RT-PCR. MALDI-TOF

MS can be applied to analyze the N-glycan profile of the freshly obtained tumor samples.

Alternatively, levels of GlcNAc-TI protein could be measured by immunoblotting cell

lysates or by immunohistochemistry on paraffin blocks of tumors. For these later two

assays, optimal assay conditions and antibodies would have to be developed. Once the

levels of gene or protein expression in the relation to patient outcome can be determined,

expression of the gene or protein can either be divided into quartiles or as done in our

study or treated as continuous variable.

In summary, we identified two novel approaches to target the aberrant N-

glycosylation pathway in malignancy. First, we identified that targeting the Na+/K

+-

ATPase and the resultant changes in [Na+] and [Ca

2+] could inhibit the N-glycosylation

pathway. In addition, our results can explain the previously observed antimetastatic

effect of Na+/K

+-ATPase inhibitors. These findings provide support for further

investigation of other cytoplasmic and Golgi ion pumps as a means of manipulating the

109

N-glycosylation pathway. Secondly, we investigated the effects of GlcNAc-TI in

malignancy. GlcNAc-TI knockdown decreased the synthesis of complex N-glycans and

inhibited the N-glycosylation-mediated processes of tumor cell migration, invasion and

metastasis. In addition, we showed that lower expression of GlcNAc-TI is associated

with lower risk of metastasis in breast cancer patients. Therefore, GlcNAc-TI inhibitors

could have therapeutic use as anti-metastatic agents and GlcNAc-TI levels might confer

important prognostic information in patients with cancer.

110

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119

Appendix1

The chemical screening of LoPAC and Prestwick library

The LoPAC library (Sigma Inc.) was screened for compounds that inhibit PHA-

induced cell death. Jurkat cells (5,000 cells/well) were seeded in 96 well plates followed

by the addition of aliquots from the LOPAC library (final concentration ~ 5 μM) with a

final DMS concentration of < 0.1%. 24 hours later PHA was added at a final

concentration of 20 µg/ml. 48 hours after the addition of PHA, cell viability was

measured by an MTS assay. Overall 1280 compounds were tested in two separate assays

each of them covering half of the library.

The following formula was used to normalize the raw data of all plates(Appendix 1

Figure 1):

A490(sample) / A490(positive control) X 100

To identify statistically significant hits, we calculated the Z-score for each hit

relative to the negative control. These Z-scores were transformed into p-values based on

the standard-normal distribution. To control for multiple-testing, we employed a false

discovery rate (FDR) correction to generate a q-value for each compound. Statistically

significant hits were selected as those with a q-value of less than 5%, at which level four

compounds were identified which are listed in Appendix 1 Table 1.

The Z-factor and Z'-factor of the second assay were calculated to be 0.44 and 0.6,

respectively. These factors are defined in the following equations:

120

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200 1400

Cel

l G

row

th (

% o

f co

ntro

ls)

samples

LoPAC HTS

3SD of Sample + 3SD of (+)control

Z = 1 –

| mean of sample – mean of (+)control |

3SD of (+)contorl + 3SD of (-)control

Z' = 1 –

| mean of (+)control – mean of (-)control |

Appendix 1 Figure 1. The high throughput screen of LoPAC library

Jurkat cells (5000) were seeded in 96 well plates and treated with aliquots of the LOPAC

chemical library (final concentration ~5 µM). Twenty four hours after treatment, L-PHA

(final concentration 20 µg/mL) was added. Forty eight hours after L-PHA addition, cell

viability was measured by an MTS assay. Viability was expressed as a percentage of buffer

treated control cells.

