1 Genetic Reprogramming of Cancer Cells: HDACs and the Bad WrapDr. Cassandra Tierney Thank you so much for inviting me to come share my research with you. Its an honor to be back at FU

2 Cancer Statistics In general, cancer affects over 600,000 people in the US each year. These charts show the top 10 cancer sites that contribute to deaths - as you can see CRC is high on the list for both CT and nation-wide Therefore it is very important to understand what contributes to the formation and progression of this cancer – specifically what is happening on a molecular level which is what my research is focused on. BUT before we get into that let’s get a little background on CC.

3 Quick Anatomy Lesson Colon, aka Large Intestine is about 5 ft in length (SI = 22-25ft) Functions to extract H20 and salt from solid waste before eliminated Location of your flora-aided fermentation of unabsorbed materials (probiotics) Cancers are more frequently found further along as waste spends more time there

4 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) FAP HNPCC A syndrome is a group of symptoms. The 2 most common inherited syndromes linked with colorectal cancers are familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). Familial adenomatous polyposis (FAP): FAP is caused by changes (mutations) a gene that a person inherits from his or her parents. About 1% of all colorectal cancers are due to FAP. People with FAP typically get hundreds or thousands of polyps in their colon and rectum, most often in their teens or as early adults. Cancer often starts in one or more of these polyps as early as age 20. By age 40, almost all people with this disorder will have cancer if surgery to remove the colon is not done. Hereditary non-polyposis colon cancer (HNPCC): HNPCC (also known as Lynch syndrome) accounts for about 3% to 5% of all colorectal cancers. It can be caused by inherited changes in a number of different genes that normally help repair DNA damage. The cancers that occur as a part of this syndrome also happen when people are fairly young. People with HNPCC may also have polyps, but they only have a few, not hundreds as in FAP. The lifetime risk of colorectal cancer in people with this condition may be as high as 80%. Women with this condition also have a very high risk of getting cancer of lining of the uterus. Other cancers are also linked with HNPCC.

5 Colon Cancer Risk FactorsAge (being over 50) Smoking Being overweight Having colorectal cancer previously Heavy use of alcohol Having a history of adenomatous polyps Having inflammatory bowel disease (such as Crohn's disease and ulcerative colitis) Family history of colorectal cancer Eating a high fat diet Having diabetes

6 Treatment Options Surgery Radiation Chemotherapy5-Fluorouracil (5-FU) and leucovorin Capecitabine (Xeloda®) Irinotecan (Camptosar®) Oxaliplatin (Eloxatin®) Combinations of the above

7 Progression of Colon CancerThis figure illustrates the various stages of CC progression Normal: Crypts, smooth surface facing lumen ACF: First visible changes, crypts are larger and contain more cells than a normal crypt Adenoma: As the cancer accumulates more changes, it can develop into an adenoma or a pre-malignant growth, polyp Finally it can become a carcinoma that can later metastasize to other parts of the body like the liver as shown here What are the underlying mechanisms that contribute to this progression? Figure The Biology of Cancer (© Garland Science 2007)

8 Cancer has genetic changesThis model depicts some of the most common genetic changes that occur in CC progression. Not surprisingly, most of the genes involved play roles in regulating cell cycle and or proliferation. In reality though, the development of CC is a but more complicated: for instance there is evidence that cancers with mutated Kras oncogene typically don’t have a mutated p53 and vice versa. SO, its more likely that the dev of cancer looks more like this, where we still see in about 90% of cancers the mutation of the APC gene, after this first initiating step any number of different genetic changes could occur leading to uncontrolled proliferation. As I mentioned, the APC gene is a critical first step in the dev of most CC and it is part of the Wnt signaling pathway that functions to regulate the expression of many genes involved in proliferation The Biology of Cancer (© Garland Science 2007)

9 Genetic mutations result in massive changes in gene expression in colon cancerJust an illustration on how massive the genetic changes can be in a patient with CC. This is what happens in order to sustain the cancer – the cancer cells have to bypass the normal controls that cells have in order to grow uncontrollably. Subsequent changes in gene expression can be attributed to epigenetic mechanisms. Epigenetics involves heritable changes in gene expression that do not affect DNA sequence.

