The Institute for Cancer Research Annual Progress Report: 2009 Formula Grant

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1 The Institute for Cancer Research Annual Progress Report: 2009 Formula Grant Reporting Period July 1, 2012 June 30, 2013 Formula Grant Overview The Institute for Cancer Research (formerly Fox Chase Cancer Center) received $3,477,323 in formula funds for the grant award period January 1, 2010 through December 31, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose Regulation of Genomic and Epigenomic Stability at CpG Sites - The purpose of this project is to understand how two base excision repair enzymes, thymine DNA glycosylase (TDG) and methyl-cpg-binding endonuclease 1(MED1)/ methyl binding domain 4 (MBD4), not only protect CpG sequences from transition mutations and ward off against endogenous deamination events, but also mediate DNA demethylation and modulate DNA methylation states, chromatin structure and gene transcription. This dual role in genomic and epigenomic stability of CpG sites is likely to be important for cancer formation, given the frequent occurrence of CpG to CpA (or TpG) point mutations and hypermethylation of tumor suppressor genes in cancer. Thus, knowledge accumulated in this project may lead to novel strategies for cancer prevention. Anticipated Duration of Project 1/1/ /31/2013 Project Overview The conversion of cytosine into 5-methylcytosine (DNA methylation) is an important epigenetic modification of the mammalian genome that is potentially mutagenic. In fact, 5-methylcytosine has a tendency to spontaneously deaminate, generating thymine, which, if not removed from the G:T mismatch prior to replication, will lead to incorporation of adenine. Indeed, deamination at CpG sites is estimated to cause nearly one-third of all mutations in both cancer and human genetic diseases. Work conducted by us and others indicate that in order to maintain genomic stability at CpG sites, two base excision repair enzymes remove the offending thymine with their DNA N-glycosylase activity, MED1 (previously identified by us and also known as MBD4) and TDG. DNA methyltransferases are known to initiate DNA methylation, whereas it is less clear how the removal of the methylation, or demethylation, takes place. Evidence in Arabidopsis and Zebrafish points to the role of the DNA repair machinery in affecting demethylation, either by The Institute for Cancer Research 2009 Formula Grant Page 1

2 direct removal of 5-methylcytosine, or by its enzymatic conversion by deaminases into thymine, which is then removed from the resulting G:T mismatch. However, it is still largely unknown how mammalian cells demethylate the DNA and what functional consequences demethylation has in mammals. Given the ample remodeling of methylation patterns at CpG sites during development, and the hypermethylation of tumor suppressor genes and other CpG islands in human cancer, it is expected that a defect in demethylating activities may lead to a situation of epigenomic instability and contribute to developmental alterations and tumor formation. The overall goal of this project is to test the hypothesis that the mammalian DNA repair enzymes TDG and MED1/MBD4 have dual functions in protection from tumorigenesis and developmental disorders: 1) Promote genomic stability at CpG sites by suppressing mutations; and 2) Maintain proper DNA methylation patterns by regulating CpG demethylation. Additional funding was secured from NIH during the first year of this grant to support the work outlined below under aims 1 and 2. This project has been expanded to logically continue the research through the addition of aims 3, 4 and 5. Therefore, the focus of the remainder of this project will be to analyze the effect of a modification we have recently identified in MED1, sumoylation, on MED1 DNA damage response and repair capacity; to characterize the structural and functional properties of the AID-GADD45a-TDG demethylating complex, that we have recently described in mammalian cells; and to examine the effects of double inactivation of MED1 and TDG. Thus, to test the general hypothesis outlined above, we now propose five Specific Aims: Aim 1: Characterize the requirement of TDG during development and its role in DNA demethylation and chromatin modification, using knock-out and knock-in mice; Aim 2: Evaluate the role of MED1 and TDG in tumorigenesis using animal models of intestinal cancer and analyzing human cancer specimens. Aim 3: Analysis of MED1 sumoylation in DNA damage response and repair; Aim 4: Characterization of the structure and function of the TDG demethylating complex; Aim 5: Characterization of the phenotype of TDG-MED1-double mutant embryos. Principal Investigators Alfonso Bellacosa, MD, PhD Professor The Institute for Cancer Research 333 Cottman Avenue Philadelphia, PA Other Participating Researchers Helene Leger, PhD - employed by The Institute for Cancer Research Expected Research Outcomes and Benefits By evaluating the contribution of pathways of base excision repair (via MED1/MBD4 and TDG) The Institute for Cancer Research 2009 Formula Grant Page 2

