Modulating Crb2 protein levels effect on tumour progression

Baijun KouDepartment of Biomedical Sciences&

School of Medicine

University of Sheffield, U.K.

European Union Crumbs Consortium

June, 2008. Marseille, France

APICAL-BASAL POLARITY IS VITAL FOR NORMAL CELLULAR FUNCTION

Defects in apical-basal polarity are associated with

developmental defects

Cancer et al.

No

rma

l tissue C

arcin

om

a tissu

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CRB2 expression in human breast cancer

Grade I Grade II Grade III

CRB2 expression in human normal & prostate cancer

CRB2 is a Crumbs family member with at least 3 isoforms (R.Walker)

Located at human chromosome 9q33.3.CRB2 is a type I transmembrane protein with multiple EGF-like domains and Laminin G-like domains.

CRB2

Extracellular

hCrb2s(1-10a exon)

hCrb2f(1-13exon)

hCrb26a(6a-13exon)

Laminin G-like domain EGF-like domain

signal peptidetransmembrane domain

cbEGF-like domain

cytoplasmic domain intracellularextracellular

Patial EGF-like domain

Three protein complexes maintain cells polarity

Alteration CRB2 affects cells proliferation and apoptosis

WT Over KD

Immunocytochemistry(Ki67)

ß-actin

CPP32

sh129

WT6a-1

31-

13 WTsh

128

WB

Overexpression knockdown

n=3 *p<0.05

0

500

1000

1500

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3500

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K-C K-6a-13 K-1-13 K-sh128 K-sh129

Cel

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popt

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K-C K-6a-13 K-1-13 K-128 K-129ki6

7 p

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ells

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n=10,*p<0.05,***p<0.0001

Altering CRB2 expression levels affects….migrationWounding assay:

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** ****p<0.01,***p<0.001 v K-C

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K-C

K6a-1

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K1-13

K128

K129

Wid

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f w

ou

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(U

)

*** ***

**p<0.01,***p<0.001 v K-C

Alteration of CRB2 affects cells invasion

8 12 16 20 24 28 32 36 40 44 48(hrs)

36hrs 40hrs 44hrs

Mitomycin(1ng/ml)

n=4 p<0.05 ,ANOVA

Ki67 antibody staining with the membrane

Altering CRB2 expression levels affects

…cadherin expression

Pan-cad

K-c 6a-13 1-13 k-c sh128 sh129

γ-tubulin

WB

Summary

Modulating Crb2 protein levels results in:

•A positive correlation with proliferation and negative with apoptosis

•A positive correlation with migration & invasion

•Interfered with cell adhesion and tight junction markers

•Crb2 depletion induces tumor suppressor protein expression

Crb2 protein is localised to the apical surface of the developing neural tube and normal adult

epithelial tissue

Neural tube in chicken embryo

Human ovarian tissue

CRB2 expression in cancer cell lines

1 2 3 4 5 6 7 8 9 10 1112 13

6a 10a

54 human cancer cell lines were screened- by RT-PCR10 cell lines express different exons of CRB2 with variable levels

Altering CRB2 expression levels affects

…morphology

phalloidin (F-actin) staining

A way to find the external partner of crumbs (EPOC)?

WB

1-9a cells w

hole lysis

1-9a protein

1-9a culture supernatant

V5 antibody

F-actin staining

1-9a protein was added

3 days in K-C cells

K-C

Altering CRB2 expression levels affects

… ß-Catenin expression

ß-Catenin

γ-tubulin

K-c 6a-13 1-13 K-c sh128 sh129

WB

WT Over-expression Knockdown

ß-Cat

DAPI

Alteration of CRB2 assembles PATJ

WB

K-C 6a-13 1-13 sh128 sh129

PATJ

γ--tublin

WT

PATJ

DAPI

3-D

PATJ

Pan-cad

DAPI

2-DOverexpression Knockdown

CRB2 knockdown induced tumor suppressor proteins expression

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

K-C

K6a-13

K1-13

K-C

K128

K129

SCRIB

-tubulin

LKB1

LGL1

WB

Tight junction

Adherin junction

Altering CRB2 expression levels affects …ability to polarization

K-C

K1-13

K128

-catenin DAPI Merge

5000 cells were cultured in matrigel coated chamber with 10% FBS RPMI 1640 for 6 days, Images were taken by Zeiss 510 confocal microscope

Future questions & work• What are the cells I have been working on?-in progress

• How do different Crb2 splice forms interact with the cell cytoskeleton (Rho

activation?)- in progress

• Can K1-9a cells enable us to identify EPOC? - in progress

• How does knockdown of Crb2 increase/stabilise Lgl1 etc?