10 Colon cancer has epigenetic changes: DNA MethylationActive gene transcription NORMAL Repetitive DNA elements/ Pericentromeric heterochromatin = unmethylated = methylated CANCER Repressed gene transcription: p16, p14, hMlh1, APC Hypomethylation of repetitive elements One epigenetic mechanism is DNA methylation. Methylation occurs at the cytosines of CpG dinucleotides and is caused by DNMT enzymes. Normal cell: PROMOTERS of actively transcribed genes are Unmethylated. Repetitive elements are heavily methylated. In cancer: Promoter CpG islands become hypermethylated, and gene expression of important tumor suppressors is repressed (age related) p16: when repressed, disruption of cell cycle P14: negatively affects p53 function hMlh1: DNA repair enzyme that cannot function when repressed - leads to increases in DNA damage APC: could contribute to activation of Wnt signaling In general, the genome becomes hypomethylated (maybe to active silent retroviral activation) - contributes to microsatellite instability

11 Colon cancer has epigenetic changes: Histone ModificationTumor suppressor gene transcription NORMAL = Acetylation = H3K4Me = unmethylated DNA = Repressive complex CANCER = H3K9Me = MBD = methylated DNA Tumor suppressor gene repression In addition to DNA methylation, histone modifications also effect gene expression. In the nucleus, DNA is wound around histone proteins and packaged into chromatin. The interaction between DNA and histones is affected by various histone modifications, including acetylation and methylation. These changes affect how tightly the DNA is wound, leading to either an ‘open’ or ‘closed’ transcriptional state. Here, we’re looking at how histone modifications can affect the expression of a tumor suppressor gene. In a normal cell, the pattern of histone modifications includes acetylation and certain methylation marks, leading to an open chromatin state and active transcription of the tumor suppressor gene. In contrast, in a cancer cell, the pattern of histone modifications becomes altered. Histones are deacetylated and the pattern of methylation changes, leading to a ‘closed’ chromatin state where DNA is more tightly wound and transcription is repressed. The presence/absence of Ac is one of the key events in chromtin remodeling. Annu.Rev.Med :

12 Frequently up-regulated in cancersHistone Deacetylase Enzymes (HDACs) HDAC3 Frequently up-regulated in cancers One enzyme responsible for altering the state of Acetylation are the HDACs HDACs are grouped into classes based on seq homology/domain structure. The reason we are interested in them is because class I HDACs are freq upregulated in cancers. HDAC8 structure complexed with TSA in active site. (class I) Class I: nuclear Class II: Nuclear/cytoplasm Class III: NAD dependent Nature Reviews Drug Discovery 5, (2006)

13 What functional role might result from HDAC over-expression?HDAC over-expression in neoplasms: transcriptional regulators that mediate gene expression changes? HDAC1 Prostate Colon Ovary Halkidou et al., The Prostate 59: (2004); J Gynecol Oncol Vol. 19, No. 3: , 2008; Zhu et al., Cancer Cell. 5: (2004). Non-malignant pre-malignant What functional role might result from HDAC over-expression? Brown is the HDAC1 staining, human prostate samples, A = non-malignant ; B = pre-malignant Red: Human colon tumor samples and matched normal. Red = HDAC2 Ovary tissue comparing normal to three types of ovarian cancer. HDAC expression varies depending on the cancer type, but a general up- regulation is seen Why is this advantageous for a cancer cell?

14 Colon cancer prognosis is poor when HDACs are over-expressedWell, we know that when CC samples are all positive to HDAC o/e, prognosis for survival is worse vs. negative samples. So, how do we figure out how HDACs are acting in CC? What role do they play for the cancer cell? Weichert et al, Clin Cancer Res 2008;14(6)

15 siRNA HDAC Knockdown in colon cancer cells to determine the relative contribution of class I HDACsIf we can decrease HDACs, can we revert back to normal cell cycle control? To do this, we used an siRNA knockdown technique in the HCT116 cell line WB confirming the knockdown of the protein.