3 to the maintenance of CpG site integrity, we expect to obtain important information of relevance to several related issues. We anticipate that our studies will uncover the mechanisms by which endogenous mutagenesis by deamination is counteracted in mammalian cells, and will have immediate implications for cancer biology. Indeed, failure of these repair systems and acquisition of a mutator phenotype at CpG sites or loss of the epigenetic control afforded by CpG methylation may be critical for cancer formation. In the future, such knowledge might suggest innovative cancer prevention or therapeutic strategies. Summary of Research Completed In the timeframe 7/1/2012-6/30/2013, we made significant progress in all the Specific Aims outlined above. Specific Aim 1. We previously confirmed the essential role of TDG and TDG glycosylase activity in development. Work conducted by us and other groups identifies TDG as a critical effector of DNA demethylation pathways initiated by enzymes of the TET family. While the prominent role of the TET-TDG axis in DNA demethylation is established, the detailed mechanisms are poorly understood. This Aim involves a determination of whether TDG is involved in global demethylation of the paternal genome after fertilization. To this end, in the past year, through appropriate crosses and the employment of our conditional TDG knock-out strain, we have generated male and female mice lacking TDG in the respective gametes. These mice will be used to establish any role of TDG in demethylation of the paternal genome. Specific Aim 2. We had previously identified a specific microrna cluster involved in the downregulation of TDG. In the past year, by using a dual luciferase assay, we mapped the sites on TDG mrna that are targeted by this specific microrna cluster. We have also continued the crosses between the TDG- and MED1-mutant mice with the APC-Min mice and FABP-Cre transgenic mice in order to generate the TDG +/- MED1 +/- FABP-Cre mice that will be interbred with the TDG +/flox MED1 +/- Apc-Min mice, in turn, to generate the experimental mice. Specific Aim 3. In the past year, we studied the functional significance of the novel modification of MED1, i.e. sumoylation, that we previously discovered. We used an in vitro system in which cell lines are transfected with epitope tagged wild type MED1 cdna or a mutant MED1 cdna in which one or more of the sumoylation sites have been mutated, along with T7-tagged SUMO1 cdna. Co-immunoprecipitation experiments conducted under stringent conditions (RIPA lysis buffer) allowed us to determine the extent of sumoylation. By using this assay we made the observation that the sumoylation pattern and extent is differentially changed in response to various DNA damaging agents, such as 5-fluorouracil, cisplatin, gamma-irradiation and N- methyl-n-nitrosourea. Specific Aim 4. We had previously described, in addition to AID and GADD45a, other components of the TDG demethylating complex, including proteins previously known to play an important role in DNA repair. In the past year, we began an examination of the functional role of these TDG partners. Since we previously set up an assay in which demethylation of a Oct4 promoter::egfp reporter takes place in transfected embryonic carcinoma P19 cells and is diminished by TDG knock-down, we established P19 derivatives in which AID and the DNA The Institute for Cancer Research 2009 Formula Grant Page 3

