Browsing by Subject "Cell Transformation, Neoplastic"
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Item The Art of Viral Oncogenesis: Lessons from Human Papillomavirus and Polyomavirus Transformed Cancers(2020-05-01T05:00:00.000Z) Zhao, Jiawei; Xu, Jian; Banaszynski, Laura; Pfeiffer, Julie K.; Wang, RichardViruses account for about 15% of all human cancer. Understanding viral oncogenesis can substantially broaden our general knowledge on the molecular mechanisms of carcinogenesis. In this dissertation, I focused on two types of DNA oncoviruses, human papillomavirus (HPV) and polyomavirus (HPyV), and identified novel mechanisms by which these two types of viruses cause human cancers. In HPV transformed cancer cells, I identified a novel circular RNA species, circE7, that spans and encodes the HPV E7 oncoprotein. I later demonstrated that circE7 translated E7 protein accounts for a substantial proportion of the E7 protein in a HPV transformed cancer cell line, and whose absence significantly impacts cancer cell proliferation in vitro and in vivo. In Merkel Cell Polyomavirus (MCPyV) transformed MCC cancer cells, I identified the activation of non-canonical NF-κB pathway activation by the MCPyV small T (ST) oncoprotein. I further demonstrated that the ectopically activated non-canonical NF-κB pathway is required for cell growth in low serum. The inhibition of non-canonical NF-κB signaling by a small peptide inhibitor also resulted in impaired cell growth in vitro and in vivo due to ER stress mediated apoptosis, suggesting a novel therapeutic intervention strategy for viral positive MCC (VP-CC) patients.Item Characterization of the Role of the PP2A-AB Gene, a Putative Tumor Suppressor, in Cell Growth and Tumorigenesis(2005-05-11) Esplin, Edward D.; Mumby, Marc C.The PP2A-Aβ gene (PPP2R1B) encodes the β isoform of the A subunit of serine/threonine protein phosphatase 2A. Mutations in PP2A-Aβ have been identified in a wide variety of human cancers. The important role of protein phosphatase 2A in down regulating cell growth suggests these mutations may contribute to cancer susceptibility and tumorigenesis by compromising the function of PP2A-Aβ and that PP2A-Aβ may act as a tumor suppressor. Screening of cancer patient DNAs revealed an association between a germline alteration of the PP2A-Aβ and breast carcinoma and identified alterations of PP2A-Aβ in lung carcinoma and ALL patient genomic DNAs. The biochemical consequences of these PP2A-Aβ mutations on PP2A function were investigated by in vitro and in vivo coimmunoprecipitations between the PP2A-Aβ subunit and the B and C subunits of PP2A. These studies showed mutations in PP2A-Aβ confer a loss of function by reducing its ability to bind the B and C subunits, destabilizing the PP2A-Aβ containing PP2A complex. The affect of the PP2A-Aβ gene on cell growth was analyzed by transfecting the PP2A-Aβ gene into cancer cell line deficient for wild type PP2A-Aβ and deriving stable cell lines. The PP2A-Aβ gene appeared to confer a relative disadvantage to transfected cells, resulting in a lower fraction of derived stable lines compared to controls. These cell lines were tested for proliferation and colony formation in soft agar. No significant difference was observed in the growth rate of PP2A-Aβ cell lines compared to controls. One of the PP2A-Aβ stable cell lines demonstrated dramatic suppression of colony formation in soft agar, but this was not confirmed in any additional PP2A-Aβ stable cell lines, leaving this finding inconclusive. The stable cell lines were also analyzed by Western blotting for changes in the Wnt signaling cascade. Cell lines expressing exogenous PP2A-Aβ are found to have lower levels of β-catenin compared to control cell lines. This suggests that the PP2A-Aβ gene is involved in regulating the Wnt signaling pathway, which is shown to be involved in cell growth control and is similarly affected by known tumor suppressor genes.Item Genetic and Molecular Studies of Endometrial Cancer(2010-11-02) Akbay, Esra; Castrillon, Diego H.Endometrial cancer, which develops from the inner lining of the uterus, is the most common cancer of the female reproductive tract. Endometrial cancer is comprised of two epidemiologic, molecularly and genetically different subtypes known as type I and type II. Because of its clinical significance and aggressive behavior, my research focused on type II endometrial cancer. Type II endometrial cancer is associated with advanced age and TP53 mutations, which suggests a link between telomeres and the development of type II tumors. Telomeres and other markers of telomere status in type I and type II tumors were analyzed, short telomeres are observed in both tumor types. However, only type II tumors are associated with critical telomere shortening in the adjacent, morphologically normal epithelium. This suggests that telomere shortening contributes to the initiation of type II, but not of type I tumors. To further explore this hypothesis, 5th generation telomerase knockout mice with critically short telomeres were analyzed and a distinctive endometrial lesion that resembles the in situ precursor of type II carcinomas, endometrial intraepithelial carcinomas were observed. Subsequently, the role