methylation framework of cell cycle gene inhibitor … framework of cell cycle gene inhibitor...

Methylation Framework of Cell Cycle Gene Inhibitor p16INK4A In Hepatocellular Carcinoma Patients

Thesis Submitted for partial fulfilments

of M.D. degree in Clinical & Chemical Pathology

By

Hany Ahmed Fouad ElghobaryM.B.B.Ch, M.Sc. of Clinical & Chemical Pathology

Faculty of MedicineCairo University

Supervised By

Prof. DR. Fatma Ahmed El-MougyProfessor of Clinical & Chemical Pathology


Prof. DR. Nabil Mostafa Khalil El-KadyProfessor of Tropical Medicine


Prof. DR. Sahar Abd El-Atty SharafProfessor of Clinical & Chemical Pathology


DR. Hany Hussein El-SayedLecturer of Clinical & Chemical Pathology



2012

Acknowledgement

First of all I would like to thank ALLAH for giving me the power to

complete this work, ALLAH the most merciful, gracious and

compassionate.

I am greatly honored to express my deep gratitude and faithfulness to

Prof.Dr.Fatma Ahmed Fathy EL-Mougy Professor of clinical and

chemical Pathology, Faculty of Medicine-Cairo University for her

encouragement, sustained support and guidance throughout the work.

I would like to express my thanks and deep regards to Prof.Dr.

Nabil Mostafa El Kady, Professor of Tropical Medicine, Faculty of

Medicine-Cairo University for hir great help throughout the work. His

fatherhood attitude was so supportive for the completion of this work.

I feel extremely grateful to Prof. Dr. Sahar Abd EL-Atty Sharaf

professor of clinical and chemical pathology, Faculty of Medicine-Cairo

University for her sincere efforts in supervising this work and for her

advices and guidance throughout the work of the thesis.

I would like to thank dr. Hany Hussein El-Sayed lecturer of

clinical and chemical pathology, Faculty of Medicine-Cairo University for

his friendly attitude and kind supervision.

I would like also to thank my colleague Dr. Walaa Ahmed Rabie

lecturer of clinical and chemical pathology, as well as Dr. Mohamad

Mahmoud Nabil, Ass. Prof. of tropical medicine for their kind care and help.

Furthermore, I dedicate the thesis to all the members of my family

for their moral support and patience during this work.

A special dedication to my wife for her never ending care. She was

always supporting me and encouraging me to continue and finish this work.

Abstract

Hepatocellular carcinoma (HCC) is currently the fifth most

common solid tumor worldwide and the third leading cause of cancer

related death.

The tumor suppressor gene p16INK4A is located on chromosome

9p21 and encodes the p16 protein, which binds selectively to CDK4 to

inhibit activation of the CDK4/cyclin D complex in the G1 phase.

Inactivation of this gene which normally inhibits progression to the

G1 phase of the cell cycle is involved in the initiation of tumors.

Several studies have shown that p16INK4A is frequently

downregulated by aberrant methylation of the 5'cytosine phosphoguanine

island within the promotor region.

This study was performed to evaluate the frequency of methylated

p16INK4A in the peripheral blood of HCC and liver cirrhosis patients and

to evaluate its role as a risk factor for HCC using methylation specific

PCR (MSP).

Methylation of p16INK4A was detected in 6.7% (2/30) of HCC

patients and 5% (1/20) of LC patients.

Results of the present study revealed that methylated p16INK4A

cases in the HCC group had the highest AFP levels.

Results of this study could not conclude hypermethylation of

p16INK4A as a risk factor for HCC.

Key word: p16INK4A- Hepatocellular- Cell Cycle Gene.

I

Table of Contents

Page

Introduction 1

Aim of work 3

Review of literature 4

Chapter one: Hepatocellular carcinoma 4

I. Introduction 4

II. Risk factors for HCC 4

III. Diagnosis of HCC: 17

A. Clinical Picture 17

B. Radiologic Investigations 19

C. Laboratory findings 21

D. Histological diagnosis of HCC 32

Chapter two: Cell cycle 33

Regulation of eukaryotic cell cycle 35

Role of cyclins and CDKs 36

Checkpoint 39

Tumor suppressor genes 40

Epigenetics 52

DNA methyltion 52

Histone modification 57

Nucleosome positioning and remodeling 58

II

Genomic imprinting 59

Noncoding RNAs 60Chapter three: p16 gene 61Gene location and mapping of the p16INK4A gene 61Cellular location of p16 INK4A

