Browsing by Subject "Gene Expression Regulation, Neoplastic"
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Item Defining Tumorigenic Contributions of Meiotic Cancer-Testis Antigens(2018-07-10) Nichols, Brandt Alan; Siegwart, Daniel J.; Cobb, Melanie H.; Westover, Kenneth D.; Whitehurst, Angelique WrightCancer Testis Antigens (CTAs) are expressed in testis and/or placenta and anomalously activated in a variety of tumors. However, the mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. A cohort of CTAs are required for recombination events during meiosis, suggesting meiotic CTAs have potential to functionally contribute to the genomic stability of tumors. To assess the tumorigenic contributions of meiotic CTAs, I employed a targeted siRNA screen for five meiotic CTAs. Depletion of SYCP1 or HORMAD1 decreased tumor cell proliferation, while SYCE1 loss resulted in elevated DNA damage. Another meiotic CTA, SPO11, is a topoisomerase that induces DNA double-strand breaks during meiosis. I found that SPO11 expression not only correlates with elevated DNA damage in a variety of tumor cells, but ectopic SPO11 expression increases DNA double-strand breaks. A chemigenomics approach identified that a meiotic CTA, HORMAD1, correlates with resistance to piericidin A in non-small cell lung cancer (NSCLC). Resistance is due to a reductive intracellular environment that attenuates the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibit elevated mutation burden and reduced survival. Differential expression profiling revealed that HORMAD1 tumors are enriched for genes essential for homologous recombination (HR). Mechanistic studies find that HORMAD1 promotes RAD51-filament formation but not DNA resection during HR. Accordingly, HORMAD1 loss enhances sensitivity to gamma-irradiation and PARP inhibition. Furthermore, HORMAD1 depletion significantly reduces tumor growth in vivo. These results suggest that HORMAD1 expression specifies a novel subtype of LUAD which has adapted to mitigate DNA damage. Altogether, these finding indicate that meiotic CTAs play functional roles in altering the genomic stability of tumors and represent potential intervention strategies to enhance sensitivity to DNA damage agents and/or immunotherapies in patients.Item Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.Item High-Resolution Array Comparative Genomic Hybridization Identifies Common Targets in Rhabdomyosarcoma(2012-08-15) Paulson, Vera Ashley; Cameron, ScottRhabdomyosarcoma (RMS) accounts for nearly 50 percent of the soft tissue sarcomas that affect children. There are two major histological variants, alveolar (ARMS) and embryonal (ERMS). Both are defined as sarcomas that show exclusive evidence of muscle differentiation, but differ in their pathogenesis and prognosis. ARMS typically occurs in adolescents, presents as disease of the extremities, has a higher risk of metastasis or treatment-resistance, and in 75% of cases, is characterized by the presence of the PAX3/7:FOXO1A translocation. ERMS is associated with a younger age at presentation, sites of disease other than the extremities, a more favorable clinical outcome, and the absence of consistent chromosomal translocations. Here we used high-density array-based comparative genomic hybridization to examine the genomes of RMS to identify common programs that drive tumor pathogenesis.Item An In Vivo Functional Genomics Screen Identifies New Regulators of Tumorigenesis in Non-Small Cell Lung Cancer(2015-11-24) Hight, Suzie K.; Shay, Jerry W.; Minna, John D.; Roth, Michael G.; White, Michael A.Cancer cells are characterized by the aberrant regulation of signaling pathways that govern responses to growth stimuli, resulting in dysregulated cellular proliferation. The accumulated genomic alterations that cause this malignant growth phenotype can also result in acquired vulnerabilities in the tumor. Loss-of-function screening using pooled short hairpin RNAs (shRNAs) is a powerful method for identifying new therapeutic targets in cancer. We have tested the use of parallel in vitro and in vivo screens using a mini-library of shRNAs to identify previously unknown acquired vulnerabilities in lung cancer. Using a mini-library of 1062 lentiviral shRNAs targeting nuclear hormone receptors and their coregulators (120 genes total), we screened the lung adenocarcinoma cell line NCI-H1819 for dependency on these genes during in vitro and in vivo growth. We identified six genes required for survival in vitro (BRCA1, CCND1, MED1, PHB, HNRNPU, and PELP1), and three genes that were required for tumor survival in vivo, but not in vitro: FOXA1, HDAC1, and NCOR2. None of these genes were mutated by full exome sequencing of H1819, however FOXA1 was found to be co-amplified with NKX2-1 on chromosome 14q. Here we report that NSCLC