of dysfunctional telomeres in endometrial cancer development was investigated in the mouse by conditionally inactivating Pot1a, a key component of the shelterin complex that stabilizes telomeres in endometrial epithelium. Inactivation of Pot1a by itself does not stimulate endometrial carcinogenesis. However, simultaneous inactivation of Pot1a and p53 results in endometrial intraepithelial carcinoma-like lesions by 9 months and metastatic tumors in 100% of the animals by 15 months. These tumors are poorly differentiated endometrial carcinomas with prominent nuclear atypia, DNA damage, and aneuploidy, resembling human type II tumors. These studies thus lend support to the hypothesis that dysfunctional telomeres play a critical role in type II endometrial carcinogenesis.Item Investigating the Effects of Radiation Exposure on Lung Carcinogenesis(2014-07-10) Kaisani, Aadil A.; Scaglioni, Pier Paolo; Shay, Jerry W.; Story, Michael; Mendelson, Carole R.Although it is the second most diagnosed cancer in both males and females, lung cancer accounts for the most cancer-related deaths worldwide. Radiation-induced carcinogenesis is a major concern for therapeutic radiation oncology as well as for astronauts on long-term missions. The large surface area of the lung makes it a prominent target for radiation exposure. From analysis of several human populations such as atomic bomb survivors, it is evident that the lung is remarkably susceptible to radiation-induced cancer. However, this data cannot be extrapolated to assess the risk in astronauts because of differences between terrestrial and space radiation. Due to lack of understanding of how the ionizing space radiation affects cellular functions and carcinogenesis, there is a need to develop in vitro and in vivo systems. Using immortalized human bronchial epithelial cells (HBECs) we have developed a novel three-dimensional (3D) culture system. When cultured on top of reconstituted basement membrane with lung fetal fibroblasts cultured below, HBECs form budding and branching structures resembling branching morphogenesis during lung development. HBECs in this culture system maintain markers of multiple cell types in the airway epithelium indicative of their multipotent potential. Studies to determine the effects of radiation on HBECs in 3D cultures are ongoing. Radiation-induced carcinogenesis may require changes to the immune system and tumor microenvironment therefore we utilized the LA1 K-ras mouse model to analyze the effects of terrestrial and space radiation. Mice irradiated with simulated solar particle events (SPE) and silicon particle radiation (types of radiation in space) had a significantly shorter lifespan in contrast to unirradiated cohorts or mice irradiated with equivalent terrestrial radiation. A significant increase in invasive carcinoma was also observed in mice irradiated with SPE and silicon particle radiation but not with terrestrial radiation. Analysis of mice 70 days post-irradiation suggests that chronic inflammation is a likely contributor to tumor progression. CDDO-EA an anti-oxidant/anti-inflammatory modulator was tested as a potential radio-protector. A significant decrease in carcinoma was observed in mice fed a CDDO-EA countermeasure diet. These observations suggest that the lung is highly susceptible to carcinogenesis upon irradiation and countermeasure compounds may mitigate this outcome.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 by ERK1/2 of Novel Substrates, Kinesins KIF2A and KIF2C(2013-04-15) Zaganjor, Elma 1981-; Albanesi, Joseph P.; Cobb, Melanie H.; White, Michael A.; Fontoura, BeatrizThe kinesin-like protein KIF2A is a microtubule-associated motor protein thatauses microtubule depolymerization by inducing a conformational change in tubulin. The depolymerase function of KIF2A is utilized in mitotic cells as it is required to establish proper, bipolar spindles. Studies in KIF2A knockout mice revealed KIF2A function in regulation of interphase microtubules as KIF2A-/- neurons exhibit abnormal axon branching. Though protein kinases are known to regulate the mitotic function of KIF2A, how KIF2A is regulated in interphase cells has not been studied. In a yeast-two hybrid screen, designed to preferentially uncover interactors of the active form of ERK2, we identified KIF2A. We find that, human KIF2A can be v phosphorylated in vitro by pERK1/2 and that the kinases interact with KIF2A in cells. Through phosphorylation prediction tools and mutagenesis we identified threonine 78 (T78) as a major pERK2 phosphorylation site on KIF2A. Inhibition of ERK1/2 prevents KIF2A from localizing at the leading edge of cells. Additionally, knockdown of KIF2A phenocopies the effect of inhibiting ERK1/2 on microtubules; both treatments result in elongated microtubules. These data suggest that ERK1/2 may regulate KIF2A localization which in turn may be important for KIF2A function in depolymerizing microtubules. The close relative, KIF2C can also bind to and be phosphorylated by pERK1/2 in vitro, but the functional significance of this event remains unknown. In a laboratory generated non-small cell lung cancer (NSCLC) in which oncogenic K-RasG12V has been overexpressed and the tumor suppressor p53 has been knocked