Each dot represents one compound. The average of positive and negative controls are shown

by red solid lines and the SD is shown by red dashed lines

121

Cell growth

(% of control)

mol weight

Structure

Name Class Action

78 586.68 Dihydroouabain Ion Pump Inhibitor

77 522.71 rac-2-Ethoxy-3-

hexadecanamido-1-

propylphosphocholine

Phosphorylation Inhibitor

72 516.55 Rottlerin Phosphorylation Inhibitor

70 386.03 Hexahydro-sila-

difenidol hydrochloride,

p-fluoro analog

Cholinergic Antagonist

The Prestwick chemical library was screened using the HTS design described

above. ~1200 compounds were tested in one run of the assay covering the whole

library(Appendix 1 Figure 2 ). The top hit compounds from this screen are shown in

Appendix 1 Table 2. An arbitrary value of >83% cell growth was used to define hit

compounds. The Z-factor and Z'-factor of the HTS of Prestwick were calculated to be

0.43 and 0.120, respectively.

Appendix 1 Table 1 The statistically significant hits of LoPAC library

To identify molecules that inhibited L-PHA induced cell death, a statistically robust

methodology was applied. For each compound in the LOPAC library, a Z-score was

calculated using the extensively replicated L-PHA control wells. These Z-scores were

converted into p-values using the standard normal distribution. As 1280 distinct compounds

were screened, a false discovery rate (FDR) correction for multiple-hypothesis testing to the

vector of p-values was applied. Statistically significant hits were defined as compounds with

a q-value (adjusted p-value) less than 0.05, indicating a false-positive rate of at most 5%. The

four statistically significant hits identified in this manner are listed here

122

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200

Cell G

row

th (

% o

f C

on

tro

l)

Samples

Prestwick HTS

Appendix 1 Figure 2 The high throughput screen of Prestwick library

Jurkat cells (5000) were seeded in 96 well plates and treated with aliquots of the Prestwick chemical

library (final concentration ~5 µM). Twenty four hours after treatment, L-PHA (final concentration

20 µg/mL) was added. Forty eight hours after L-PHA addition, cell viability was measured by an

MTS assay. Viability was expressed as a percentage of buffer treated control cells.

Each dot represents one compound. The average of positive and negative controls are shown by red

solid lines and the SD is shown by red dashed lines

Each dot represents one compound. The average of positive and negative controls are shown by red

solid lines and the SD is shown by red dashed lines.

123

Cell Growth(% of

Control)

Name Action

104% Berberine Chloride Weak antibiotic properties

94% Lasalocid Sodium Salt Anticoccidial agent

85% Mefloquine hydrochloride Blocker of gap junction channels

Cx36 and Cx50

84% Lovastatin Potent Anticholesterimic Agent

Appendix 1 Table 2 The top hits of the Prestwick library

An arbitrary value of >83% cell growth was used to define hit compounds from the

Prestwick library.

124

Appendix 2

The spectra of MALDI-TOF data presented in chapter 2

In order to investigate the effect of Na+/K

+-ATPase inhibition on N-glycan profile of the

malignant cells, Jurkat cells (1 X 108) were treated with 100 nM digoxin, 2 µM

Swainsonine or buffer control. After incubation, cells were lysed in 35 mM Tris, 8M

urea, 4% CHAPS, 65 mM DTT, pH 8.0 followed by sonication and freezing. Equal

amounts of protein for each sample were dialysed with cassettes (Slide-A-Lyzer,

molecular weight cutoff of 7,000, Pierce, Rockford, IL) in 10 mM ammonium

bicarbonate/0.02% SDS. Following dialysis, samples were concentrated via vacuum

centrifugation. Samples were deglycosylated at 37ºC for 48 hours with PNGase F (New

England Biolabs, Ipswich, MA). Oligosaccharides were purified via C18 and porous

graphitized carbon solid phase extraction. The composition of N-glycans was

semiquantified using mass spectrometry via MALDI-TOF, using DHB as matrix, on a

MALDI-CFR (Shimadzu Biotech, Columbia, MD) as previously described(94). The

intensities of the peaks were arbitrarily normalized to the level of

(Hexose)2(HexNAc)2)Deoxyhexose1. The MALDI-TOF and annotation of the spectra

was performed in Dr. Reindold’s facility at The Glycomics Center, Division of

Molecular, Cellular, and Biomedical Sciences, University of New Hampshire.