cell lines and tumor samples have significantly higher FOXA1 expression compared to normal lung epithelial cells and tissues, and that 14q-amplified NSCLC cell lines are preferentially dependent on FOXA1 for clonogenicity and in vivo growth. Integrative transcriptomic and cistromic analyses identified a subset of direct FOXA1 targets as positive regulators of key growth signaling pathways, and negative regulators of several growth inhibitory mechanisms. This regulatory function appears to be partially independent of NKX2-1, and combing de novo motif discovery with expression analyses has identified several other putative FOXA1 coregulators. Our findings provide new insight into the functional consequences of 14q amplification in lung adenocarcinomas and suggest exploration of new transcriptional networks for potential therapeutic vulnerabilities.Item Natural Products as Selective Chemotherapeutic Agents and as Chemical Probes to Understand Biological Processes(2013-09-26) Carrasco, Yazmin Paulina; Tambar, Uttam; MacMillan, John; Roth, Michael G.; Ready, Joseph M.Aware of the important role that terrestrial microbial natural products play in the discovery of therapeutics and the decrease in rate of discovery of new natural products in the pharmaceutical industry, there is an immediate need to explore novel sources of microbial natural products with biological relevance. Marine bacteria are an excellent source of bioactive metabolites. With this idea in mind our laboratory has developed new techniques to isolate >600 species of marine actinomycetes and has created a natural product fraction library from these bacterial strains. My research efforts have focused on the isolation of natural products that exhibit selective activity against a panel of tumor derived cell lines that include lung, colon, melanoma, pancreatic cancer and glioblastoma. In parallel to structure elucidation efforts of our active metabolite, the determination of its mode of action was ongoing in the laboratory of Dr. White. Through the use of a new screening tool, called FUSION (Functional Signature Ontology) we were able to determine that our active compound had similar activity to inhibitors of the TBK1 signaling pathway. Additionally, a novel polyketide was isolated from the marine-derived bacteria Salinispora arenicola, believed to be a key intermediate in the biosynthetic pathway of saliniketal, an inhibitor of ornithine decarboxylase induction. In addition, we have utilized the natural product leptomycin B (LMB), as a chemical tool to understand its inhibition of CRM1, a protein involved in export of cargo from the nucleus to the cytoplasm and a possible chemotherapeutic target. This work was done in collaboration with Dr. Chook. We observed that LMB irreversibly binds to CRM1 due to the hydrolysis of its lactone moiety causing stabilization of the protein-LMB complex. In contrast, compounds that lack the capability of being hydrolyzed by CRM1 are reversible inhibitors of CRM1. These research findings probe the question of designing molecules capable of reversibly inhibiting CRM1 and this perhaps will lead to reduced toxicity. This work illustrates the importance that natural products have not only as therapeutic agents for the treatment of diseases such as cancer but also as important chemical tools to understand complex biological processes.Item Probing the Molecular Requirements for Breast Cancer Cell Motility(2014-11-20) Dang, Tuyen Thi Thanh; Brekken, Rolf A.; Pearson, Gray W.; Cobb, Melanie H.; Whitehurst, Angelique WrightInvasion of breast cancer cells into the stroma is an early step in metastasis. How invasive ability is conferred in breast cancer cells is poorly understood. It has been shown that the tumor microenvironment can assist in the progression of cancer. The mechanism by which the microenvironment, specifically the mammary fibroblast, contributes to the invasion of breast cancer cells is unknown. To elucidate the function of mammary fibroblasts in breast cancer invasion, we co-cultured and co-injected breast cancer cells with mammary fibroblasts. We have found that mammary fibroblasts can induce invasion of motile basal-subtype breast cancer cells better than non-motile luminal subtype breast cancer cells. Based on these observations, the intrinsic motility of breast cancer cells was the determinate for invasion. To discover novel regulators of motility, we screened 879 distinct miRNAs using a wounding assay as a readout for migration. To prioritize, we determined which of the miRNAs inhibited migration and were expressed in non-motile breast cancer cells. This approach led us to focus on miR203a. We discovered that miR203a inhibits the expression of the transcription factor deltaNp63alpha, which leads to a decrease in cell migration. To determine