University of Pittsburgh Annual Progress Report: 2008 Formula Grant Reporting Period July 1, 2011 June 30, 2012 Research Project 1: Project Title and Purpose Small Molecule Inhibitors of HIV Nef Signaling - The project focuses on discovery of small molecules to target a unique human immunodeficiency virus type 1 (HIV-1) protein, called Nef, which is essential for progression from a state of HIV infection to full-blown acquired immune deficiency syndrome (AIDS). Using in vitro and cell-based screens, the investigators aim to identify two classes of drug-like compounds with potent activity against HIV replication. These studies could eventually provide new tools for understanding Nef interactions with host cell proteins and identify novel lead compounds for anti-hiv therapeutics. 1/1/2009-3/31/2011 Research Project 2: Project Title and Purpose Alterations in Bioenergetics and Mitochondrial Function in Tumor Cells - Cells generate the energy they need to function and divide through two different metabolic pathways, mitochondrial oxidative phosphorylation and glycolysis. Due to their rapid growth, tumor cells have unique energy demands. However, the mechanism by which tumor cells regulate energy production is poorly understood. This project will examine the balance between these two energy producing pathways in tumor cells. The cells to be examined are associated with specific genetic alterations in critical genes (oncogenes, like Myc and Ras) or include critical genes that have been mutated (tumor suppressor genes, like p53). This project also addresses how shifts in energy demands can increase genome instabilities like mutations. Understanding bioenergetic shifts of specific tumor types may improve therapeutic approaches. 1/1/2009-12/31/2010 University of Pittsburgh 2008 Formula Grant Page 1
Research Project 3: Project Title and Purpose Clinical Trials in Melanoma - The University of Pittsburgh Cancer Institute s (UPCI) Clinical Research Service (CRS) provides valuable resources for researchers and clinicians seeking to improve clinical research as a means to elevate patient care standards and treatment efficacy. This project will evaluate the clinical response of melanoma patients at various disease stages to novel therapeutic regimens designed to be specific to their treatment needs. 1/1/2009-12/31/2010 Research Project 4: Project Title and Purpose Therapeutic Vaccine Trials in Recurrent Prostate Cancer - The University of Pittsburgh Cancer Institute s (UPCI) Clinical Research Service (CRS) provides valuable resources for researchers and clinicians seeking to improve clinical research to elevate patient care standards and treatment efficacy. This project will conduct two novel vaccine trials in patients with recurrent prostate cancer: (1) a safety and efficacy study of a new vaccine targeted against mucin-1 (MUC-1) in conjunction with poly-iclc (polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) in patients with recurrent and/or advanced prostate cancer and (2) a safety and efficacy evaluation of alpha-type-1 dendritic cell (DC)-based vaccines loaded with allogeneic prostate tumor cells in combination with androgen ablation in patients with recurrent prostate cancer. 1/1/2009-12/31/2010 University of Pittsburgh 2008 Formula Grant Page 2
Research Project 5: Project Title and Purpose Research Infrastructure: Bridgeside Point II Building Renovations - Bridgeside Point II (BPII), the University of Pittsburgh s newest research facility, is nearing completion. This project will address final construction and laboratory fit-out in the building. By establishing this additional space, the Department of Orthopaedic Surgery, the Department of Microbiology and Molecular Genetics, and the McGowan Institute for Regenerative Medicine will be able to expand their research programs, recruit additional faculty members, and accommodate researchers changing needs. 12/17/2009 9/30/2010 Research Project 6: Project Title and Purpose Estrogen Receptor Genetics in Breast Cancer - Breast cancer remains a major health issue in the United States, with approximately 210,000 new cases and 40,000 deaths per year. Breast cancer can be subdivided into estrogen receptor (ER) positive and negative disease. Breast cancers that express ER are often less aggressive and respond to anti-hormone therapy; however, a significant fraction of patients with ER positive disease do not respond to therapy. New sequencing technologies have recently allowed an unprecedented view of ER action in breast cancer. This project will investigate how genetic alterations in ER and the ER pathway affect response to the hormone and whether the ER itself is involved in generating genetic changes in breast cancer. Better knowledge of ER action should translate into improved treatment of ER positive breast cancer. 3/30/2011 12/31/2012 Project Overview ER is a major determinant of breast cancer initiation, progression, and response to anti-hormone therapy. Recent advances in molecular techniques have allowed an unprecedented view into how this hormone receptor orchestrates its biological response in normal cells and tumors. This project is based on the use of novel next-generation sequencing techniques, which have provided a unique insight into genetic changes in ER in human breast cancers and new ideas about how ER may effect genetic changes in breast cancer. This project will use an array of new University of Pittsburgh 2008 Formula Grant Page 3