• Does Crb2 levels effect tumour formation in mice?- in progress

• Are the results repeatable in other cell lines?-in progress

• Determine role of CRB2 in cancer progression

AcknowledgementsAcknowledgements

Penny RashbassPenny Rashbass( Supervisor)( Supervisor)

Rob WalkerRob WalkerJean IllesJean IllesThorsten BoroviakThorsten BoroviakMariyam MurtazaMariyam Murtaza

Funding: Yorkshire Cancer Research

CollaboratorsCollaboratorsSimon CrossSimon CrossJim CattoJim CattoRichard BryantRichard BryantSteve WinderSteve WinderDuncan BakerDuncan BakerKim ReevesKim ReevesNicky BrownNicky Brown

EU Crumbs consortium EU Crumbs consortium

Ki67 antibody staining with the membrane

Counting 100 cells per field n=4, p>0.05 After adding mitomycin 36hrs

k-c k6a-13 k1-13 k128 k129

Histo- microarray-immunostaining of CRB2

Oesophageal (82ca /39con)

Ovary(58ca/29con)

Lung(84ca/4con)

Prostate(121ca/44con)

Breast(336ca)

Colorectal(112ca)

Brain(43ca/4con)

Baijun Kou

Summary-1 CRB2 expression was profiled in 7 different epithelial tissues - 786 cancer & 116 non-cancer

samples. In normal & benign adenoma - CRB2 is usually apical In breast, colorectal & oesophageal adeno

- CRB2 expression levels In prostate, ovary, brain & oesophageal squamous-

CRB2 expression levels CRB2 tends to become cytoplasmic

In general, within tumour samples, there is a negative correlation between grade &/or stage and CRB2 expression levels.

• Need to be done: Quantification of CRB2 expression in human cancer

tissues by q-PCR.

Depleting Crb2 expression levels affects …polarity & adhesion

Depleting Crb2 expression levels affects …proliferation

Depleting Crb2 expression levels affects …differentiation

Western blot analysis of CRB2& F-actin expression of cytoskeleton after alteration of CRB2 level

ß-actin

C 1-9a 6a-13 1-13 C sh128 sh129

overexpression knockdown

CRB2

WT 1-9a 6a-13 1-13 sh128 sh129

Gibson & Perrimon 2003

Complexes maintaining cell polarity

Functions & binding partners Include:•Polarity•Adhesion•Signalling•Vesicular trafficking•Proliferation•Actin cytoskeleton•microtubules

Crumb2 knockdown induces cancer suppressor genes expression and induces cells to epithelial phenotype

LGL1

Scribble

5000 cells were cultured in matrigel coated chamber with 10% FBS RPMI 1640 for 4 days, Images were taken by Zeiss 510 confocal microscope

WT Overexpression Knockdown

β-cat

Crb2

DAPI

γ-tublin

K-c 6a-13 1-13 k-c sh128 sh129

6 days culture

Tissues Cancer/non-ccancer grade stage mislocalization local invasion lymph node vascular /distance Expression ratio metastasis metastasis

Oesophageal squa adeno squa adeno NR NR almost all NR NR NR (including non-cancer) Prostate NR NR Cancer>non-cancer NR all ( ) NR Ovary NR NR(grade) PR

Lung NR almost all cancer NR NR trend NR Breast NR (tumor size)NR apical-cytoplasma-lose with grade increase Colon NR apical-cytoplasma-lose with grade increase Brain all

CRB2 expression in epithelial carcinomas

mCrb2 overexpression in kelly cell lines

1 2 3 4 5 6 7 8 9 10 1112 13

Have used mouse cDNA constructs

1-13

6a-13

1-9a

Robert Walker

CRB2 RNA interference(RNAi) through MISSION shRNA

• MISSION shRNA clones are sequence-verified shRNA plasmids(pLKO.1-puro).

• pLKO.1 contains puromycin selection marker for stable transfection.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.