16 Important growth regulatory genes are repressed by HDAC3 in colon cancer cellsCell Cycle Inhibitor Differentiation Gene Regulator Since we know HDACs can affect gene expression, we examined the mRNA levels of several genes involved in cell proliferation and differentiation. P21= cell cycle inhibitor, VDR = hormone receptor that functions to induce differentiation, or apoptosis, TLE1 = negative regulator of Wnt pathway As you can see the knockdown of an individual HDAC was sufficient to induce the expression of some of these genes. One interesting result here was that the most robust activation for all these genes took place when you knocked out HDAC3. So we were interested in exploring this idea further, and decided to look at the gene expression changes that occur on a global level through use of a microarray. Growth Signal Repressor

17 Micoarray Data Analysis: HDAC3 has the largest effect on gene regulation in colon cancer cellsIllumina bead array, covering about transcripts Quality control, where we compare all the expression data of some sample to that of another: if they are replicates of each other you’d expect a very high correlation as seen here. When we compared control samples to the HDAC knockdown samples, we still see a fairly high correlation which basically says we are not getting a lot of changes when we knock down any one HDAC. BUT what was interesting again, was that when we looked at HDAC3, it had the most changes in comparison to all the other HDAC knockdowns. Taking this analysis a step further, we chose to do pathway analysis using IPA software

18 Ingenuity Pathway Analysis of Microarray Data Following HDAC3 knockdownAll–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. Did this for all the knockdown data, but since our earlier experiments pointed to a prominent role for HDAC3 –we were particularly interested in what pathways this enzyme was affecting. Gene List Fold change ± 1.5-fold Interactive Pathway Models

19 Ingenuity Pathway Analysis Results: siHD3 G1-S Phase Checkpoint Control p CDC42EP Cyclin A SKP MDM CDC25C And it turns out, one of the affected pathways in HDAC3 knockdown cells is the G1-S checkpoint: Obviously a major player in cell cycle control So this is very interesting to us and we wanted to examine the role HDAC3 plays in regulating proliferation and differentiation. Skp2 = one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which function in phosphorylation-dependent ubiquitination. cdc25C = It directs dephosphorylation of cyclin B-bound CDC2 and triggers entry into mitosis. CDC42EP3 = They bind to, and negatively regulate the function of, CDC42.

20 Summary I Knockdown of individual HDACs in colon cancer cells affects gene expression Individual enzymes show a degree of specificity to target genes Identified role for HDAC3 in regulation of genes involved in cell cycle control To what degree is HDAC3 up-regulated in colon cancer? How does up-regulation of HDAC3 affect gene expression?

21 HDAC3 expression is variable in human colorectal adenocarcinomas and cell culture models : : Low Intermediate High First, we needed to examine the expression levels of this enzyme. The variability also translates into different cell culture models of CC. We compared HDAC expression for three different CC and one lung cancer model that is know to overexpress HDAC3 about 90% of the time SW480 – has particularly high levels of HDAC3 Moreover, these cells are unable to express p21 in response to butyrate, unlike other CC cells like the HT29s So in order to study how HDAC3 is affecting these cells, we chose to create a stable knock-down cell line.

