Jonathan R. Pollack - US grants

DESCRIPTION (provided by applicant): Genomic DNA amplifications, which lead to the increased expression of oncogenes, frequently contribute to the development of cancer. Characterizing these DNA copy number changes is important for advancing our understanding of cancer, as well as for its diagnosis and treatment. The objectives of this study are to characterize recurrent gene amplifications in breast cancer, to understand their role in tumor development and progression, and to investigate their utility as prognostic markers and therapeutic targets. The achievement of these goals relies on three recent technical developments: array-based comparative genomic hybridization (array CGH), tissue microarrays, and RNA interference (RNAi). In array CGH, tumor and normal genomic DNA are differentially labeled and co-hybridized to a microarray containing thousands of different genes. Fluorescence ratios at each element on the array provide a high resolution, "gene-by-gene" map of DNA copy number alteration across the cancer genome. Tissue microarrays consist of hundreds of different tissue biopsies from individual tumors sectioned onto a single microscope slide. Fluorescence in situ hybridization (FISH) on tissue microarrays permits the highly parallel detection of a single gene's amplification, and its correlation with clinical outcome data, for each of the specimens on the array. Finally, RNAi is the sequence-specific, post-transcriptional gone silencing initiated by double-stranded RNA that is homologous to the gene targeted. RNAi methods will permit the identification of oncogene(s) within recurrent regions of amplification, the investigation of their functional role in tumorigenesis, and an evaluation of their potential utility as targets for cancer therapy. The specific scientific aims of this proposal are: (1) to identify and map at high resolution recurrent gene amplifications in breast cancer by array CGH; (2) to evaluate gene amplifications as prognostic markers using FISH on tissue microarrays; and (3) to determine the functional role of, and therapeutic potential of targeting, identified gene amplifications in breast cancer using RNAi techniques.

[unreadable] DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth-most common cause of cancer-related death in the United States. Understanding the genetic abnormalities underlying pancreatic cancer pathogenesis is critical for developing robust clinical assays for early detection, as well as for mechanistic therapeutic strategies. The long term objectives of this study are to identify pathogenetic gene amplifications and deletions in pancreatic cancer, and to functionally characterize the underlying oncogenes and tumor suppressor genes. Array-based comparative genomic hybridization (array CGH) on pancreatic cancer cell lines defined previously unrecognized DNA amplifications and deletions, harboring novel candidate oncogenes and tumor suppressor genes. These studies identified two candidate cancer genes in particular whose altered copy number and expression may contribute to pancreatic cancer: SMURF1, which modulates TGFp signaling, and DKK3, likely involved in WNT signaling. A major goal of this proposal is to characterize the function of SMURF1 and DKK3 in pancreatic cancer development and progression through studying tumor growth in cell culture and in mice. A second major goal is to extend array CGH studies to primary pancreatic tumors, thereby discovering additional candidate pancreatic cancer genes. These studies will further knowledge of the role of gene amplification and deletion in pancreatic cancer, in particular in regards to the TGFp and WNT pathways, and may provide new strategies for therapeutic intervention. The specific scientific aims of this proposal are: (1) to characterize the role of gene amplification in the pathogenesis of pancreatic cancer, focusing on SMURF1, a modulator of TGFp signaling; (2) to characterize the role of gene deletion in the development of pancreatic cancer, focusing on DKK3, a potential modulator of WNT signaling; and (3) to identify additional novel DNA amplifications and deletions in pancreatic cancer, by array CGH analysis of primary pancreatic tumors. [unreadable] [unreadable] [unreadable] [unreadable]

The overall objective of the Tissue Procurement Facility (TPF) is to procure and provide needed tissue specimens to Stanford University Cancer Center investigators to support their cancer-related research. TPF activities and services include collecting and banking freshly-frozen tumor and normal tissues from excess surgical material and from autopsy, providing fresh tumor tissue for viable cell studies, processing and banking serum specimens from cancer patients, maintaining a tissue database with links to clinicopathological data, providing histological staining and pathological review, and coordinating patient consent and assuring regulatory compliance. As a centralized Shared Resource, the TPF adds value through experience, efficiency, standardization, accountability, protection of patient confidentially, and timely completion of research. An advisory board provides recommendations on facility policies and activities, and performs scientific and logistical review of service requests. In FY2005 the TPF, and satellite repositories, have provided tissue specimens to Cancer Center members representing 8 out of 10 Programs. Highlights of the research supported by the Facility include the discovery of clinically-relevant gene-expression subtypes of breast and prostate cancers. The TPF also provides a portal to a "virtual bank" linking inventories of specialized "satellite" repositories, including collections of hematological and neurosurgical specimens. Future plans include an expansion of universal-consent based tissue procurement, increased efforts to bank both paraffin tissue blocks and viable cells, and more detailed clinical annotation of tissue specimens.

