Browsing by Subject "Carcinogenesis"
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Item Examining the Regulation and Function of Cancer Testis Antigens(2015-12-01) Pavlovsky, Ashly Ann; Brekken, Rolf A.; Whitehurst, Angelique Wright; Johnson, Jane E.; Cobb, Melanie H.Cancer Testis Antigens (CTAs) are a class of genes whose expression is generally restricted to the testis, but are reactivated in cancer cells. The function and regulation of many CTAs are unknown, however several CTAs have been shown to impact tumor cell fitness and correlate with poor prognosis. Our lab became interested in CTAs after a pan-genomic-loss-of-function RNAi screen identified several CTAs as chemo-sensitizers. Acrosin Binding Protein (ACRBP) was identified in this screen and further analysis confirmed ACRBP's function as a microtubule stabilizer that protects cancer cells from the mitotic defects attributed to paclitaxel treatment. Because ACRBP is not expressed in normal somatic tissue, we are interested in how ACRBP is reactivated in cancer cells. In mouse spermatogonium, the ACRBP promoter is bound by Cyclic-amp Response Element Modulator (CREM) at a conserved Cyclic-amp response element (CRE). Depletion of its close family member CREB results in a loss of ACRBP expression in cancer cells, while overexpression of CREB induces ACRBP expression. Interestingly, this regulation appears to be phospho-independent and seems to apply to other CTAs. Another CTA, Chondrosarcoma Associated Genes 1, was identified by a loss-of-function RNAi proliferation screen as a supporter of melanoma cell proliferation. CSAG1 is highly expressed in Melanoma lines with little to no expression in normal tissues. Additional CSAG1 siRNA experiments have validated the screen data confirming CSAG1's involvement in melanoma cell proliferation. Additionally, CSAG1 loss-of-function reduces long-term melanoma cell viability and induces senescence in cancer cells. Consistent with this data, overexpression of CSAG1 enhances colony-forming ability in cancer cells. These data suggest that CSAG1 supports cancer cell viability. Further studies will help elucidate how CSAG1 supports tumor cell fitness. Understanding the regulation and function of CTAs may provide new insight into novel cancer therapeutics.Item Identifying Novel Regulators of LIN28B Through a Genome-Wide CRISPR/Cas9 Screen(2017-01-17) Budhipramono, Albert; Hao, Zhu; Nguyen, LiemLIN28 is a family of RNA-binding proteins that are well-conserved across species. It is well-known to regulate developmental timing by inhibiting the biogenesis of the let-7 microRNAs. Numerous studies have shown that LIN28 is dysregulated in a wide spectrum of cancer types, especially pediatric cancers such as hepatoblastoma and Wilms' tumor. Our laboratory previously showed that reactivation of LIN28B, one of the two LIN28 homologs, is sufficient to drive liver cancer, and that LIN28B deletion is detrimental to tumor development. LIN28B exerts its oncogenic function by inhibiting the maturation of let-7 precursors, as well as directly binding to and enhancing translation of growth-promoting mRNA targets, such as members of the IGF2BP family. While our work and others established that LIN28B functions as an oncogene, the identity of factors that regulate LIN28B expression during normal development and cancer remains elusive. As LIN28B is a driver of oncogenesis in various cancers, understanding its regulation in the process of oncogenesis will help uncover novel therapeutic targets. Here, we show an original approach for identifying regulators of the human LIN28B gene utilizing the CRISPR/Cas9 genome engineering system. Traditional transgenic approaches to study gene function often fail to capture transcriptional regulation at distal promoter and enhancer sequences. Using the CRISPR/Cas9 system, we knocked a GFP reporter sequence into the endogenous locus of LIN28B in human cancer cell lines, engineering a fusion LIN28B-GFP protein. This approach is unique in that GFP expression will be altered not only by changes in regulation at the coding sequence, mRNA and protein levels, but also changes at distal regulatory sequences. To identify unknown regulators of LIN28B, we will perform a genome-wide CRISPR/Cas9-mediated knockout screen in human cells expressing the fusion LIN28B-GFP protein. Using a genome-scale library with 76,441 sgRNAs, we will knock out 19,114 genes individually and assess their effects on LIN28B levels by measuring GFP expression. sgRNAs that are enriched in the high GFP-expressing population suggest genes that normally function as inhibitors of LIN28B. On the other hand, sgRNAs that are depleted suggest activators of LIN28B. Through this screen, we hope to gain further insight into how LIN28B is regulated in normal development and cancer. Furthermore, identifying regulators of LIN28B can provide novel avenues for developing cancer therapeutics.Item The Role of Autophagy in Early Development and Tumor Suppression Using a Zebrafish Model System(2013-06-25) Lee, Eunmyong; Brugarolas, James B.; Abrams, John M.; Cleaver, Ondine; Levine, Beth; Amatruda, James F.Autophagy is an evolutionarily conserved lysosomal degradation pathway which involves the sequestration of cytoplasmic components into a double membraned structure called the autophagosome. By using genetically manipulated autophagy-deficient models, important roles for autophagy in development and tumorigenesis have been suggested. Genetic analyses indicate that autophagy is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryonic development. To address this question, the role of autophagy in early development was examined using zebrafish, a model system for studying vertebrate tissue and organ morphogenesis. Active autophagy was observed in multiple tissues during early embryonic development, as evidenced by the presence of autophagosomes in electron microscope images or GFP-LC3 puncta in autophagy reporter fish line Tg(cmvItem 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 [UT Southwestern Medical Center News](2013-02-06) Lyda, Alex