22 Short hairpin RNA stable knockdown of HDAC3 in SW480 cells accentuates expression of the p21 cell cycle inhibitor shHD3 0 B T B T B T B T shCNTL HDAC3 HDAC2 Actin p21 Stable knockdown: a plasmid delivery system of shRNA integrates into the genome for a permanent silencing of target. Here I’m showing the confirmation of the HDAC3 KD in three separate clones in comparison to cells that received an empty vector As you can see we were able to KD HDAC3, without a rise in other HDACs, detailed here by HDAC2 levels staying constant. More over, the stable HDAC3 knockdown was able to re-establish the p21 response to butyrate as well as TNF treatment. Global effects? What is the impact of stable HDAC3 knockdown in SW480 cells on global gene expression? 0 = no treatment B = Butyrate T = TNFα

23 Illumina Beadarray AnalysisMultiple Pathways Affected by HDAC3 knockdown Wnt Signaling Cell Differentiation Vitamin D Signaling We’ve already done an array on the HCT116 cells, but now we were interested in looking at a cell line that overexpresses HDAC3 and what occurs in a stable KD line. So, again we performed our quality control analysis. This time we observed a broad spread in gene expression changes with HDAC3 knockdown. And after pathway analysis, we found effects on several oncogenic signaling pathways including TGF beta, and the Wnt pathway. We found some interesting results for the Wnt pathway, where some changes were contributing to inhibition of its signaling, whereas others were pointing to its activation and we were interested to see what was going on with these cells. HDAC3 overexpression prevents cell cycle arrest and differentiation of colon cancer cells.

24 HDAC3 knockdown suppresses nuclear translocation of b-catenin: the master regulator of cell proliferation shCNTL shCNTL + BA shHD3 ICC: -catenin (Red) Nuclei (Blue) HDAC3 High Pan-HDAC Inhibitor HDAC3 knockdown Nuclear Cytosolic Actin shCNTL shHD3 TO do that we decided to use immunofluorescence to assess the localization of B-catenin within the cell. Important to remember that these cells are APC mutant! Meaning they do all of this without changing that aspect of the signaling....sooo how are they doing it? HDAC3 High HDAC3 knockdown

25 HDAC3 knockdown increases expression of TLE1 and TLE4: two b-catenin inhibitorsSW480 – shCNTL, shHD3 Stable knockdown, increases TLEs – repressors of the TCF transcription factor, and decreases B-catenin target gene – c myc Preferential binding of TLE to TCF transcription factor C-myc Cyclin-D TCF/LEF Groucho/TLE -catenin

26 ~ As Early as the 1980s it was suggested that VitD intake/sunlight exposure was inversely related to incidence of CRC. ~ Since then several other studies have shown high intakes of vitamin D and calcium as being inversely related to CRC. ~What I’m showing you here is a recent paper that uses mice as a model organism. Researchers had three groups of mice that were each fed a different diet. One was the standard diet, the second was a diet high in fat, low in fiber and many other nutrients including calcium and vitD, the third was this same high fat low nutrient diet with Calcium and VitD supplemented back in. What they have found is that the mice that this new western diet have very high frequency and incidence of colon tumors. However, when they supplement that diet with cal and vitamin d, the incidence and frequency go down significantly. SO, how does Vitamin D effect gene expression to reduce colon cancer? Yang, K. et al. Cancer Res 2008;68:

27 Vitamin D and Colon CancerLiver: 25(OH)D3 CYP27A1 Skin: Pre-Vitamin D3 Kidney: 1, 25(OH)2D3 CYP27B1 Co-activators D3 RXR VDR Apoptosis, Cell Cycle Arrest, Differentiation, Anti-angiogenesis Let’s briefly go over some background information on how vitamin D and colon cancer are connected. Research has shown that the 1, 25dihydroxyD3 is the active metabolite and can regulate cell proliferation and differentiation This happens when 1, 25 binds to VDR and modulates gene expression. Currently, clinical trials are being done to see how good D3 treatment could be in prevention of CRC. VDRE

28 HDAC3 knockdown restores VDR expressionBoth in stable and transient knockdowns Also able to induce expression E-cadherin

29 And makes cells more sensitive to D3 treatmentWe are also able to induce a VDR target gene, E-cadherin following D3 treatment only in cells with reduced HDAC3

30 BUT, HDAC3 does not affect apoptosis in SW480 cells

31 Summary II HDAC3 expression varies in human colon adenocarcinomas and cell culture models HDAC3 over-expression increases activity of b-catenin to stimulate Wnt signaling and proliferation HDAC3 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 butyrate What is the effect of HDAC3 on histone acetylation?