DESCRIPTION (provided by applicant): The main aim of the proposed work is to understand the biology of tumors occurring in young sporadic colorectal cancer (CRC) patients. Colorectal cancer (CRC) is one of the most common lethal cancers in USA and Europe. Studies on CRCs have mainly focused on either older patients (above 50 years) or younger patients (below 50 years) with a familial predisposition. Two major forms of familial cancer syndromes have been well studied viz. the Hereditary Non-polyposis Colorectal Cancer (HNPCC) caused mainly due to germline inactivation of mismatch repair genes and the Familial Adenomatous Polyposis (FAP) caused mainly due to the germline inactivation of the Adenomatous Polyposis Coli tumor suppressor gene. Studies on sporadic CRCs occurring in the young have however been limited. Several recent reports have indicated an increase in the proportion of young sporadic CRC patients in developing countries in Africa and Asia, including India, as compared to USA and Europe. These patients account for almost 1/4th of the total CRC patients and often succumb to aggressive metastatic disease. Moreover, the tumor in these patients is usually localized to the rectum, as against similar young patients from the USA and Europe, in which case the tumor is predominantly localized to the colon. There are therefore several indications that suggest that the tumors occurring in the young may be distinct from similar tumors occurring in the elderly. In the present grant application, we propose a comprehensive comparative characterization of sporadic CRCs occurring in the young with those occurring in the elderly, from India. A multi-pronged strategy would be used including 1) screening for mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene, 2) determination of status of Wnt signaling through studies on beta-catenin and transcript profiling of Wnt target genes, 3) screening for microsatellite instability, 4) identification of recurrent DNA copy number alterations and 5) analyses of genome-wide gene expression profiles. The present study therefore is not only expected to yield valuable insights into the molecular basis for young sporadic CRC but may also provide inputs into cellular pathways governing tumor initiation and progression in general. Public Health Relevance: Colorectal cancer (CRC) is one of the most common and lethal cancers in the USA. Non-hereditary young CRC patients often are ill fated, and this type of cancer is not understood well. In our present study, we expect to define the detailed behavior of this class of tumors, which is expected to aid in designing more efficient therapies in the future.

DESCRIPTION (provided by applicant): Prostate cancer is the most frequently diagnosed cancer among men in the United States. Yet, the vast majority of men with prostate cancer will not die from their disease, reflecting the clinically-indolent behavior of their tumors. Indeed, most clinicians now agree that prostate cancer is both over-diagnosed and over-treated, a situation exacerbated by widespread prostate specific antigen (PSA) screening, and leading to significant treatment-associated morbidity. A key clinical question then is which men with prostate cancer could be spared unnecessary and potentially harmful therapy. Addressing this question requires a more detailed molecular understanding of clinically-indolent prostate cancer. In prior published studies, we identified three previously unrecognized molecular subtypes of prostate cancer based on distinct patterns of gene expression. Notably, one of these subtypes, subtype-1 , exhibited clinically-favorable behavior, and we speculate might represent a class of indolent tumors not requiring therapeutic intervention. Our more recent studies using array-based comparative genomic hybridization (array CGH) now indicate a distinct genetic basis underlying the different subtypes, including specific deletion within chromosome cytobands 5q21 and 6q15 in the clinically-favorable subtype-1 tumors. These findings at once implicate novel tumor suppressor genes (TSGs) in the pathogenesis of clinically-favorable prostate cancer, and define their location. The goal of our proposed research is to discover the pathogenic TSGs at 5q21 and 6q15 underlying a clinically-favorable prostate cancer subtype. The specific aims are (1) To delimit the boundaries of recurrent deletion at 5q21 and 6q15 by array CGH;and (2) To screen remaining candidate TSGs for DNA mutations and promoter hypermethylation. These studies will further our knowledge of the molecular pathogenesis of prostate cancer. Our findings may also suggest novel gene-based markers for the diagnosis of clinically- favorable tumors, leading to improved treatment stratification and clinical management of men with prostate cancer.

