Prostate cancer is the second most common cancer in American men, and it has been estimated that about 1 man in 6 will be diagnosed with prostate cancer during his lifetime. The prostate-specific antigen (PSA) serum test is widely used to screen men for prostate cancer, however the test has a false-positive rate of nearly 70% and leads to many unnecessary biopsies being performed. Anterior gradient 2 (AGR2) is a secreted protein produced by prostate cancer cells in primary tumors, and due to its cancer-specific property, AGR2 can potentially be used to develop a urine test for prostate cancer diagnosis. The expression of AGR2 in cancer patients were studied by frozen section immunohistochemistry, sandwich ELISA, tissue microarray, and statistical analysis. The results show that AGR2 can be a suitable biomarker used to develop a test in screening prostate cancer for the following reasons: (1) it is highly expressed in cancer; (2) it is found in almost all tumors; (3) it is expressed by pre-malignant high-grade prostatic intraepithelial neoplasia (HGPIN) lesions. Since AGR2 is specific to cancer cells and is secreted into urine, we find the clinical application of AGR2 to be viable and that a urine test used for prostate cancer diagnosis is possible. AGR2 is detected at picograms per millimeter level for cancer patients.

Cancers contain large numbers of mutations. This reservoir of genetic variation can be used by cancer to produce new phenotypes in response to environmental barriers. In order to accumulate these mutations we have proposed that cancers express a mutator phenotype, i.e. an increased rate of mutation. Previous results in the lab demonstrate that sequential selection of cancer cell lines (colon cancer cells HCT116 and SW620) for novel phenotypes enriches for cell lineages with elevated mutation rates. After three rounds of sequential selection for resistance to non-mutagenic drugs (diphtheria toxin, puromycin, staurosporine), both cancer cell lines exhibit an increase in mutation rate. Specifically a 3-fold for HCT116 and 6-fold for SW620 was measured. I will extend these experiments using additional selection conditions (e.g., elevated temperature, hypoxia and nutrient deprivation) and will determine mutation rate of the resulting cell lines using the PIGA gene mutation assay. The random mutation load will be characterized using Duplex Sequencing. We believe the process of sequential selection favors mutator lineages as has been observed in bacteria. However, because an increased rate of mutation is potentially deleterious to a cell, we propose the existence of a mutation limit beyond which cells are no longer viable, i.e. the cells undergo apoptosis. I will screen mutagenic nucleosides for their potential to increase the mutation rate of cancer cells beyond this threshold. Targeting cancer’s error threshold may represent a novel and innovative therapeutic strategy for the treatment of human malignancies.

Breast cancer tumors are classified within different subtypes based on the expression of three types of cell surface receptor proteins.  These proteins connect the extracellular environment with cellular activities via signaling pathways.  To elicit cell responses, signaling pathways employ protein phosphorylation as a regulatory mechanism, as adding a phosphate group to a particular protein can switch its activity on or off.  However, normal signaling can go awry if mutations in signaling proteins lead the cell to believe its components are always phosphorylated and active.  Consequently, the entire signaling pathway functions abnormally, which can lead to uncontrolled cell growth characteristic of cancer.  Given its potential role as a molecular mechanism of disease, our research focuses on obtaining a global view of the phosphoproteome, or the cell’s entire set of expressed, phosphorylated proteins, of 10 breast cancer cell lines.  To generate lists of phosphoproteins present in these cell lines, we are concurrently extracting and analyzing cell line phosphopeptide samples via mass spectrometry, an analytic technique that produces characteristic spectra for individual phosphopeptides that are then matched to a library of known protein spectra through the use of computer algorithms.  In our preliminary results, we have generated large-scale data sets identifying thousands of phosphorylated proteins present in two cell lines and generated side-by-side comparisons of the proteins expressed in each line.   By identifying common phosphorylated proteins across all of the cell lines, our goal is to deduce a molecular signature for breast cancer.  This signature can be used in future studies to design a universal protein inhibitor drug as an alternative anti-cancer therapy.   By identifying the phosphoproteins exclusive to each cell type, we can uniquely characterize specific proteins active in one type of breast cancer which may facilitate development of inhibitor drugs as a form of personalized medicine.