4 repair proteins have been knocked down by specific lentiviral shrna infection. Experiments are in progress to determine whether downregulation of AID and the DNA repair proteins reduces the efficiency of demethylation of the transfected reporter. Specific Aim 5. In the past year, from interbreeding of double heterozygous TDG +/- MED1 +/- mice, we have successfully generated experimental TDG-MED1-double mutant embryos. Preliminary observation of the first litters indicates that TDG-MED1-double mutant embryos have a more severe phenotype than TDG-single mutant embryos. Research Project 2: Project Title and Purpose Estrogen Receptor-Mediated Gene Silencing in Tumorigenesis - The role of estrogen in contributing to women s cancers, especially those of the breast and uterus, is well established at the epidemiologic level. The molecular mechanism through which inappropriate timing and/or excessive levels of estrogen exposure initiate or promote human tumorigenesis remains largely unknown, however. The purpose of this study is to test the hypothesis that the activated estrogen receptor, which functions as a transcription factor, is also capable of silencing genes, through promoter hypermethylation, that normally function to suppress tumorigenesis in human cells subject to regulation by estrogen. In addition, we will explore the molecular mechanism of this methylation-dependent gene silencing. Duration of Project 1/1/ /31/2012 Project Overview The broad objective of this project is to test the hypothesis that the activated estrogen receptor silences the expression of critical tumor suppressor genes, and further, to test whether this methylation-mediated gene silencing involves the recruitment of DNA methyl-transferases (DNMTs) and/or histone deacetylase (HDAC) proteins to the promoter regions of target genes. A substantial body of preliminary data has been generated to support these hypotheses. These objectives will be tested through five specific aims. Aim one will be to confirm that treatment of estrogen receptor (ER)-positive human breast cancer cells with estrogen results in the downregulation of expression of the genes encoding lipocalin 2 (LCN2) and corticotropin releasing hormone (CRH). Aim two will be to determine whether this down-regulation of expression is associated with hypermethylation of the promoter regions of these two genes, to map the specific methylation sites, and to determine the time course of methylation in relation to gene silencing. Aim three will be to test whether this phenomenon is associated with the physical association of activated ER with DNMT1 and/or DNMT3B, and the physical association of HDAC1 with this complex in the promoter regions of LCN2 and CRH. Further, we will test whether deacetylation and methylation occur at H3-K9 in conjunction with these biophysical events. In aim four, RNAi approaches will be used to test whether knockdown of DNMTs prevents the ER-activation associated methylation and silencing of LCN2 and CRH. Finally, aim five will be to determine whether exposure of human breast cancer cells to recombinant LCN2 or CRH has a growth inhibitory or apoptotic effect, confirming the physiologic relevance of this phenomenon. These The Institute for Cancer Research 2009 Formula Grant Page 4

5 aims will be accomplished using standard techniques of cell culture, qrt-pcr, western blotting, real-time MS-PCR, bisulfite sequencing, chromatin immunoprecipitation (ChIP)-PCR, stable transfection of shrna expression vectors, and cell proliferation and apoptosis assays. Principal Investigator Jeff Boyd, PhD Senior Vice President, Molecular Medicine Executive Director, Institute for Personalized Medicine The Institute for Cancer Research 333 Cottman Ave Philadelphia, PA Other Participating Researchers Eric Ariazi, PhD - employed by The Institute for Cancer Research Expected Research Outcomes and Benefits Successful completion of the aims of this research project will provide additional insight into the mechanism through which estrogen contributes to human tumorigenesis, e.g., breast and endometrial cancers. Such an understanding will allow for a more informed approach to the use of this hormone pharmacologically, in terms of risk-benefit analysis. Furthermore, a fundamental understanding of the molecular mechanism through which estrogen contributes to the development or progression of women s cancers is essential in order to develop more effective preventive strategies for these cancers, in light of the long-term exposure of females to this hormone over a reproductive lifetime. Finally, proof of the hypotheses proposed in this project will lead to a new model for the role of hormone receptor transcription factors in human carcinogenesis, which would provide greater insight into the process of tumorigenesis in a wide range of cancers not limited to those associated with estrogen exposure. Summary of Research Completed We tested the hypothesis that ERα can silence gene expression via DNA methylation. We took the approach that genes repressed in the presence of ERα may be silenced by DNA methlyation, and therefore loss of ERα expression would allow these silenced genes to re-express through decreased methylation. To identify genes silenced in the presence of ERα and de-repressed upon loss of ERα, or ERα inversely associated genes, we used gene expression microarrays to compare several antihormone-resistant ERα-negative/low T47D-based cell lines (estrogen deprivation-resistant T47D/ED2 andt47d/ed3 cells, and fulvestrant-resistant T47D/FUL cells) to wild-type ERα-positive T47D/A18 cells. The nearly entire loss of ERα at the RNA and protein level due to long-term estrogen deprivation or fulvestrant treatment in these antihormoneresistant T47D-based cells is shown in Fig. 1. To further select for genes inversely regulated by ERα, we compared genes down-regulated in T47D/ED2 cells treated with 1 nm 17β-estradion (E2) and termed T47D/ED2/E2 cells, compared to T47D/ED2 cells not treated with E2. Genes whose expression inversely associated with ERα expression across all models were then cross- The Institute for Cancer Research 2009 Formula Grant Page 5