how deltaNp63alpha controlled migration, we next determined which genes and miRNAs require deltaNp63alpha for expression. From these analyses, we discovered that deltaNp63alpha is required for the expression of miR205, the transcription factor Slug and the tyrosine kinase Axl. Additional functional analyses revealed that Slug, Axl and miR205 contributed to migration through their actions within parallel signaling pathways. Furthermore, using, an orthotropic xenograft model, we determined that deltaNp63alpha and Slug functionally contribute to the induction of collective invasion in vivo. Together, our results suggest a deltaNp63alpha dependent signaling through miR205, Axl, and Slug can lead to the induction of breast cancer invasion.Item Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.Item The Role of the WWTR1(TAZ)-CAMTA1 Gene Fusion in Epithelioid Hemangioendothelioma(2021-05-01T05:00:00.000Z) Driskill, Jordan Harrison; Dellinger, Michael T.; Cleaver, Ondine; McFadden, David G.; Pan, DuojiaEpithelioid hemangioendothelioma (EHE) is a devastating and mysterious vascular cancer which has no known definitive treatment. Due to a lack of valid animal or cell-based models of EHE, progress toward understanding and treating this cancer has been severely limited. However, recent studies have determined that 90% of patients exhibit a lone, characteristic in-frame gene fusion, TAZ(WWTR1)-CAMTA1. While expression of the TAZ-CAMTA1 fusion protein has been validated as a biomarker of EHE, it remains unknown whether this genetic abnormality is a passenger or a driver of EHE. In this project, I present the first genetically-engineered mouse model (GEMM) of EHE, showing that the expression of the TAZ-CAMTA1 protein in endothelial cells is sufficient to drive the formation of EHE-like tumors in the lungs of mice. Furthermore, I demonstrate that the cessation of TAZ-CAMTA1 expression leads to the regression of these vascular tumors. I also demonstrate that TAZ-CAMTA1 transforms the MS1 endothelial cell line and that subcutaneous transplantation of these cells into nude mice leads to the formation of solid, progressive EHE-like vascular tumors that have the capacity to metastasize to the lung. Utilizing these two novel models of EHE, I unravel the gene program of TAZ-CAMTA1 and demonstrate that TAZ-CAMTA1 drives a gene signature similar to TAZ, the key effector of the Hippo pathway. Expression of an activated TAZ in endothelial cells is also sufficient to drive EHE-like vascular tumors in mice, and genetic blockade of the transcriptional partners of TAZ, the TEAD family of transcription factors, prevents the formation of TAZ-CAMTA1-induced vascular tumors. Next, I show that TAZ-CAMTA1 induces an angiogenic and regenerative-like gene program in endothelial cells. I validate that TAZ-CAMTA1 exhibits gain-of-function activities by having increased resistance to proteasomal degradation and increased nuclear enrichment over TAZ. Lastly, I show that TAZ-CAMTA1 still maintains its binding to the Hippo pathway proteins which are known to negatively regulate TAZ. In summary, I generate two novel models that pinpoint TAZ-CAMTA1 as the key driver of EHE and utilize these models to suggest several new lines of investigation for the treatment of patients with EHE.Item Studies of the Molecular Features of Brd4-Nut and P300 That Contribute to Condensate Formation and Transcriptional Regulation(2023-05-01T05:00:00.000Z) Kosno, Martyna Olga; Kohler, Jennifer J.; Tu, Benjamin; Henne, W. Mike; Brekken, Rolf A.; Liszczak, Glen; Rosen, Michael K.Aberrant formation of biomolecular condensates has been proposed to play a role in several cancers. The oncogenic fusion protein Brd4-Nut drives aberrant gene expression and forms condensates in Nut Carcinoma (NC). It has not been clear how these condensates form and whether they modulate gene expression. Here, I dissected the molecular features of Brd4-Nut and a histone acetyltransferase (HAT), p300, and analyzed their contribution to condensate formation and transcriptional changes. I determined that a minimal fragment of Nut (MIN) in fusion with Brd4 is necessary and sufficient for binding to p300, and for condensate formation. A Brd4-p300 fusion protein also forms condensates and drives a transcriptional profile similar to Brd4-Nut(MIN). The intrinsically disordered regions, transcription factor - binding domains, and HAT activity of p300 all collectively contribute to condensate formation. Conversely, only HAT activity appears to be necessary to mimic the transcriptional profile of cells expressing Brd4-Nut. My results suggest that interaction of Brd4-Nut with p300 is important for aberrant condensate formation, and that multiple, yet distinct, regions of p300 contribute to condensate formation and transcriptional regulation.