Cancer genomic analyses performed by our team have allowed us to identify frequently mutated genes in Small Cell Lung Cancer (SCLC). Among these genes, MLL2 was found to undergo loss of function mutations suggesting it could act as a tumor suppressor. MLL2 is a histone methyl transferase that transfers a methyl group to a histone on a DNA strand. This process regulates chromatin state and transcription. More specifically, MLL2 methylates the lysine 4 of histone H3 leading to the activation of gene expression. Interestingly, MLL2 was also recently found mutated in squamous cell lung carcinoma, non-Hodgkin lymphoma, medulloblastoma and cell renal carcinoma pointing out that it could be a novel major tumor suppressor. To investigate this hypothesis, both in vivo (mouse models) and in vitro (cell based assays) will be used. The mouse model consists of a conditional, tissue specific inactivation of two prototype tumor suppressors namely p53 and Rb, which are found highly mutated in SCLC. The role of MLL2 will be studied in this mouse model by generating triple mutants (Rb, p53, and MLL2) mice. The survival of control SCLC mice (p53 and Rb inactivated) will be compared to the survival of the triple mutant (MLL2, Rb, and p53) mice. This critical experiment should enlighten us on the functional role of MLL2 during SCLC development. Molecular and histopathological analyses will also be performed. Additionally, in vitro approaches with human and mouse cells will be used to identify and understand the biological processes and pathways regulated by MLL2. Altogether, it should help us decipher the role of MLL2 in SCLC and it might ultimately lead to the identification of novel treatments and/or preventive options for SCLC.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Soft tissue sarcoma is a cancer that develops in the muscle, fat, fibrous tissue, blood vessels, or other supporting tissue. RMS is categorized into two major subtypes: alveolar RMS (ARMS) and embryonal RMS (ERMS). Most ARMS express the PAX3-FOXO1 fusion oncoprotein generated by the 2;13 chromosomal translocation. Among RMS tumors, PAX3-FOXO1-positive ARMS commonly gives the worst prognosis. Sphingolipids are defined by a common structural feature (a long-chain (sphingoid) base). Sphingosine is the most common sphingoid base composed of an 18 carbon-chain. The overall goal of this study is to investigate roles of sphingosine on growth, apoptosis (programmed cell death), and MYCN oncogene expression in PAX3-FOXO1-positive ARMS cells (RH30 cell line). The specific aims of this study are to determine: 1) effects of sphingosine on apoptosis; 2) effects of sphingosine on MYCN expression; 3) effects of MYCN overexpression on growth inhibitory and pro-apoptotic activities of sphingosine. Our previous results have indicated that sphingosine caused time and dose-dependent inhibition of the growth and death of RH30 cells, suggesting sphingosine as a potential chemotherapeutic agent against ARMS. Sphingosine, at a concentration that can induce apoptosis, down-regulates the MYCN expression as early as two hours. In the present study, the data from flow cytometry demonstrates that sphingosine induces apoptosis identified by a well-known apoptosis marker, AnnexinV+/PI+. Experiments are in progress to determine whether the growth inhibitory effects of sphingosine are decreased in RH30 cells overexpressing MYCN. This will test a hypothesis that MYCN is a mechanism which mediates the growth inhibitory and pro-apoptotic effects of sphingosine on PAX3-FOXO1-positive ARMS cells. Thus, this experiment will determine if MYCN is a possible target for chemotherapeutic agents in ARMS.

Genome-wide association studies indicate that the sequence polymorphism rs6983267 located in the 8q24 risk locus correlates with increased risk for colorectal, prostate, breast and ovarian cancers. This region is particularly interesting because it is located in a ‘gene desert’ in which no expressed genes are known; however, several reports suggest that it functions as an enhancer that regulates the expression of the Myc oncogene located more than 300 kilobases away from rs6983267. The enhancer region also contains a binding site for CTCF, a factor known for its contributions to long-distance chromosomal interactions and gene regulation. Here, we aim to elucidate the specific function of CTCF located close to the rs6983267 sequence variation in DLD1-colorectal cells. We test the hypothesis that the CTCF element at the 8q24 risk region is required for a physical interaction between the enhancer of the 8q24 risk locus and the Myc gene. This hypothesis predicts that deletion of the CTCF binding site will yield reduced Myc expression. In our study we introduced a series of genomic modifications with recombinant adeno-associated virus (rAAV) to generate site-specific deletions and to examine CTCF’s role in long-range chromosomal interactions at the 8q24 locus. Mutant cell lines were developed with modifications on either one or both 8q24 alleles in the colorectal cancer cell line DLD1. Cell growth characteristics, gene expression and chromosome conformation capture (3C) experiments will be performed to better understand the functional and physical interaction between the 8q24 risk locus and the Myc gene.

Angiogenesis is the development of new blood vessels from pre-existing vasculature and is a hallmark of Kaposi’s sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals. The etiological agent of KS, Kaposi’s sarcoma-associated herpesvirus (KSHV), induces angiogenic phenotypes in endothelial cells during latent infection. Interestingly, KSHV infection leads to down-regulation of transforming growth factor-beta 2 (TGF-β2), an anti-angiogenic cytokine. In addition to six viral genes, KSHV also encodes 17 recently discovered microRNAs (miRNAs) during latent infection. These endogenously encoded RNAs of 19-23 nucleotides post-transcriptionally regulate gene expression through basepairing interactions with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs) and either mark them for degradation or prevent ribosomal binding. These viral miRNAs appear to directly contribute to pathogenesis and angiogenesis by targeting host gene expression. We screened the miRNAs of KSHV to determine if any are involved in the down-regulation of TGF-β2 during KSHV infection. We found that two KSHV miRNAs, miR-K3 and miR-K8, are independently sufficient to downregulate TGF-β2. To determine if these miRNAs directly target the 3’UTR of TGF-β2 as opposed to altering genes that affect TGF-β2 expression, we used a TGF-β2 3’UTR luciferase reporter construct. We also used competitive inhibitors of miR-K3 and miR-K8 to determine if these miRNAs are necessary for the down-regulation of TGF-β2. By identifying specific targets of KSHV miRNAs such as TGF-β2, we can improve our understanding of how KSHV is able to maintain latent infection and target host gene expression to alter endothelial cells.