6 referenced with those de-repressed by the demethylating agent decitabine (5-aza- 2 deoxycytidine) in parental wild-type T47D/A18 cells (Fig. 2). To improve identification of ERα inversely associated genes, we have extended the exposure of T47D/ED2/E2 cells to E2 from 24 weeks as in the prior report to 38 weeks. We did so because the parental T47D/ED2 cells contained only 5% the ERα levels as in wild-type T47D/A18 cells, and therefore a longer E2 exposure period may be necessary to allow ERα to repress by methylation additional target genes. This allowed us to find additional genes significantly repressed by E2 at 38 weeks exposure that were not significantly repressed at 24 weeks. Thus, we identified more candidate ERα targeted genes for methylation that were not previously identified, such as TM4SF1, FBLN1, S100A2, S100A9, MAGEA10 and PLAU (Fig. 2) To extend our studies to more than one cell type, we incorporated fulvestrant-resistant MCF7/FUL cells into the gene expression microarray analyses. Where the T47D-based models lost nearly all ERα expression, the MCF-7/FUL cells ultimately retained ERα but only after initially losing most ERα at 8 weeks of derivation and then re-gaining ERα expression by 21 weeks of derivation (Fig. 1). Thus our final analysis to identify ERα inversely associated genes that are also regulated by DNA methylation involved finding the intersection of 1) genes upregulated in 3 independent antihormone-resistant ERα-negative/low T47D-based cells compared to wild-type ERα-positive T47D/A18 cells; 2) genes down-regulated by E2 in T47D/ED2/E2 cells vs. parental T47D/ED2 cells; 3) genes up-regulated in ERα-low MCF7/FUL week 8 vs. wild-type ERα-positive MCF7/WS8 cells; 4) genes down-regulated in ERα-positive MCF7/FUL week 21 cells vs. ERα-low MCF7/FUL week 8 cells; and 5) genes up-regulated by decitabine in wild-type ERα-positive T47D/A18 cells. These analyses refined the list of candidate ERα-targets for methylation to 34 genes in more than one cell type. To validate that our gene expression analyses identified ERα-targeted genes for methylation, we examined TRIM29's methylation status by pyrosequencing of bisulfite DNA. A review of the literature indicated TRIM29 decreases E2-stimulated growth by reducing ERE-dependent transcriptional activity in MCF-7 cells, and high TRIM29 associates with increased relapse-free survival in early-stage (I or II) ER-positive breast cancer patients younger than 55 (premenopausal) but not in patients older than 55 (post-menopausal). Using pyrosequencing of bisulfite-treated DNA, methylation of CpG sites spanning +26 to +61 relative to TRIM29 s transcriptional start site (TSS) were decreased in ERα-negative/low T47D/FUL, T47D/ED3 and T47D/ED2 cells relative to wild-type ERα-positive T47D/A18 cells. Also, these CpG sites showed increased methylation in T47D/ED2/E2 cells versus T47D/ED2 cells (Fig. 3). We previously found similar results for LCN2 and hence TRIM29 was the second gene to be validated. CpG methylation of LCN2 and TRIM29 was also examined in MCF7 cell type. Both LCN2 and TRIM CpG methylation was increased in ERα-positive MCF-7/FUL cells at 21 weeks derivation relative to wild-type ERα-positive MCF-7/WS8 cells (Fig. 3). This likely occurred because MCF-7/FUL increased the rate of ERα synthesis or decreased the rate of ubiquitin-mediated ERα degradation to compensate for fulvestrant-mediated ERα degradation (Fig. 1). Hence, methylation of LCN2 and TRIM29 reflected increased ERα turnover in the MCF7/FUL compared to wild-type MCF7/WS8 cells. The Institute for Cancer Research 2009 Formula Grant Page 6