The overall objective of the Tissue Procurement Facility (TPF) Shared Resource is to procure and provide needed tissue specimens to Stanford Cancer Center investigators to support their cancer-related research. TPF activities and services include collecting and banking freshly frozen tumor and normal tissue from excess surgical material and from autopsy, providing fresh tumor tissue for viable cell studies, processing and banking blood specimens (plasma, serum, leukocyte DNA) from cancer patients, maintaining a tissue database with links to clinicopathological data, providing histological staining and pathological review and coordinating patient consent and ensuring regulatory compliance. As a centralized shared resource, the TPF adds value through experience, efficiency, standardization, accountability, protection of patient confidentially and timely completion of research. An oversight committee provides recommendations on facility policies and activities, and performs scientific and logistical review of service requests. Though largely supported by the Cancer Center and the School of Medicine, operating costs are defrayed by modest user fees. In the past 12 months alone, the TPF has provided over 450 fresh and frozen tissue specimens to 25 Cancer Center investigators from eight Cancer Center programs, to support cutting-edge cancer research in diverse areas, including cancer genomics, cancer stem cells and molecular imaging. The TPF also provides a portal to a virtual bank linking inventories of specialized satellite repositories, including collections of hematological and neurosurgical specimens. Future plans include increasing specimen capture to meet growing investigator needs, and expanding a new paraffin block collection.

PROJECT SUMMARY/ ABSTRACT (ADMINISTRATIVE CORE) The broad goals of the Administrative Core are to coordinate activities among the various Center Members and programs, to train and inspire future leaders in the field, and to establish productive interactions with the greater urologic research community outside of Stanford University. To accomplish those goals, the Administrative Core will provide clerical support to both the Scientific Research Project and Educational Enrichment Program. The Core will also coordinate collaborations with NIDDK-sponsored Centers, Programs, and other outside investigators, and maintain a Website to highlight Center activities and facilitate Resource sharing. Within the outstanding biomedical research environment at Stanford University, 11 participating Departments and Institutes will provide a rich infrastructure to support the Core's educational mission. The Educational Enrichment Program will leverage interdisciplinary research environments for trainee research experiences, and bring creative educational approaches to the Center. Focusing on the theme of precision medicine, the Educational Enrichment Program will sponsor Seminars, hand-on Workshops, and trainee Hybrid talks that cover novel methods and approaches with potential application to benign urology research. Through all of the above, the Administrative Core will realize the Center's vision and enhance the broader urologic research community.

DESCRIPTION (provided by applicant): SWI/SNF is a multi-subunit chromatin remodeling complex that repositions nucleosomes to control access to DNA, thus regulating gene expression. Recently, we discovered focal DNA deletions and deleterious mutations that target SWI/SNF subunits in more than one-third of human pancreatic cancers, a frequency approaching that of TP53 mutation. SWI/SNF re-expression studies support a growth-suppressive function, and our preliminary data nominate polycomb repressive complex 2 (PRC2) antagonism and TGF? signaling as possible downstream effector pathways. SWI/SNF has also been reported to function in DNA damage repair, and our preliminary data show SWI/SNF loss sensitizes pancreatic cells to DNA damage. Building from these findings, the broad goals of the proposed studies are to define the pathways and mechanisms by which SWI/SNF alterations contribute to pancreatic cancer, and to identify therapies that might selectively target SWI/SNF-deficient pancreatic cancers. To achieve these goals, in Aim 1 we will investigate the role of PRC2 antagonism as a mediator of SWI/SNF growth suppression, by molecular studies of PRC2 in SWI/SNF-deficient and reconstituted pancreatic cancer cells, and in primary pancreatic tumors. In Aim 2, we will similarly assess the role of TGF? signaling in mediating SWI/SNF growth suppression. In Aim 3, we will determine whether residual SWI/SNF complexes (remnants of SWI/SNF alteration) contribute to growth phenotypes in pancreatic cancer cells. Finally, in Aim 4 we will evaluate possible therapies selective to SWI/SNF-deficient pancreatic cancers, starting with DNA damaging agents (to exploit the reported role of SWI/SNF in DNA damage repair) in cell viability assays. Completion of these studies should establish the pathways and mechanisms by which SWI/SNF alterations drive pancreatic cancer, and define therapeutic strategies for SWI/SNF-deficient pancreatic cancers. Given that SWI/SNF alterations are commonplace in pancreatic cancer, that almost nothing is known of their consequence (for example compared to TP53 mutations), and that pancreatic cancer is such a devastating disease, the proposed studies are expected to make a high-impact contribution to the field. Moreover, findings are likely to be extendable to other cancer types with SWI/SNF mutations.