32 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 To asses the amount of AC in HDA3 high and KD cells: we used specific antibodies to certain Ac marks. Basically one came up different between HD3 high and KD cells: H4K12 ** remember Increased acetylation = increase in gene expression (open chromatin) shCNTL shHD3

33 Acetylation in Normal Mouse IntestineH4K12 Acetylation H4K12Ac overlaps with proliferating cells in normal intestine tissue In normal mouse intesting, H4K12ac is associated with proliferating cells with normal control systems happening. H4K12Ac PCNA merge Co-localization Statistics 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)

34 HDAC3 overexpression in mouse colon tumors results in a reduction in histone H4 K12 acetylationNormal/Tumor IHC: HDAC3 H4K12Ac PCNA Merge T T T Acetylation in tumor tissue that has high HDAC3 is lowered, contributing to closed chromatin, and decreasing expression of important regulatory genes Overlap with proliferating cells continues even in tumor tissue Decreased Ac = Increased HDAC3, closed chromatin and regulating genes turned off. N N N Co-localization Statistics 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)

35 Histone H4-K12 acetylation allows access to differentiation factorsDifferentiation signal Normal K12ac K12ac K12ac K12ac K12ac Tumor Differentiation signal No response to differentiation signals because chromatin in closed up HDAC3 + + + + +

36 Summary III Acetylation at H4K12 is is the primary target of to HDAC3 overexpression High HDAC3 in tumors reduces H4K12Ac Low acetylation levels on histone H4K12 may prevent the activation of genes involved in proliferation control and differentiation

37 Summary & Future ObjectivesHDACs (including HDAC3) are over-expressed in human and APCmin 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 case We see that with our evidence from the tumor array, and cell cultures. Acetylation levels are reduced – specifically H4K12Ac is altered when HDAC3 is high. The microarray points to several different cancer pathways being affected by HDAC3 expression. Looking at transient KD of all class I HDACs, we see that only decreases in HDAC3 are able to restore VDR expression. HDAC3 status of a cancer my help select/rule out a Vitamin D therapy option. Our data also suggest one way to use HDAC inhibitors and Vitamin D treatment in conjunction with each other. This could be through pharmalogocial HDACi for specific HDACs OR through a dietary agent that can decrease HDAC activity like short chain fatty acids like butyrate, or Sulfurafans that come from brocclli or other. In fact a recent study has shown in humans that HDAC activity can be decreased after a broccoli sprout meal – so the potential is there, more research and clinical studies will be needed to see if the two are compatible.

38 Acknowledgements Dr. Charles Giardina Dr. Lawrence HightowerDr. Colleen Spurling Giardina, Lynes, Zwiefach, and Lee labs

39 Questions?

40 H4K12 Acetylation is correlated with S-phaseshHD3 cells histone H4 histone H3 Time after G1 release: hrs H3K9Me3 H4K12Ac HDAC2 PCNA Lovastatin (60μM) x 33hrs Cells accumulate in G1 To further examine this phenomenon, we synchronized HDAC3 knockdown cells using the lovastatin treatment and mevalonic acid release. We extracted protein over time and examined PCNA, HDAC2, and Acetylation As you can see, as the cells enter into S phase, PCNA increases, as well as the acetylation of Histone H4 Mevalonic Acid (releases the G1 block)

41 Histone acetylation as an integral component of cellular regulation in intestinal epitheliumH4K12Ac Open Chromatin Cell Maturation/ Differentiation HDAC3 normal HDAC3 High H4K12Ac Closed Chromatin Transformed state De-Differentiation TAC= intermediate cell type, not pluripotent, but not fully differentiated, programmed for expansion

42 Colon Cell DifferentiationStem Cell Notch Signaling Wnt Signaling Colon Cell Differentiation Secretory Lineage Absorptive Lineage Hes1 Math 1 Gfi1 Ngn3 Elf3 Goblet Cell Enterocyte Enteroendocrine Paneth Cell Modified from Clevers H Annu Rev Physiol.