63

Function of p16 INK4A 63

Role of p16 INK4A in HCC 68

Molecular mechanism of p16INK4A DNA methylation in HCC 69

Subjects and methods 71

Results 93

Discussion 106

Summary 120

Conclusion & Recommendation 124

References 125

III

List of tables

Page

82 Carrier RNA and Buffer BL volumesTable 1

82Bisulfite Reaction componentsTable 2

83 Bisulfite Conversion Thermal Cycler ConditionsTable 3

87 PCR reaction mixtureTable 4

93 Presenting symptoms & signs in subjects of different

groups

Table 5

94 Radiological findings of patients in different groupsTable 6

94 Site and number of hepatic focal lesion (HFL) in

HCC cases

Table 7

95 Haematological testsTable 8

95 Biochemical testsTable 9

96 Number and percentage of viral and bilharzial

markers in different groups

Table 10

96 Comparisons of lab data between different groupsTable 11

99Number & percentage of p16INK4A methylated cases

among different groups

Table 12

102Important data of subjects with methylated p16INK4ATable 13

IV

List of figures

Page

6Cellular signaling pathways implicated in

hepatitis B virus (HBV) X protein-related

hepatocarcinogenesis.

Figure 1

7A hypothetical model depicting potential

genetic and epigenetic changes induced by

HBV infection

Figure 2

9Cellular signaling pathways implicated in

hepatitis C virus (HCV)

Figure 3

14Main oncogenic pathways associated with

hepatocarcinogenesis

Figure 4

33Schematic representation of the cell cycleFigure 5

37Mammalian cell cycle regulation by

CDK/cyclin holoenzymes and CKIs

Figure 6

38 Rb and cell cycle machinery.Figure 7

41Regulation of the G1/S transition by the

Cdk4/6–cyclin D/INK4/Rb pathway.

Figure 8

44Schematic representation of alterations in

p53/ARF and RB/INK4A pathways in HCC.

Figure 9

45 The roles of p53 in growth arrest and

apoptosis

Figure 10

48Function of p21Figure 11

59Nucleosome and histone modificationsFigure 12

61Genomic structure, mutations, and transcripts

of the INK4b (p15) and INK4a (p16/p19ARF)

locus.

Figure 13

V

Page

64 A simplified view of the pRB−E2F pathwayFigure 14

78QIAamp Spin ProcedureFigure 15

94Number of focal lesions in HCC groupFigure 16

97correlation between AFP & ASTFigure 17

98correlation between AFP & ALPFigure 18

98ROC curve for AFPFigure 19

99Number & percent of p16INK4A methylated

cases among groups

Figure 20

101Detection of products of MSP for aberrant

p16INK4A methylation in HCC subjects

Figure 21

101Detection of products of MSP for aberrant

p16INK4A methylation in LC subjects

Figure 22

102Detection of products of the unmethylated

(quality control) primer

Figure 23

VI

List of abbreviations

microgramµgmicrogram/litreµg/LmicrolitreµlAflatoxin B1 AFB1 Alpfa fetoproteinAFPAlpha-1- fucosidase AFUalbuminALBAlkaline phosphataseALPAlanine aminotransferaseALTAcivator proteinAP-1Apoptotic Protease Activating Factor 1 Apaf

Adenomatous Polyposis ColiAPCAnaphase-promoting complexAPCAlternative reading frame ARFapoptosis signal-regulating kinase 1ASK1 Aspartate aminotransferaseAST Activated transcription factorATFActivating Transcription Factor 2. ATF-2 Adenosine triphosphateATP Bcl-2-associated X protein BaxBase pairbpBreast Cancer BRCABub1-related kinase 1; Bub1, budding uninhibited by benzimidazoles 1

BubR1

C1q receptor C1qR Calcium Ca++ Caspase 3CASP 3Complete blood countCBCCyclin D2 CCND2Cluster of differentiation CDCell devision cycle 2cdc2 Cyclin-dependent kinase inhibitor 2A CDKN2A cyclin-dependent kinases CDKsComplementary deoxyribonucleic acidcDNA CDK interacting protein/Kinase inhibitory protein CIP/KIPcyclin-dependent kinase inhibitory protein-1CIP-1Cyclin kinase inhibitor CKIcentimeter cm