PROJECT SUMMARY Odontogenic tumors represent a spectrum of entities with differing morphologies, clinical presentations and treatments, despite commonalities in association with tooth development and anatomy. The complexity of this classification system is challenging for most pathologists, who rarely see these tumors, and the treatment options are largely limited to the extent of surgery, which can result in disfigurement, create functional problems with speech and swallowing, and is not always curative. Recent studies suggest that there are recurrent genomic events in at least some of these tumors. We have discovered activating mutations in SMO (most commonly SMO-L412F), part of the Hedgehog pathway, and in the mitogen-activated protein kinase (MAPK) pathway (most commonly BRAF-V600E) in over 80% of ameloblastomas. Others had previously found PTCH1 germline inactivation, also part of the Hedgehog pathway, in keratocystic odontogenic tumors associated with Gorlin syndrome (nevoid basal cell carcinoma syndrome). These pathways are druggable and we hypothesize that a new paradigm for the diagnostic classification and treatment of odontogenic tumors can be generated with genetic analysis. We will conduct whole-exome sequencing and transcriptome sequencing of odontogenic tumors, focusing on ameloblastoma, keratocystic odontogenic tumors and other morphologically-similar diagnoses, to identify driver mutations and oncogenic pathways. Using both the mutation and expression profiles, we will create a novel classification system for odontogenic neoplasms. We will create multiple new ameloblastoma cell lines from patient material bearing recurrent driver mutations, including BRAF, SMO, KRAS, and FGFR2. These new cell culture models will enable us to examine the response to gene targeted therapies, as well as to anticipate likely mechanisms of therapy resistance.

PROJECT SUMMARY Benign Prostatic Hyperplasia (BPH) is the benign enlargement of the prostate gland that occurs in older men, obstructing bladder outflow. The resultant lower urinary tract symptoms, such as urgency, frequency and incomplete emptying, have considerable morbidity, and carry annual healthcare costs in the Billions. Current BPH treatments are not very effective because the drugs target normal prostate physiology but not BPH pathophysiology, which is still poorly understood. Next generation, disease-targeted therapies will require a more detailed knowledge of BPH pathogenesis. In genomic studies of BPH clinical samples, we discovered a singular, massive overexpression of Bone Morphogenetic Protein 5 (BMP5) in BPH. BMPs, members of the TGF-beta superfamily, have important roles in tissue morphogenesis, though BMP5 itself has not been extensively studied. In pilot single-cell analyses, we identified a poorly characterized fibroblast cell type enriched in BPH stroma as the principle cell expressing BMP5. Remarkably, addition of recombinant BMP5 to prostatic cells in culture alters their gene-expression profiles to more closely resemble the profiles that we observe in the BPH clinical specimens. From these data, we hypothesize that BMP5 orchestrates key molecular and cellular changes underlying the BPH disease process. As such, therapies that abrogate BMP5 signaling may provide a new precision approach to prevent or treat BPH. Towards that goal, the proposed studies aim to Define the prostatic cell type and triggers of BMP5 production in BPH; Define the cell types and receptors for BMP5 signal transduction in BPH; and Determine whether blockade of BMP5 signaling reverses BPH disease phenotypes. Study findings will provide important new insight into the prostatic cells and signaling orchestrated by BMP5, and key validation data towards new disease-targeted therapies for BPH.

ABSTRACT ? PROJECT 1 Benign Prostatic Hyperplasia (BPH) is the benign enlargement of the prostate gland that occurs in older men, obstructing bladder outflow. The resultant lower urinary tract symptoms, such as urgency, frequency and incomplete emptying, have considerable morbidity, and carry annual healthcare costs in the Billions. Current BPH treatments are not very effective because the drugs target normal prostate physiology but not BPH pathophysiology, which is still poorly understood. New disease-targeted therapies will require a more detailed knowledge of BPH pathogenesis. In genomic studies of BPH clinical samples, we discovered a stromal gene signature that correlated with BPH symptoms, and an enrichment of fibroblasts overexpressing signaling proteins BMP5 and CXCL13. Fibroblast BMP5 enhanced prostate epithelial proliferation and drove gene expression profiles to mimic BPH tissue. From these data, we hypothesize that BPH is driven (at least in part) by pathogenic fibroblast cell subset(s), where defining those subsets will provide important new opportunities for disease targeted therapies. Towards that goal, the proposed studies aim to Define the fibroblast subsets in BPH versus normal prostate; Determine the key interactions between BPH fibroblast subsets and prostate epithelium that drive prostate enlargement; and Distinguish between BPH origins in embryonic re-awakening versus injury response. Study findings will provide new understanding of the contribution of prostate stroma to BPH pathogenesis, and identify new strategies for targeted treatment.