43 HDAC3 levels affect differentiationSecretory Lineage Absorptive Lineage Hes1 Math 1 Gfi1 Elf3 Goblet Cell Enterocyte HDAC3 levels affect differentiation shCNTL shHD3 HES1 MATH1 HDAC3 High HDAC3 KD TFF3 MUC2 Actin shHD3 shCNTL p21 MUC2 TFF3 p21 Switching gears a little, we are going to look at a gene that contributes to colon cell differentiation , VDR. I mentioned earlier that this gene controls the expression of other genes involved in differentiation as well as cell cycle and apoptosis HDAC3 High HDAC3 KD

44 VDR controls expression of genes important to Cell cycle arrest and differentiationVDRE Co-activators RXR VDR D3 Apoptosis, Cell Cycle Arrest, Differentiation, Anti-angiogenesis Skin: Pre-Vitamin D3 Liver: 25(OH)D3 CYP27A1 Kidney: 1, 25(OH)2D3 CYP27B1 VDR is low in normal cells, increases as a response to early tumor activities...then at the higher grades, is absent/very low and can contribute to de-differentiation

45 Nuclear versus cytoplasmic VDR expression in normal colonic epithelium, ACF, polyps, colon cancers of defined differentiation, and tumors metastasizing to regional lymph nodes VDR is low in normal cells, increases as a response to early tumor activities...then at the higher grades, is absent/very low and can contribute to de-differentiation Nuclear versus cytoplasmic VDR expression in normal colonic epithelium, ACF, polyps, colon cancers of defined differentiation, and tumors metastasizing to regional lymph nodes. In all instances, immunohistochemistry was performed and chromogen quantified as described in Materials and Methods. Columns, means; bars, ±SE. Inset, nuclear to cytoplasmic ratio of VDR expression as determined by quantitative immunohistochemistry. Abbreviations: NL, normal colonic epithelium; T, tubulovillous; V, villous; TV, tubulovillous; W, well differentiated; M, moderately differentiated; P, poorly differentiated; LN, lymph node. Matusiak D et al. Cancer Epidemiol Biomarkers Prev 2005;14: Well to poorly differentiated Normal ©2005 by American Association for Cancer Research

46 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 treatment

47 shHD3 cells have corrected lineage markers 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 acetylation Take Home Message shHD3 cells have corrected lineage markers They undergo cell cycle arrest Restore some histoneacetylation to allow for open chromatin

48 Epigenetics Heritable changes that occur in gene expression/phenotype that are NOT due to changes in DNA sequence DNA Methylation Histone Modifications Methylation occurs at the cytoside residue in CpGdinucleotides and is caused by the action of DNMT enzymes Histone modifications can include things like acetylation, P, Me, and Ub

49 Histone Proteins: generally transcriptionally repressiveOctamer: H2A, H2B, H3, H4 147bp of DNA High positive charge to bind negatively charged DNA Highly conserved from yeast to humans The longest human chromosome is about 82,000um, but the average eukaryotic nucleus diameter is only 6um IN order to fit all this DNA into the nucleus, eukaryotes compact chromatin using histone proteins so that this same chromosome becomes about 1um in diameter. Histones are made up.. Generally block transciption by compacting DNA..but when the tails are modified, DNA can relax Tails are subject to post-translational modifications, one of which is histone Acetylation