down, we found an increase in expression of KIF2A and KIF2C. This increase could be suppressed by inhibiton of the effector pathway RAS-RAF-MEK1 but not the PI3K pathway. As it is accepted that cancer cells have more dynamic microtubules that give them a migratory advantage, we hypothesized that upregulation of KIF2A and KIF2C also promote migration. Indeed, knockdown of KIF2A and KIF2C resulted in reduced migration in cancer cell lines. Microarray studies that had been performed on lung cancer lines revealed upregulation of KIF2A and KIF2C in cancers, suggesting that these proteins may be significant factors in the development of lung cancer. Finally, KIF2A and KIF2C regulate lysosomal dynamics. This regulation has an impact on signaling, particularly for mTORC1 which requires lysosomal localization for its activity.Item The Role of LKBI Tumor Suppressor in Endometrial Cancer(2009-09-04) Contreras, Cristina Maria; Castrillon, Diego H.The LKB1 gene contributes to endometrial carcinogenesis in both the mouse and human. Although this role was not anticipated based on the tumor spectrum observed in Peutz Jegher women, many tumor suppressors have been found to have a much broader role in cancer than would have been predicted on the tumor spectrum associated with the hereditary syndrome. The studies that comprise this dissertation work first demonstrate that in addition to gastrointestinal polyposis, mice heterozygous for LKB1 develop endometrial cancers. To develop a better model of LKB1-driven endometrial carcinogenesis not prone to death from GI obstruction, two model systems were developed based on conditional ablation of the LKB1. In the first, intrauterine delivery of a CRE-expressing adenovirus was employed. A striking 65% of mice in this system, developed highly invasive endometrial tumors by 9 months. Although this system allowed the demonstration and exploration of the role of LKB1 as an endometrial tumor suppressor, the model suffered from technical limitations stemming from its recombination inefficiency. Then, in collaboration, a much more efficient transgenic model was developed using the promoter region of SPRR2F to drive CRE expression specifically in the endometrial epithelium. With this model, female mice with homozygous endometrial LKB1 inactivation developed diffuse malignant transformation of their entire endometrium with rapid extrauterine spread and death, by 7 months, suggesting that LKB1 inactivation was sufficient to promote the development of invasive endometrial cancer. This much improved model also permitted the exploration of theory that LKB1-deficient tumors should be hypersensitive to rapamycin. Collectively these studies demonstrate that LKB1 is a uniquely potent endometrial tumor suppressor but also suggest that clinical responses to mTOR inhibitors may be linked to LKB1 inactivation.Item The Role of Polyploidy in the Liver and Its Implications for Cancer Therapy(2018-04-13) Zhang, Shuyuan; O'Donnell, Kathryn A.; Zhu, Hao; Brugarolas, James B.; Yu, HongtaoThe description of liver polyploidy dates back to the 1940s, but its functional roles are still largely unknown. Numerous observations and studies have suggested that liver polyploidy may participate in multiple biological processes, including regeneration, stress response, and cancer. However, little evidence has established direct causal links between polyploidy and the observed phenotypes, mainly due to the lack of appropriate tools to specifically manipulate ploidy levels without causing other permanent changes. Specifically, whether polyploidy promotes or inhibits cancer is still under debate. Inspired by a phenomenon we observed in somatically mutated mouse livers, where homozygous Apc deletions were more difficult to obtain due to hepatic polyploidy, we aimed to build inducible tools to manipulate liver ploidy levels in vivo and systematically study the role of polyploidy in liver cancer. By toggling the weaning time and levels of Anln or E2f8 genes to change liver ploidy levels, we found that liver tumorigenesis was inversely correlated with initial polyploidy levels, suggesting a tumor suppressive role for polyploidy. Moreover, the additional alleles in polyploid cells led to a reduced likelihood of loss of heterozygosity (LOH), which largely contributed to the tumor suppressive effect. These results revealed an important function of polyploidy in mammalian livers and also led us to seek related therapeutic strategies for treating liver cancer. Since hepatocyte polyploidization mainly occurs through cytokinesis failure, we hypothesized that inhibiting cytokinesis could be an effective strategy to suppress liver tumorigenesis while preserving normal liver function. Therefore, we inhibited cytokinesis via Anln knockdown in multiple models and found that liver tumor development was significantly suppressed but normal liver function and regeneration capacity were not impaired. These results suggest that cytokinesis inhibition via Anln knockdown is potentially a safe and efficacious strategy for suppressing liver cancer. Overall, we uncovered an important role of polyploidy in the liver and explored its potential applications in liver cancer therapy.Item [UT News](1987-04-01) Rutherford, Susan