7 Taken together, our data indicate that ERα targets at least LCN2 and TRIM29 for DNA methylation mediated silencing. Figure 1. ERα expression in estrogen deprivation (ED) -resistant and fulvestrant (FUL)-resistant T47D and MCF-7 cells. (A) ERα mrna expression measured by qrt-pcr. (B) ERα protein expression measured by Western blotting. Also shown are the 46 and 36 kda ERα splice variant isoforms. Western blots were visualized using an Odyssey Infrared Imaging System (Licor). The Institute for Cancer Research 2009 Formula Grant Page 7

8 Figure 2. Gene expression microarray identification of genes inversely associated with ERα and induced by decitabine (DAC). Candidate ERα-targeted genes for methylation were induced by DAC in wild-type T47D/A18 cells and inversely associated with ERα expression across all antihormone-resistant models. Global transcriptional profiling was carried out using Agilent 4x44K v2 microarrays and one color-labeled (cyanine 3-CTP) c-rna. Differentially expressed genes were determined by pairwise comparisons using SAM (significance analysis of microarrays) at a false discovery rate (FDR) of 0%. The Institute for Cancer Research 2009 Formula Grant Page 8

9 Figure 3. Validation of TRIM29 and LCN2 methylation status by pyrosequencing. (A) Decreased TRIM29 CpG methylation in ERα-negative/low T47D/FUL, ED3, and ED2 cells vs. ERα-positive T47D/A18 cells. (B) IncreasedTRIM29 CpG methylation upon 1 nm E2 reexposure in T47D/ED2/E2 cells compared to parental T47D/ED2 cells. (C) Increased TRIM29 and (D) increased LCN2 CpG methylation in MCF-7/FUL cells vs. MCF-7/WS8 cells. Note that MCF-7/FUL cells show increased ERα turnover to compensate for FUL-mediated ERα degradation as in Fig. 1. CpG site positions relative to the gene s transcriptional start site (TSS) are indicated. The Institute for Cancer Research 2009 Formula Grant Page 9

10 Research Project 3: Project Title and Purpose Markers of Adult Epithelial Stem Cells - The purpose of this project is to identify novel stem cell markers in adult epithelial tissues. Currently, no markers are available that consistently and robustly enable the in vivo identification and purification of adult stem cells from normal tissues or tumors. Moreover, the difficulty associated with in vivo identification of stem cells has hampered attempts to conduct functional analyses of genes thought to regulate stem cell function or contribute to the formation of tumor cells with self-renewing properties. We aim to determine the molecular identity and function of a new epithelial stem cell marker in the fly and assess its functional conservation in mammalian epithelial tissues. The information provided in this study will be a foundation for the development of agents that promote or target stem cell function for use in replacement therapies and anti-cancer treatment. Anticipated Duration of Project 1/1/ /31/2013 Project Overview Substantial progress has been made in the identification of markers used to isolate and identify stem cells from adult tissues. In some cases, markers are relatively stem-cell specific (e.g. Oct4). In other cases, broadly expressed proteins exhibit differential levels of expression in stem cells vs. non-stem cell daughters. Despite these advancements, purification of adult tissue stem cells, identification of stem cells in situ, and the functional relevance of stem cell markers currently used in isolation protocols remain challenges for this field. Our goal is to identify molecules that control stem cell identity and maintenance. Since identifying novel signals that regulate stem cell function is technically challenging in mammalian tissues, we are focusing on Drosophila ovarian Follicular epithelium Stem Cells (FSCs) as a model system for this analysis. In this system, epithelial stem cell formation can be visualized directly in vivo. Moreover, genetic mutational analysis allows us to pinpoint functional roles for specific genes in the cellular events required for stem cell commitment and self-renewal. The striking conservation of stem cell control signals in mammals and flies suggests that novel stem cell regulatory mechanisms identified in the fly system will generally be applicable for understanding epithelial stem cell regulation in mammalian tissues. To identify novel stem cell regulators, we have initiated a screen for antibodies that specifically recognize FSCs in situ. Using this approach, we identified one antibody that recognizes a nuclear protein that is highly upregulated in FSCs and their differentiated progeny cells. We propose that this follicle cell-nuclear antigen (FC-NA) is a novel epithelial stem cell marker that is important for promoting FSC commitment and function. To test this hypothesis, we will 1) determine the molecular identity of FC-NA using biochemical approaches and molecular genetics, 2) define how FC-NA regulates FSCs in vivo through mutational analysis and imaging, and 3) define the protein expression patterns of FC-NA in mammalian epithelial tissues using immunostaining techniques. This project has the potential to uncover novel stem cell markers The Institute for Cancer Research 2009 Formula Grant Page 10