50 HDAC Inhibitors promote cell cycle arrest and cell differentiationCell cycle arrest and differentiation HDAC inhibitor: BA TSA SAHA Cancer cells in culture MCF-7 Breast Cancer Cells Munster, Pamela et al. (2001), Cancer Research 61 +SAHA Well, some of the evidence we have comes from inhibition experiments, that use pan-HDAC inhibitors and study the effects of cancer cells. What has been determined is that inhibition causes cancer cells to undergo cell cycle arrest and differentiation. Here I’m just showing one example of that in breast cancer cells that have been treated with SAHA and stained for milk proteins Both stained for same things. MFG (milk fat globule protein = green). MFMG (milk fat membrane globule protein = red) both are only present in differentiated cells normally. BA= butyrate TSA = Trichostatin Acid SAHA=suberoylanilide hydroxamic acid

51 What are the roles of individual HDACs in mediating growth HDAC inhibition causes cell cycle arrest, differentiation and apoptosis Untreated HCT116 Colon Cancer cells injected into nude mice HDAC inhibitor given by IV 5x/week X 3 weeks Increasing concentrations of HDAC inhibitor Here is another example using HCT116 cells that have been injected into nude mice, then fed an HDACi as indicated here by arrows, and what we see is a general reduction in tumor load compared to untreated mice. So far, I’ve only talked about non-specific inhibitors that will decrease the activity of all HDACs or a single class of HDACs, but there is some evidence that individual HDAC enzymes have specificity in the genes they regulate – so I wanted to know what would happen in colon cancer cells when a single HDAC was inhibited. treatments started when HCT116 tumors reached a mean size of 50 mm3 and nude mice were injected 5 times per week for 3 weeks, for a total of 15 doses. Drugs given by IV Annual Review of Pharmacology and Toxicology. Vol. 45: , 2005 What are the roles of individual HDACs in mediating growth and differentiation in cancer cells?

52 First hit: Wnt signaling pathwayFrizzled Groucho/TLE HDAC TCF/LEF C-myc Cyclin-D LRP5/6 -catenin P TrcP Ub Cadherin -catenin cytoskeleton Axin APC GSK-3B CK1 Wnt GSK-3 Disheveled In the wnt signaling pathway, an extracellular wntligand stimulates transcription of genes involved in cell proliferation/differentiation through the action of the TF, b-catenin. In absence of Wntligand, b-catenin is present in the cell and are involved in cell-to-cell adhesion complexes on the PM. The amount of bcatenin in the cell is tighly regulated by destruction complexes that target bcatenin for proteasomal degradation. This includes the APC and axin proteins. Genes that are activated by bcatenin are not transcribed and repressed by a complex that includes groucho/tle and HDACs. In contrast, when Wntligand is present - Axin is recruited to Wnt receptors at the membrane and the degradation complex does not form. bcatenin accumulates in the cytoplasm and translocates into the nucleus and activates wnt-responsive genes. The reason why this pathway is so impt in cancer devt is because many of these Wnt-responsive genes are involved in the promotion of cell proliferation. As I mentioned before, mutations in the wnt pathway are just the first in a series of gene expression changes that results in cc. Initial mutations, like those in the APC gene, are genetic whereas subsequent changes in gene expression can be attributed to epigenetic mechanisms. Epigenetics involves heritable changes in gene expression that do affect DNA sequence. APC = critical regulator of Wnt signaling and is the first genetic change that occurs in CC. C-myc = oncogene Cyclin D = promotes progression through cell cycle

53 Effects on Wnt pathway components – transient knockdownHT29 – siHD3±BA HCT116 – siHD3±BA shCNTL – siHD3, ± BA How does HDAC3 affect the cell cycle and cell growth? BUT when you do a transient knockdown of HDAC3, more complicated – we do see an increase in the repressors, but also in c myc Some researchers have shown that HDACi can over-stimulate the Wnt pathway and induce cell death! This may be going in here – short term, HDAC siRNA can promote Wnt signaling and only cells with a robust inhibitory response say of TLEs can survive this overstimulation.

54 Influence of HDAC3 on cell cycle and growthHDAC3 KD HDAC3 High G1 arrest in cells with low HDAC3 Likewise, high HDAC3 can make cells resistant to growth inhibition through the use of BA HDAC3 High HDAC3 KD