VII

Cytosine phosphoguanineCpGc-AMP response element bindingCREBComputed tomography CTCytochrome C Cyto CDistilled waterD.WDeoxyadenosine TriphosphatedATPDirect bilirubinDBilDeleted in colorectal carcinomaDCCDes-gamma-caboxy prothrombinDCPDeoxycytidine TriphosphatedCTPDamage specific-DNA binding proteinDDBDeoxyguanosine TriphosphatedGTPDeoxy ribonucleic acidDNADNA methyltransferasesDNMTsdeoxynucleotide triphosphatesdNTPs dimerization partner 1 DP1Deoxythymidine triphosphatedTTP E-cadherinEcadEthylene diamine tetraacetateEDTAEnzyme linked immunosorbent assayELIZAEstrogen receptors ERExtracellular Signal-Regulated Kinase ERKFibrosis scoreFFine needle aspiration biopsyFNABgram gGap 0 phase G0 phaseGap 1 phaseG1 phaseGap 2 phase G2 phaseGrowth Arrest and DNA Damage-inducible GADDGamma glutamyl transferaseGGTGolgi protein 73GP73Glipican 3GPC3Gene associated with Retinoid-Interferon-induced Mortality

GRIM

glutathione S-transferase promoterGSTP1 Helicobacter pyloriH.pyloriHistone acetyl transferaseHATHaemoglobin Hb Hepatitis B envelope antigen HBe Ag Hepatitis B surface antigenHBs Ag

VIII

Hepatitis B virusHBVHepatitis B x protein HBxHepatocellular carcinoma HCC Hydrochloric acid HCLHepatitis C virusHCVhistone deacetylasesHDAC Histone demethylaseHDMT Hepatic focal lesionHFLhepatocyte growth factorHGFhuman herpes virus-8HHV-8Hypoxia inducible factor 1α HIF-1 αHuman immunodeficiency virusHIVhistone methyltransferaseHMTHigh performance liquid chromatographyHPLChepatic stellate cells HSChuman telomerase hTERT insulin-like growth factor IIIGF II Inhibitor of Kinase 4AINK4AInsulin resistance IRc-Jun NH-terminal kinaseJNK kilobytekbkilodaltonKDaKaposi's sarcoma-associated herpesvirus KSHV litreLLiver cirrhosisLCLens culinaris agglutininLCAlow-density lipoproteinLDLLarge hepatitis B envelopeLHBeMole MMitosis phaseM phase mitogen-activated protein kinase MAPKMilli absorbance unitmAUmurine double minute (mdm2) MDM2milligram mgMagnesium chloride MgCl2minuteminMicro ribonucleic acidmiRNAmillilitremlmillimolemMMatrix metalloproteinases MMPs

IX

Magnetic resonance imaging MRIMessenger ribonucleic acidmRNAMethyl Specific Polymerase Chain ReactionMSPmultiple tumor suppressor 1 MTS-1Non alcoholic fatty liver diseaseNAFLD Non alcoholic steatohepatitisNASHNon coding ribonucleic acidncRNANuclear factor kappa betaNF-κB nanogramngNumber No.Degrees centigrade/Celsiusº Copen reading frame ORFprobabilityp

،p53 Inducible Ribonucleotide Reductase P53R2 Peripheral blood PBProthrombin concentrationPCProliferating cell nuclear antigenPCNAPolymerase chain reactionPCRPlatelet derived growth factorPDGFpicogrampgPhosphoinositide 3-KinasePI3K Proteins induced by vit K absence PIVKA2Protein kinase CPKCplateletsPltsPeroxisome Proliferator Activated Receptor PPARPhosphorylated retinoblastoma pRBC-terminally phosphorylated Smad3 pSmad3C linker-phosphorylated Smad3 pSmad3L Prothrombin timePTphosphatase and tensin homologue deleted on chromosome ten

PTEN

correlationrRapidly Accelerated Fibrosarcoma RAF Rat sarcoma rasRetinoblastoma 1Rb1Restriction landmark genomic scanningRLGSRibonucleic acid RNA Receiving operating characteristicROCReactive oxygen speciesROSRevolutions per minuterpm

X

secondsSynthesis phase S phaseStress activated protein kinaseSAPKSquamous cell carcinoma antigenSCCAStandard deviationSDsoluble Glipican 3sGPC3Suppressors of Cytokine Signaling 1SOCS-1Serine Protease 1 SP1 Stimulatory protein 1 Sp1 Silencing RNA complexSrc Sterol Regulatory Element Binding Protein SREBPSWItch/Sucrose NonFermentable SWI/SNF Tris borate EDTATBETotal bilirubinTBilTATA box binding proteinTBPTransforming growth factor-αTGF-α Transforming growth factor betaTGF-βUltrasound US United states of AmericaUSAVascular endothelial growth factorVEGFVon Hippel-LindauVHLwild-type p53-activated fragment 1 WAF 1 wild-type activating fragment-1 WAF-1White blood cellsWBCsWorld health organization WHO Wild type p53-induced phosphatase 1 Wip1/Ppm1d Wnt inducible signal pathway protein WISP-2 X gravity X g

Introduction & Aim of work

1

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common and the

third most fatal malignancy worldwide (Eric et al., 2010). It showed a

dramatic increase of its incidence worldwide over the last three decades

and is associated with a very poor prognosis. Except for patients accessible

for surgical treatment, the 5-year survival is less than 3% (Antal et al.,

2010). The burden of hepatocellular carcinoma (HCC) has been increasing

in Egypt, with a doubling in the incidence rate in the past 10 years (Anwar

et al., 2008).