11 with broad application in stem cell purification and identification as well as in functional analysis of normal and tumor stem cells. Principal Investigator Alana M. O Reilly, PhD Assistant Professor The Institute for Cancer Research 333 Cottman Avenue Philadelphia PA Other Participating Researchers None Expected Research Outcomes and Benefits The promise of stem cell research is two-fold. On the one hand, stem cell replacement therapies may provide cures for a diverse group of disorders, including cancer, diabetes, and traumatic injury. On the other hand, the identification of cancer cells with self-renewing properties that promote tumor growth and evade conventional anti-cancer therapies may lead to the development of potent new approaches to cancer treatment. Substantial progress has been made in the development of in vitro technologies that allow stem cells to be grown in culture and stimulated with factors that promote their differentiation into specific cell types. Far less progress has been made in identifying adult stem cells and their mechanisms of regulation in functioning adult tissues. In this project, we aim to identify new markers of adult epithelial stem cells. Our preliminary results indicate that an unidentified nuclear protein is highly upregulated in epithelial stem cells in the Drosophila ovary, suggesting that this protein may be an important new stem cell marker. If the results of the experiments proposed support our hypothesis that this nuclear antigen is a novel regulator of epithelial stem cell function in flies and in mammals, a new tool for the identification, purification, and in vivo analysis of stem cells will be available. In addition, this study has clear implications for understanding the role of specific genes in promoting stem cell self-renewal and, importantly in the genesis of epithelial stem cells from uncommitted precursor cells. Conservation of the normal developmental mechanisms described in this study may have an important impact on the ability to recognize newly forming cancer cells with stem cell properties. Such a discovery would open new therapeutic avenues for treating cancer in its earliest stages. Finally, this tool may advance our understanding of the mechanisms regulating stem cells in their normal environments, information that is critical for the success of stem cell replacement therapies. The Institute for Cancer Research 2009 Formula Grant Page 11

12 Summary of Research Completed Epithelial stem cell proliferation is controlled by the environment We recently published work in the Journal of Cell Biology describing the role of dietary cholesterol in stimulating stem cell proliferation. Surprisingly, the initiation of stem cell proliferation after a period of nutrient restriction occurred independently of insulin signaling, which is known to promote stem cell proliferation in other populations. Instead, cholesterol binding to its receptor in the Hh producing cells of the fly ovary triggers S6-kinase-mediated phosphorylation of the Boi and Hh release. Hh is then delivered to the stem cell niche where it accumulates quite specifically within the stem cells to drive proliferation. This mechanism is novel for several reasons. First, it is the first description of a nutrient-responsive signaling pathway that directly controls stem cell proliferation rather than triggering production of insulin as an initial step in the signaling. Secondly, the mechanism is novel since nutrient-regulated growth factor sequestration and release have not been previously described. Finally, the identification of the Hh binding protein Boi as a rheostat to tailor stem cell divisions to the levels of available nutrients is a new discovery with important implications for stem cell regulation in other systems. The next question we have begun to address is the mechanism of S6 kinase activation via the cholesterol receptor. The cholesterol receptor is a steroid hormone receptor that is known to control transcription of cholesterol scavenging proteins. However, the response to feeding is too rapid for the mechanism to occur through transcriptional regulation. We have identified a second steroid hormone receptor that is expressed within the Hh producing cells and is required for Hh release, suggesting that the two receptors might act together to control S6 kinase activation. We also generated new data on how Boi phosphorylation alters the conformation of the Boi protein to trigger Hh release. Finally, we identified a new component of this signaling pathway that is essential for controlling stem cell proliferation and Hh release in a genetic screen. The analysis of the detailed mechanisms of interactions between these proteins under different environmental conditions is currently underway. An interesting puzzle that arose from this project is based on the observation that increasing stem cell proliferation is associated closely with rapid stem cell loss. To better understand this phenomenon, we designed a screen for genes that are expressed within follicle stem cells and are important for determining stem cell lifetime. We isolated a group of 6 proteins that function together within the same complex inside cells to maintain stem cell lifetime. The mechanism of cooperation between these 6 genes and the Hh signaling pathway is currently being pursued. Mechanistic conservation of Hh sequestration and release in human cancer The idea that growth factors can be sequestered and released in response to specific cues is exciting in terms of identifying a novel, general mechanism of growth factor regulation. Importantly, many epithelial cancers depend on Hh signaling to maintain stem-like cells within the tumors or to influence the surrounding environment to protect the tumors from drug treatment. Currently, we are focusing on the role of Hh sequestration and release in pancreatic cancers using several different models. First, the expression patterns of components of this The Institute for Cancer Research 2009 Formula Grant Page 12