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14

50

15

00

Ma

ss/C

ha

rge

34

mV

[su

m=

17

17

49

mV

] P

rofi

les

1-5

00

0 S

mo

oth

Av 1

0 -

Ba

seli

ne

10

0

Dig

oxin

, P

NG

ase

, C

18

, P

GC

, ce

llu

l, u

nre

d/n

at

Da

ta:

9-9

-06

Dig

oxin

_0

10

00

1.A

14

9 S

ep

20

06

14

:13

Ca

l: D

avid

9 S

ep

20

06

13

:48

Kra

tos

PC

Axim

a C

FR

plu

s V

2.3

.4:

Mo

de

re

fle

ctr

on

, P

ow

er:

14

0,

P.E

xt.

@ 1

00

0 (

bin

10

8)

917.1

1

918.0

8

1485.1

31257.1

01486.0

6933.1

1

1079.1

11258.0

61282.1

3919.0

81419.0

61487.0

31283.1

01080.1

3934.0

81259.0

61054.1

11013.0

91420.9

91284.1

11081.1

2920.1

1935.0

61216.0

91339.1

11136.1

41378.0

91260.0

91175.0

61056.1

31015.1

31444.1

31241.1

9995.1

0969.1

91036.1

31360.0

31319.0

81492.9

91467.3

11403.1

3

(Hex) 2

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 3

(HexN

Ac) 2

(Hex) 3

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 4

(HexN

Ac) 2

(HexN

Ac)1

+

(Man)3

(Glc

NA

c)2

(Hex) 4

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 2

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 1

(Deoxy

he

xose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 2

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 3

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 2

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

MA

LD

I-T

OF

sp

ectr

um

, D

igo

xin

-tre

ate

d c

ells

, P

NG

ase

rele

ase

d g

lyca

ns, 1

/2

128

0

10

20

30

40

50

60

70

80

90

10

0

%In

t.

15

00

16

00

17

00

18

00

19

00

20

00

21

00

22

00

23

00

24

00

25

00

26

00

27

00

28

00

29

00

30

00

Ma

ss/C

ha

rge

12

mV

[su

m=

58

09

6 m

V]

Pro

file

s 1

-50

00

Sm

oo

th A

v 1

0 -

Ba

seli

ne

10

0

Dig

oxin

, P

NG

ase

, C

18

, P

GC

, ce

llu

l, u

nre

d/n

at

Da

ta:

9-9

-06

Dig

oxin

_0

10

00

1.A

14

9 S

ep

20

06

14

:13

Ca

l: D

avid

9 S

ep

20

06

13

:48

Kra

tos

PC

Axim

a C

FR

plu

s V

2.3

.4:

Mo

de

re

fle

ctr

on

, P

ow

er:

14

0,

P.E

xt.

@ 1

00

0 (

bin

10

8)

1485.1

3

1486.0

6

1487.0

3

1581.0

81743.9

2

1905.8

2

1689.0

4

1540.0

71892.0

02540.1

51702.0

31503.1

32174.8

31622.1

71810.0

11758.9

02028.9

42904.6

21936.2

72488.7

22378.4

42615.7

72854.4

22123.7

32980.3

01880.7

0

(Hex) 4

+

(Man) 3

(Glc

NA

c) 2

(Hex) 3

(HexN

Ac) 1

+

(Man) 3

(Glc

NA

c) 2 (H

exN

Ac) 3

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 5

+

(Man) 3

(Glc

NA

c) 2

(Hex) 2

(HexN

Ac) 2

(Deoxyhexose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 6

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 4

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

MA

LD

I-T

OF

sp

ectr

um

, D

igo

xin

-tre

ate

d c

ells

, P

NG

ase

rele

ase

d g

lyca

ns, 2

/2

129

0

10

20

30

40

50

60

70

80

90

10

0

%In

t.