It is commonly associated with chronic liver diseases caused by

infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV),

excessive alcohol consumption, aflatoxin, and certain metabolic diseases

(Oranous et al., 2011).

Although alpha-fetoprotein (AFP) measurement and ultrasonography

are useful surveillance tests for detecting HCCs at a stage at which they

may be treated, both tests have limitations: AFP shows low sensitiviyy and

specificity , while the results of ultrasonograpy are dependent on the skill

of those performing the examination and on the condition of the patient

(Ahmed et al., 2010).

Inactivation of tumor suppressor genes and activation of oncogenes

initiated by genetic and epigenetic differences may play an important role

in carcinogenesis (Oranous et al., 2011).

The P16INK4A gene is a tumor suppressor that acts as a negative

regulator of the cell cycle by binding to and inhibiting cyclin-dependent

kinase 4 (CDK4). Reduced expression of the p16INK4A gene results in

uncontrolled division of cells. Several mechanisms that lead to p16INK4A


2

inactivation have been described, including point mutations, homozygous

deletions, and promoter hypermethylation (Matsuda, 2008). The later

having been shown to occur more frequently in HCC patients (Csepregi et

al., 2010).

The tumor suppressor gene p16INK4A is located on chromosome 9p21

and encodes the p16 protein, which binds selectively to CDK4 to inhibit

activation of the CDK4/cyclin D complex in the G1phase of the cell cycle

(Thorgeirsson and Grishan,2002) .

Inactivation of p16 INK4A gene resulting from its methylation leads to

disruption of cell cycle control, a finding which has been reported in HCC

(Shim et al., 2003).

However, reports on p16 INK4A methylation in HCC were remarkably

diverse (Zang et al., 2011) (Patopova et al., 2011).

Aberrant methylation of the p16INK4A promotor has also been

reported in early preneoplastic lesions in the lung, colon, oesophagus and

pancreas. These findings suggest that loss of p16INK4A function, often due

to promotor methylation, may be an early event in the pathogenesis of

various types of tumors (Kaneto et al., 2006).


3

Aim of work

The present study was performed to estimate the frequency of

methylated p16INK4A in the peripheral blood of patients with liver cirrhosis

(LC) and HCC to evaluate its role as a risk factor for hepatocellular

carcinoma (HCC).

Hepatocellular Carcinoma

4

Chapter 1


I. Introduction:

Hepatocellular carcinoma (HCC) is one of the leading causes of

worldwide cancer mortality, with an estimated 1 million deaths annually

and a 5-year survival rate of less than 5% (El-Serag, 2007). The incidence

of HCC varies considerably depending on, geographical location and the

cause of liver disease. HCC incidence of 4 to 15 per 100,000 has been

reported in Western countries, compared with 120 per 100,000 in Asia and

Africa (Hashem, 2011).

In most countries, HCC accounts for 70%–85% of primary liver

cancer cases (Ahmed et al., 2008), with the burden of disease expected to

increase in coming years (Alan et al., 2010).

II. Risk factors for HCC:

HCC is a complex disease associated with many risk factors and

cofactors (Gomaa et al., 2008). In most patients, HCC is preceded by

cirrhosis of the liver (Llovet, 2005) and, unsurprisingly, common causes of

cirrhosis have been identified as key risk factors for HCC. Of particular

importance is chronic infection with HBV or hepatitis C virus (HCV).

Indeed, it has been estimated that HBV is responsible for 50%–80% of

HCC cases worldwide, whereas 10%–25% of cases are thought to be a

result of HCV infection (Block et al., 2003). Antiviral therapy resulting in

viral suppression is known to significantly decrease the risk for HCC in

patients infected with HBV (Liaw et al., 2004).

Cancer emerges when immunological control fails and transformed

cells develop resistance against cell death signaling pathways. The same


5

mechanisms underlie the poor responsiveness of HCC towards

chemotherapy (Schattenberg et al., 2011).

Age of 50 years or older, male gender, and advanced fibrotic stage

were associated with an increased risk of HCC. Liver fibrosis seems to be a

crucial factor in determining carcinogenesis (Sandra et al., 2010).