13 signaling pathway and the levels of Hh release are being measured in human adenocarcinoma cell lines. In the past year, we have established conditions for these assays and are now prepared to test the effects of altering cholesterol levels on the function of the pathway. In addition, we are collaborating with Fox Chase Cancer Center colleagues to assess the role of this pathway in the development of pancreatic lesions in a mouse model of pancreatic adenocarcinoma. Novel stem cell markers: At the beginning of this project, we initiated a genetic screen for genes that are expressed in follicle stem cells. During the past year, we have continued this screen and now have identified 3 novel genes that are expressed in follicle stem cells themselves. In addition to stem cellspecific markers, the screen has also uncovered 8 genes that are expressed specifically in cells of the stem cell niche and in the growth factor producing cells that control stem cell function from a distance. Importantly, insertion of a transcriptional activator into the promoters of these genes allows us to manipulate gene expression within follicle stem cells without affecting most other cells in the fly. Research Project 4: Project Title and Purpose Role of a Novel Zinc Finger Factor in Hematopoietic Development - The purpose of this project is to understand the role of a novel erythroid factor (G4) in hematopoietic development using the zebrafish model system. Since the molecular basis for G4 function in controlling erythropoiesis is completely unknown, we will determine where g4 fits in the hierarchy of transcription factors controlling hematopoietic development, identify its human ortholog, and perform structure/function analysis to identify the domains of g4 that are essential for its function. Results from these studies will provide insights into how normal blood cell development is controlled and how those processes are perturbed in cancer. Duration of Project 1/1/ /31/2011 Summary of Research Completed This project ended during a prior state fiscal year. For additional information, please refer to the Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's Tobacco Settlement/Act 77 web page at Research Project 5: Project Title and Purpose Reducing the Burden of Hepatocellular Carcinoma: Identification of New Targets against Hepatitis B virus (HBV) for Drug Development A reduction in the burden of liver cancer depends on the development of effective therapies that prevent or arrest the growth of hepatocellular carcinomas. The purpose of the project is i) to develop rapid screening assays for primary cultures derived from hepatocellular carcinoma for drugs and drug combinations that induce cell death and to identify serine and tyrosine kinases that are required for the survival of The Institute for Cancer Research 2009 Formula Grant Page 13

14 tumor cells in the presence and absence of already available anticancer drugs and ii) to identify novel targets for antiviral therapy against hepatitis B virus. Duration of Project 1/1/2010 6/30/2012 Summary of Research Completed This project ended during a prior state fiscal year. For additional information, please refer to the Commonwealth Universal Research Enhancement C.U.R.E. Annual Reports on the Department's Tobacco Settlement/Act 77 web page at The Institute for Cancer Research 2009 Formula Grant Page 14