technologies to comprehensively interrogate ER in breast cancer cell lines. In the first aim, we will study the effect of germline single nucleotide polymorphisms (SNPs) and cancer-specific somatic mutations on ER function. Preliminary studies using next-generation sequencing in human breast tumors have identified numerous genetic changes in ER, and we will examine these changes with specific regard to their effects on estrogen-mediated cancer phenotypes, such as proliferation and invasion. In the second aim, we will examine how alterations in the deoxyribonucleic acid (DNA) sequence that ER binds might affect subsequent hormone response. Target sites will be identified by chromatin immunoprecipitation and sequencing; and changes will be characterized, especially related to hormone response. In the third aim, we will examine whether ER binding sites are associated with regions of DNA translocations in breast cancer cells and whether ER can itself direct genomic translocation in cells with reduced DNA repair activity. This project will provide significant new information regarding ER action in breast cancer and will likely have a major impact in improving prediction and response to hormone therapy. Principal Investigator Adrian V. Lee, PhD University of Pittsburgh Magee-Womens Research Institute 204 Craft Avenue Room B705 Pittsburgh, PA 15213 Other Participating Researchers Steffi Oesterreich, PhD - employed by University of Pittsburgh Expected Research Outcomes and Benefits Two-thirds (~130,000) of breast cancers detected in the United States each year are ER-positive and are treated with anti-hormone therapy. However, not all tumors respond to this therapy, and a majority will eventually develop resistance. This project will investigate genetic changes in ER and their role in causing differential responses to anti-hormone therapy. In Aim 1, we expect to find that somatic mutations in ER render the tumor resistant to anti-hormone therapy, indicating the need for alternative therapies. Similarly, germline polymorphisms in ER are likely to modulate the response to anti-hormone therapy; and some may actually render tumors hypersensitive to therapy, in which case the tumors may require anti-hormone therapy alone, thereby sparing the patient unnecessary toxic therapy. In Aim 2, we predict that genetic changes in ER binding sites will correlate with sensitivity or resistance to hormone therapy. Finally, we expect Aim 3 to produce novel data regarding how ER affects genomic translocations. The discovery of specific ER-directed genomic translocations will have major implications in breast cancer, as they may produce novel gene products that can act as tumor-specific diagnostic and therapeutic targets (similar to the oncogene fusion BCR-Abl in chronic myelogenous leukemia). In summary, this project will provide novel and critical information regarding ER function in breast cancer and is likely to have a major impact on hormone treatment of breast cancers. University of Pittsburgh 2008 Formula Grant Page 4
Specific Aim 1: Identify and characterize single nucleotide polymorphisms (SNPs) and mutations in the estrogen receptor (ER) in human breast cancer As described in the prior progress report (July 2011), we had sequenced a number of breast tumors and cell lines and identified a total of 60 DNA sequence variants (DSV). This number was thought to include some false positives since it decreased drastically when we applied more stringent criteria to the sequence mapping algorithm. Specifically, using stringency with a cutoff with at least two variant reads, variants in at least 20 percent of the reads, and requiring evidence for the variants in both strands, we identified four previously reported SNPs (rs2077647, rs46432, rs1801132, rs2228480) and two previously reported somatic mutations (H6Y and N532Y). In collaboration with Dr. Soon Paik s and Dr. Katherine Pogue-Geile s group from the National Surgical Adjuvant Breast and Bowel Project (NSABP, Pittsburgh, PA), we developed mass-spectrometry (MS) assays (Sequenom) for these DSVs and controls. In addition, we included 10 DSVs in the estrogen receptor (ESR1), which had previously been reported as somatic mutations in breast (and other) cancers. Applying MS-based analysis to breast tumors and cell lines, we were able to confirm the previously reported SNPs and one previously reported somatic mutation (H6Y). This mutation was found in one breast tumor and one cell line. None of the other reported somatic mutations could be confirmed in tumors or cell lines. We then set out to determine the phenotype of the H6Y DSV. Using site-directed mutagenesis, we introduced the change into estrogen receptor 1 complementary DNA (ESR1 cdna) and tested a number of phenotypes, including transcriptional activity and effect on growth and cell cycle. These functional assays on the ESR1 H6Y DSV failed to identify any differences to wildtype receptor. The lack of a phenotype and the infrequent occurrence of this DSV do not support a major driver role for ESR1 H6Y in human breast cancer. This analysis suggests that ESR1 mutations are an extremely rare event in ER+ primary breast cancer. These studies have been submitted to the Annual San Antonio Breast Cancer symposium, and we are currently writing up the manuscript (Oesterreich et. al., Lack of Frequent ESR1 Mutations in Human Breast Cancer ). Specific Aim 2: Examine genome-wide binding of ER in breast cancer cell lines and identify SNPs and/or mutations in ER binding sites that affect hormone response In the last progress report, we briefly discussed that we finished the ESR1 (and steroid receptor co-activator 1 [SRC1]) ChIP-seq analysis in an estrogen responsive osteosarcoma cell line. These studies led to the identification of estrogen regulation of a prostaglandin transporter that had not previously been implicated in estrogen-mediated tumor growth or metastasis. We have, therefore, begun a collaboration with Dr. Pawel Kalinksi (University of Pittsburgh Cancer Institute [UPCI], Pittsburgh, PA), an expert in the study of prostaglandin E2 (PGE2), to follow up on these studies. We have then applied a similar protocol to Michigan Cancer Foundation (MCF-7) breast cancer cells, and have successfully set up the ESR1 ChIP protocol. We are University of Pittsburgh 2008 Formula Grant Page 5