90

09

50

10

00

10

50

11

00

11

50

12

00

12

50

13

00

13

50

14

00

14

50

15

00

Ma

ss/C

ha

rge

15

mV

[su

m=

73

47

8 m

V]

Pro

file

s 1

-50

00

Sm

oo

th A

v 1

0 -

Ba

seli

ne

10

0

Sw

an

son

ine

, P

NG

ase

, C

18

, P

GC

, ce

llu

l, u

nre

d/n

at

Da

ta:

9-9

-06

Sw

an

son

ine

_0

10

00

1.A

15

9 S

ep

20

06

14

:33

Ca

l: D

avid

9 S

ep

20

06

13

:48

Kra

tos

PC

Axim

a C

FR

plu

s V

2.3

.4:

Mo

de

re

fle

ctr

on

, P

ow

er:

14

0,

P.E

xt.

@ 1

00

0 (

bin

10

8)

917.1

4

1257.1

0

1403.0

9

1079.1

4

1258.1

0918.1

11419.0

9

1420.0

8

1080.1

3

1013.1

21054.1

11405.0

51259.1

0

919.1

11095.1

11241.1

21055.1

21014.1

11216.0

91378.0

91242.1

11444.1

31096.1

0935.0

91282.1

31175.0

9920.1

11485.1

61015.1

31056.1

31243.1

01036.1

31380.0

71435.2

91337.1

2995.1

01218.0

21098.1

01298.9

71264.4

0969.2

51136.1

71077.0

71198.0

21360.0

31159.5

4

(Hex) 2

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 3

(HexN

Ac) 2

(Hex) 3

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 4

(HexN

Ac) 2

(HexN

Ac)1

+

(Man)3

(Glc

NA

c)2

(Hex) 4

(HexN

Ac) 2

(Deoxyhexose) 1

(Hex) 2

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 1

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2 (H

ex) 2

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 3

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 2

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

MA

LD

I-T

OF

sp

ectr

um

, S

wa

nso

nin

e-t

reate

d c

ells

, P

NG

ase

rele

ase

d g

lyca

ns,1

/2

130

0

10

20

30

40

50

60

70

80

90

10

0

%In

t.

15

00

16

00

17

00

18

00

19

00

20

00

21

00

22

00

23

00

24

00

25

00

26

00

27

00

28

00

29

00

30

00

Ma

ss/C

ha

rge

4.4

mV

[su

m=

21

75

1 m

V]

Pro

file

s 1

-50

00

Sm

oo

th A

v 1

0 -

Ba

seli

ne

10

0

Sw

an

son

ine

, P

NG

ase

, C

18

, P

GC

, ce

llu

l, u

nre

d/n

at

Da

ta:

9-9

-06

Sw

an

son

ine

_0

10

00

1.A

15

9 S

ep

20

06

14

:33

Ca

l: D

avid

9 S

ep

20

06

13

:48

Kra

tos

PC

Axim

a C

FR

plu

s V

2.3

.4:

Mo

de

re

fle

ctr

on

, P

ow

er:

14

0,

P.E

xt.

@ 1

00

0 (

bin

10

8)

1743.9

2

1581.0

8

1581.9

7

1606.1

1

1607.0

8

1905.8

2

1540.0

7

1460.1

41622.1

72133.7

61768.0

7

1702.0

31486.0

62082.4

41809.9

71760.0

81598.0

72540.1

51936.2

72447.1

21689.1

62175.5

62028.9

42615.7

72904.5

12980.3

0

(Hex) 4

+

(Man) 3

(Glc

NA

c) 2

(Hex) 3

(HexN

Ac) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 2

(HexN

Ac) 1

(Deoxyhexose) 1

+

(Man) 3

(Glc

NA

c) 2

(HexN

Ac) 3

(Deoxyhe

xose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 5

+

(Man) 3

(Glc

NA

c) 2

(Hex) 2

(HexN

Ac) 2

(Deoxyhexose) 1

+

(Man) 3

(Glc

NA

c) 2

(Hex) 6

+

(Man) 3

(Glc

NA

c) 2

MA

LD

I-T

OF

sp

ectr

um

, S

wa

nso

nin

e-t

reate

d c

ells

, P

NG

ase

rele

ase

d g

lyca

ns,2

/2

investigation of the effects of inhibiting n-glycosylation ... · investigation of the effects of...

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