Genetic Reprogramming of Cancer Cells: HDACs and the Bad Wrap - PowerPoint Presentation

Slide1

Genetic Reprogramming of Cancer Cells: HDACs and the Bad Wrap

Dr. Cassandra TierneySlide2

Cancer Statistics

http://www.cdc.govSlide3

Quick Anatomy LessonSlide4

How do you get Colon Cancer?

Nearly all colon cancers begin as noncancerous (benign) polyps, which slowly develop into cancer.

About 25% of patients have a familial component (

genetics)

FAPHNPCCSlide5

Colon Cancer Risk Factors

Age (being over 50)

Having colorectal cancer previously

Having a history of adenomatous polyps

Family history of colorectal cancerEating a high fat dietSmokingBeing overweightHeavy use of alcoholHaving inflammatory bowel disease (such as

Crohn's disease and ulcerative colitis)Having diabetesSlide6

Treatment Options

Surgery

Radiation

Chemotherapy5-Fluorouracil (5-FU) and leucovorin

Capecitabine (Xeloda®)Irinotecan

(Camptosar®)Oxaliplatin (Eloxatin®)

Combinations of the aboveSlide7

Progression of Colon Cancer

Figure 11.6

The Biology of Cancer

(© Garland Science 2007)Slide8

The Biology of Cancer

(© Garland Science 2007)

Cancer has genetic changesSlide9

Genetic mutations result in massive changes in gene expression in colon cancerSlide10

Colon cancer has

epigenetic

changes: DNA Methylation

Active gene transcription

Repetitive DNA elements/

Pericentromeric heterochromatin

NORMAL

CANCER

Repressed gene transcription:

p16, p14, hMlh1, APC

Hypomethylation of repetitive elements

= unmethylated

= methylatedSlide11

Colon cancer has

epigenetic

changes: Histone Modification

Annu.Rev.Med. 2008. 59:491-504

Tumor suppressor gene transcription

NORMAL

= Acetylation

= H3K4Me

= unmethylated DNA

= Repressive complex

CANCER

= H3K9Me

= MBD

= methylated DNA

Tumor suppressor gene

repressionSlide12

Histone

Deacetylase

Enzymes (HDACs)

Nature Reviews Drug Discovery 5, 769-784 (2006)

HDAC3

Frequently up-regulated in cancersSlide13

HDAC over-expression in

neoplasms

: transcriptional regulators that mediate gene expression changes?

HDAC1 Prostate

Colon

Ovary

Halkidou et al., The Prostate 59:177-189 (2004);

J Gynecol Oncol Vol. 19, No. 3:185-190, 2008;

Zhu et al.,

Cancer Cell

. 5: 455-463. (2004).

What functional role might result from HDAC over-expression?

Non-malignant

pre-malignantSlide14

Colon cancer prognosis is poor when HDACs are over-expressed

Weichert et al, Clin Cancer Res 2008;14(6)Slide15

siRNA

HDAC Knockdown in colon cancer cells to determine the relative contribution of class I HDACsSlide16

Cell Cycle Inhibitor

Differentiation Gene Regulator

Growth Signal Repressor

Important growth regulatory genes are repressed by HDAC3 in colon cancer cellsSlide17

Micoarray

Data

Analysis: HDAC3 has the largest effect on gene regulation in colon cancer cellsSlide18

Ingenuity Pathway

Analysis of Microarray Data Following HDAC3 knockdown

All–in-one, web-based application

Analyze, integrate, understand gene expression data from microarray analysis

Ingenuity Knowledge Base: repository of molecular interactions, regulatory events, gene to phenotype associations and chemical knowledge from published literature compiled by scientists

Used as starting point for exploration and a tool to interpret experimental results in the context of larger biological systems.

Gene List

Fold change ± 1.5-fold

Interactive Pathway

ModelsSlide19

Ingenuity Pathway Analysis Results: siHD3

G

1

-S Phase Checkpoint Control

p21 +2.5

CDC42EP2 +1.6

Cyclin A2 -1.5SKP2 -1.8MDM4 -1.7CDC25C -1.6Slide20

Summary I

Knockdown of individual

HDACs

in colon cancer cells affects gene expressionIndividual enzymes show a degree of specificity to target genes

Identified role for HDAC3 in regulation of genes involved in cell cycle controlTo what degree is HDAC3 up-regulated in colon cancer?How does up-regulation of HDAC3 affect gene expression?Slide21

12 : 13 : 12

Low

Intermediate

High

HDAC3 expression is variable in human colorectal

adenocarcinomas

and cell culture modelsSlide22

Short hairpin RNA stable knockdown of HDAC3 in SW480

cells accentuates expression of the p21 cell cycle inhibitor

0 = no treatment

B = Butyrate

T = TNFα

shCNTL

shHD3

0 B T 0 B T 0 B T 0 B T

HDAC3

HDAC2

Actin

p21

What is the impact of stable HDAC3 knockdown in SW480 cells

on global gene expression?Slide23

Illumina

Beadarray

Analysis

Multiple Pathways Affected by HDAC3 knockdown

Wnt

Signaling

Cell Differentiation

Vitamin D Signaling

HDAC3

overexpression

prevents cell cycle arrest and differentiation of colon cancer cells.Slide24

shCNTL

shCNTL + BA

shHD3

ICC:

-catenin (Red)

Nuclei (Blue)

Nuclear

Cytosolic

Actin

shCNTL

shHD3

HDAC3

knockdown suppresses nuclear translocation of b-catenin: the master regulator of cell proliferation

HDAC3 High

Pan-HDAC

Inhibitor

HDAC3 knockdown

HDAC3

High

HDAC3 knockdownSlide25

HDAC3 knockdown increases expression of TLE1 and TLE4: two b-catenin inhibitors

SW480 – shCNTL, shHD3

C-myc

Cyclin-D

TCF/LEF

Groucho

/TLE



-

cateninSlide26

Yang, K. et al. Cancer Res 2008;68:7803-7810Slide27

Vitamin D and Colon Cancer

VDRE

Co-activators

RXR

VDR

D3

Apoptosis, Cell Cycle Arrest,

Differentiation, Anti-angiogenesis

Skin:

Pre-Vitamin D

3

Liver:

25(OH)D

3

CYP27A1

Kidney:

1, 25(OH)

2

D

3

CYP27B1Slide28

HDAC3 knockdown restores VDR expressionSlide29

And makes cells more sensitive to D3 treatmentSlide30

BUT, HDAC3 does not affect apoptosis in SW480 cellsSlide31

Summary II

HDAC3 expression varies in human colon

adenocarcinomas

and cell culture modelsHDAC3 over-expression increases activity of b-

catenin to stimulate Wnt signaling and proliferationHDAC3 turns off VDR expression and makes cells resistant to the growth effects of vitamin D

High HDAC3 does not affect apoptotic response of cells to butyrateWhat is the effect of HDAC3 on histone acetylation?Slide32

Influence of HDAC3 on histone

acetylation

: selective targeting of histone H4 lysine 12

shCntrl

shHD3

- BA - BA

H4K5Ac

Histones

Histone H2B

H4K12Ac

H4K8Ac

H4K16Ac

Histone H4

*

shHD3

shCNTLSlide33

Acetylation in Normal Mouse Intestine

H4K12Ac

PCNA

merge

H4K12 Acetylation

H4K12Ac overlaps with proliferating cells in normal intestine tissue

Pearson's

Coefficient: r=0.918

Overlap Coefficient: r=0.939

Manders

' Coefficients (original):

M1=0.997 (fraction of A overlapping B)

M2=0.994 (fraction of B overlapping A)

Co-localization StatisticsSlide34

Normal/Tumor

IHC: HDAC3

T

N

HDAC3

overexpression

in mouse colon tumors results in a reduction in histone H4 K12

acetylation

H4K12Ac

PCNA

Merge

Pearson's Coefficient: r=0.969

Overlap Coefficient: r=0.976

Manders' Coefficients (original):

M1=0.998 (fraction of A overlapping B)

M2=0.999 (fraction of B overlapping A)

T

T

T

N

N

N

Co-localization StatisticsSlide35

K12ac

K12ac

K12ac

K12ac

K12ac

Histone

H4-K12

acetylation

allows access to differentiation factors

+

+

+

+

+

Differentiation signal

Differentiation signal

HDAC3

Normal

TumorSlide36

Summary III

Acetylation at H4K12 is

is

the primary target of to HDAC3

overexpression

High HDAC3 in tumors reduces H4K12AcLow acetylation levels on histone H4K12 may prevent the activation of genes involved in proliferation control and differentiation Slide37

Summary & Future Objectives

HDACs (including HDAC3) are over-expressed in human and

APC

min

tumors/Acetylation is reduced.

HDAC3 knockdown effects genes involved in cancer

HDAC3 specifically functions in negative regulation of VDR

Inhibition of HDAC3 may sensitize cancers to vitamin D treatment

Pharmacological

Dietary: SCFA (

butyrate)

SFN

(Broccoli

)

Mouse study underway to determine if this is the caseSlide38

Acknowledgements

Dr. Charles

Giardina

Dr. Lawrence HightowerDr. Colleen

SpurlingGiardina, Lynes,

Zwiefach, and Lee labsSlide39

Questions?Slide40

histone H4

histone H3

Time after G1 release:

0 12 14 18 20 22 24 hrs

H3K9Me3

H4K12Ac

HDAC2

PCNA

H4K12 Acetylation is correlated with S-phase

shHD3 cells

Lovastatin (60μM) x 33hrs

Cells accumulate in G

1

Mevalonic Acid

(releases the G

1

block)Slide41

Histone acetylation as an integral component of cellular regulation in intestinal epithelium

H4K12Ac

Open Chromatin

Cell Maturation/

Differentiation

HDAC3 normal

HDAC3 High

H4K12Ac

Closed Chromatin

Transformed state

De-DifferentiationSlide42

Stem Cell

Notch Signaling

Wnt

Signaling

Secretory Lineage

Absorptive Lineage

Hes1

Math 1

Gfi1

Ngn3

Elf3

Goblet Cell

Enterocyte

Enteroendocrine

Paneth Cell

Modified from

Clevers H. 2009 Annu Rev Physiol.