A. Hepatitis B Virus:

Human hepatitis B virus is a member of the hepadnavirus family; it

is a DNA enveloped virus, which is remarkably stable to organic solvents,

and also heat- and pH-resistant (Gripon et al., 2002).

HBV CarcinogenesisThe geographic distribution patterns of HCC and hepatitis B virus

(HBV) almost coincide with each other (Marchio et al., 2000), as with

many malignancies, the carcinogenesis of HCC is a multi-step process

involving a number of different genetic alterations that ultimately lead to

malignant transformation of the hepatocyte (Laurent-Puig et al., 2001).

The role of HBV in tumor formation appears to be complex and may

involve both direct and indirect mechanisms. Integration of HBV DNA into

the host genome occurs at early steps of clonal tumor expansion, and it has

been shown to induce direct insertion mutagenesis of cancer-related genes

in a number of cases (Chemin and Zoulim, 2009)

The X gene of the viral genome codes for functionally active

transactivator proteins, including the X protein (HBx), which is oncogenic

in transgenic mice (Chan et al., 2008). HBx transactivates several

cytoplasmic signaling pathways, including PKC (protein kinase C), Smad

and Wnt and by binding to nuclear transcription factors, including cAMP

response element binding (CREB) and activating transcription factor 2

(ATF-2) (Feitelson and Lee, 2007; Feitelson et al., 2009).


6

Figure 1 Cellular signaling pathways implicated in hepatitis B virus (HBV) X protein-related hepatocarcinogenesis. Bolded boxes indicate key driving forces for carcinogenesis (Tsai and Chung, 2010).

Prolonged expression of the viral regulatory protein HBx and the

large envelope protein (LHBe) may contribute in deregulating the cellular

transcription program and proliferation control, and sensitize liver cells to

carcinogenic factors (Liu et al., 2008).

Chronic liver inflammation and hepatic regeneration induced by

cellular immune responses may favor the accumulation of genetic

alterations in infected hepatocytes (Liu et al., 2006).

Genetic studies have provided insight into the mechanisms

underlying viral associated hepatocarcinogenesis (Paterlini-Brechot et al.,

2003; Huang et al., 2005; and Park and Chung., 2007). It has been shown

that the rate of chromosomal alterations is significantly increased in HBV-

related tumors compared with tumors associated with other risk factors.

HBV might therefore play a role in enhancing genomic instability

(Murakami et al., 2005). Inactivation of tumor suppressor gene p53 by

mutations and allelic deletions is found more frequently in tumors

associated with HBV infection (Hytiroglou and Theise, 2006).


7

Nishida and Goel, 2011 suggested that viral proteins affect the

function of the p53 protein and contribute to HCC formation. For example,

HBx protein itself reportedly binds to p53 and disturbs its capacity for

DNA binding, transcription and induction of apoptosis. Also, HBV-related

tumors harbor a low rate of oncogene β-catenin mutations (Liu et al.,

2008). Together, these data strongly support the notion that chronic HBV

infection might trigger specific oncogenic pathways, thus playing a role

beyond stimulation of host immune responses and chronic necro-

inflammatory liver disease (Liu et al., 2008).

Figure. 2. A hypothetical model depicting potential genetic and epigenetic changes induced by HBV infection and integration of the viral genome that may promote hepatocarcinogenesis. (1) HBV genome tends to integrate within the host cell-genome, which may result in insertional mutations of critical cellular genes. However no consistent pattern of insertional mutation has been noticed among different HCC patients HCC liver samples (Feitelson, 2006). (2) HBV encoded protein X (HBx) directly interacts with different cellular nuclear proteins and affects cellular processes (such as DNA damage repair) resulting in high mutational rates due to a compromised DNA damage repair capacity and integrity of cellular genome (Lupberger andHildt ,2007) . (3 and 4) It is hypothesized that being the DNA of a foreign pathogen, the HBV genome may be targeted for methylationmediated silencing by host surveillance machinery and the gradual spread of DNA hypermethylation may affect nearby genes or enhancers. Alternatively, activation of the host surveillance mechanism may exhibit long-range effects resulting in hypo-/hyper-methylation of other parts of the host genome. (5–7) HBV genome exists as a minichromosome in the host cell and uses host cellular machinery for its transcription and replication, which is further regulated epigenetically by histone modifications and DNA methylation (Guo et al., 2009). HBX may interact with and affect histone modification enzymes (Zheng et al., 2009) Hijacking/interfering with the host cellular transcriptional/ epigenetic machinery may result in an aberrant epigenetic state of the host cell genome making it susceptible to neoplastic transformation (Herceg and Paliwal, 2011).

methylation framework of cell cycle gene inhibitor … framework of cell cycle gene inhibitor...

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