currently performing additional ChIP studies, and expect to send the samples out for sequencing in the next few months. Meanwhile, a number of groups have published ChIP-seq studies with ESR1. To address whether germline DSVs in ESR1 binding sites affect response to hormone therapy, we have used an in silico approach. Therefore, in collaboration with Drs. Xinghua Lu and Takis Benos (Center for Translational Bioinformatics, University of Pittsburgh), we downloaded publicly available ChIP-seq datasets and identified DSVs. As an example, for the MCF-7 cell line, we currently have access to nine ER ChIP-seq data sets. Focusing on the overlapping (i.e., high confidence) estrogen-induced ESR1 binding sites, we identified ~ 5,200 binding sites. Cross-referencing these sites with the single nucleotide polymorphism database (dbsnp) and the 1,000 Genomes Project showed that approximately 85 percent of these sites harbor known SNPs. The remaining 15 percent are either sequencing artifacts (which is unlikely, given the Genome Analysis Toolkit [GATK] workflow stringency criteria we applied to our analysis), or they are low-frequent SNPs that have not yet been deposited into public databases; finally, they might actually be somatic mutations in ER binding sites. We are currently testing the latter, most exciting possibility by screening COSMIC (a public database containing information on mutations in cancer specimens) and by using other publicly available data sets. In parallel, we are currently performing ER ChIP-seq studies in unique models that have not been studied by others, such as models for invasive lobular cancer (ILC). We have already performed gene expression array studies in these cells (-/+ estrogen, six-hour and 24-hour time points). The UPCI Biostatistics Facility is currently analyzing the gene expression results. We will then combine the data sets, with the expectation to identify DSVs in ILC that alter hormone response in these cells. We have also requested tumor samples from the University of Pittsburgh Health Sciences Tissue Bank and are currently isolating DNA and ribonucleic acid (RNA), with the goal of confirming DSVs; we are also correlating gene expression changes in clinical samples. Finally, we will be able to correlate these changes with response to endocrine therapy, since the tumors we are using are extremely well curated with respect to clinical information. Thus, we have made significant progress on this aim we now have all methods set up and have performed genome-wide assays, which will aid in the identification of relevant DSVs in ER binding sites. Specific Aim 3: Determine the role of ESR1 in orchestrating genomic instability and translocations ESR1 and other nuclear hormone receptors regulate gene transcription by bringing DNA enhancer elements (that are situated a long distance away) into proximity with promoters. This looping of DNA is a relatively unique action of nuclear hormone receptors but may represent a potential Achilles heel, with nuclear hormone receptor action in cancer cells with reduced DNA repair capacity potentially increasing chromosomal translocations. Consistent with this hypothesis, we identified a gene, PLAC1, which is a fetal placental gene but is re-expressed at very high levels in MCF-7 breast cancer cells. In this cell line, PLAC1 is under estrogen regulation, something that is inconsistent with its role in placental development. We hypothesized that PLAC1 may have an ESR1 binding site translocation upstream of its promoter University of Pittsburgh 2008 Formula Grant Page 6
to confer de novo estrogen regulation. We performed paired-end sequencing of DNA and identified a genomic translocation of chromosome 6 upstream of the PLAC1 gene on the X chromosome; this region of chromosome 6 harbors three ESR1 binding sites validated by ESR1 ChIP. If this is a MCF-7 specific event, then we would hypothesize that PLAC1 would be estrogen regulated only in MCF-7 breast cancer cells. Consistent with this hypothesis, we did, indeed, find that PLAC1 is highly estrogen regulated in MCF-7, but not in five other breast cancer cell lines, each of which showed estrogen regulation of classical genes (PR [progesterone receptor] and PS2) (Fig 1). Further studies are investigating the biological role of estrogen regulation of PLAC1 in MCF-7 cells and the identification and validation of other ESR1- mediated translocations. Figure 1: Cell line specific regulation of PLAC1. ER-positive breast cancer cell lines were cultured in 5 percent charcoal-stripped serum and then stimulated with estradiol (1nM) for 24 hours. RNA was isolated and Q-RT-PCR performed for A) PLAC1, B) PS2 and C) PR. Bars represent the average of triplicate wells +/- S.E.M, and values represent fold control normalized to expression in the absence of estradiol. University of Pittsburgh 2008 Formula Grant Page 7