Colon Cell DifferentiationSlide43

Secretory

Lineage

Absorptive Lineage

Hes1

Math

1

Gfi1

Elf3

Goblet Cell

Enterocyte

MUC2

TFF3

shCNTL

shHD3

HES1

MATH1

TFF3

MUC2

Actin

shHD3

shCNTL

p21

p21

HDAC3 levels affect differentiation

HDAC3

High

HDAC3

KD

HDAC3

High

HDAC3

KDSlide44

VDRE

Co-activators

RXR

VDR

D3

Apoptosis, Cell Cycle Arrest,

Differentiation, Anti-angiogenesis

Skin:

Pre-Vitamin D

3

Liver:

25(OH)D

3

CYP27A1

Kidney:

1, 25(OH)

2

D

3

CYP27B1

VDR controls expression of genes important to Cell cycle arrest and differentiationSlide45

Nuclear versus cytoplasmic VDR expression in normal colonic epithelium, ACF, polyps, colon cancers of defined differentiation, and

tumors

metastasizing to regional lymph nodes

Matusiak D et al. Cancer Epidemiol Biomarkers Prev 2005;14:2370-2376

©2005 by American Association for Cancer Research

Normal

Well to poorly differentiatedSlide46

Summary IV

HDAC3 over-expression reprograms colon cancer cells, increasing the expression of some genes while repressing others

High HDAC3 expression in cancer cells can cause lineage infidelity

High HDAC3 expression reduced VDR and rendered cells resistant to 1, 25 dihydroxyvitamin

D3 treatmentSlide47

Normal HDAC

Histone Acetylation

Open Chromatin

Differentiation

Post-mitotic

Normal

HighHDAC3

De-Acetylation

Closed Chromatin

Gene Modulation

Maintain de-differentiated state

Cancer/pre-cancer

HDAC Inhibition During Cancer

High HDAC3

Specific Inhibitor

Re-expression of

lineage-specific markers

Cell Cycle arrest

Restoration of some histone acetylationSlide48

Epigenetics

Heritable changes that occur in gene expression/phenotype that are NOT due to changes in DNA sequence

DNA

Methylation

Histone ModificationsSlide49

Histone Proteins: generally

transcriptionally

repressive

Octamer

: H2A, H2B, H3, H4

147bp of DNA

High positive charge to bind negatively charged DNA

Highly conserved from yeast to humansSlide50

HDAC Inhibitors promote cell cycle arrest and cell differentiation

Cancer cells in culture

Cell cycle arrest and differentiation

HDAC inhibitor:

BA

TSA

SAHA

MCF-7 Breast Cancer Cells

Munster, Pamela et al. (2001),

Cancer Research

61

+SAHA

BA= butyrate

TSA =

Trichostatin

Acid

SAHA=

suberoylanilide

hydroxamic

acidSlide51

HDAC inhibition causes cell cycle arrest, differentiation and apoptosis

HCT116 Colon Cancer cells injected into nude

mice

HDAC inhibitor given by IV 5x/week X 3 weeks

Untreated

Increasing concentrations of HDAC inhibitor

Annual Review of Pharmacology and Toxicology. Vol. 45: 495-528, 2005

What are the roles of individual

HDACs

in mediating growth

and differentiation in cancer cells?Slide52

First hit

:

Wnt

signaling pathway

Frizzled

Groucho/TLE

HDAC

TCF/LEF

C-myc

Cyclin-D

LRP5/6



-catenin

P

P



TrcP

Ub

Ub

Ub



-catenin

Cadherin



-catenin

cytoskeleton

P

P

Axin

APC



-catenin

GSK-3B

CK1

C-myc

Cyclin-D

TCF/LEF



-catenin



-catenin



-catenin



-

catenin

Frizzled

Wnt

GSK-3



CK1

Axin

APC



-catenin

Cadherin



-catenin

LRP5/6

Disheveled

PSlide53

Effects on Wnt pathway components – transient knockdown

HT29 – siHD3±BA

HCT116 – siHD3±BA

shCNTL – siHD3, ± BA

How does HDAC3 affect the cell cycle and cell growth?Slide54

Influence of HDAC3 on cell cycle and growth

HDAC3 High

HDAC3 KD

HDAC3 High

HDAC3 KD