Browsing by Subject "PI3K"

Regulation of cell growth is controlled by a variety of factors, including a number of oncogenes and tumor suppressors. PTEN is an inositol phosphatase that regulates cell growth by hydrolyzing the phospholipid products of PI3K. PTEN is mutated in a number of cancers, leading to its characterization as an important tumor suppressor. Recent data indicate that PTEN may also perform important functions that are independent of its phosphatase activity, most notably within the nucleus. Studies in this thesis addressed a novel role for PTEN in the regulation of the cellular response to hypoxia.

PTEN overexpression significantly increased hypoxic gene expression independent of its catalytic activity, while shRNA-mediated silencing of PTEN significantly inhibited hypoxia-mediated HRE-luciferase activity. Nuclear-localized PTEN was more effective in promoting HRE activity than nuclear-excluded PTEN. These results suggested a scaffolding function of PTEN in the hypoxic nucleus. To identify specific gene targets regulated by PTEN in hypoxia, a custom oligo-array consisting of 46 hypoxia-responsive genes was utilized following both gain- and loss-of- PTEN function. Based on real-time quantitative results, PTEN positively regulated genes involved in metabolism (PFKFB3, PFKFB4, ALDOA, PGK-1), oxygen supply (VEGFA, EPO), cell growth (Tgf-a, TERT, cyclin D1, BNIP3), motility (E-cadherin) and transcription (DEC2). A single missense mutation at isoleucine 224 (I224M) of PTEN, however, abrogated the ability of PTEN to regulate the hypoxia response without affecting its lipid phosphatase activity. PTEN has previously been shown to bind to the co-activator p300 and to affect p53 acetylation and stabilization. As p300 is also a co-activator for the HIF proteins, we hypothesized that PTEN's association with p300 would promote the HIF/p300 complex to positively regulate hypoxic gene transcription. Overexpression of PTEN-WT extended the half-life of p300 and histone acetyltransferase activity of p300 in hypoxia, while overexpression of PTEN-I224M or PTEN silencing decreased both. In vivo, these effects resulted in a significant increase in hypoxic area in PTEN-null tumors compared to tumors expressing endogenous levels of PTEN, suggesting an inability to mount a hypoxia response necessary for revascularization of the tissue. PTEN's effect on p300 extended to other functions of p300 outside of the hypoxia response, most notably p300's role in p53 stability and p53-mediated gene transcription. Overexpression of PTEN resulted in an increase in p53 reporter activity following DNA damage (mitomycin C treatment). PTEN silencing or overexpression of PTEN-I224M resulted in abrogation of these effects. Taken together, these findings demonstrate that PTEN is required for the hypoxia response and they suggest that PTEN acts as a scaffold for p300 and the HIF machinery in the hypoxic nucleus independent of its canonical lipid phosphatase activity. These results may have important implications for the treatment of tumors in which PTEN is lost or mutated. The potential use of PTEN-I224M as a therapeutic is also discussed

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 95% of the adult population. While infection is typically asymptomatic, in some individuals, particularly the immunocompromised, EBV is the causative agent of several cancers including lymphomas and epithelial cancers. Specifically, gastric carcinoma, nasopharyngeal carcinoma, and lymphoepithelioma-like carcinoma which occurs across multiple sites in the body, most notably, in the lung. EBV-associated gastric cancer (EBVaGC) is a unique subset of gastric cancer that makes up 10% of all GCs worldwide. EBVaGCs display an 80% rate of activating PIK3CA mutation and are also the most hypermethylated of any tumor type, displaying what is known as a CpG island hypermethylator phenotype (CIMP). EBV infection of B cells can be easily modeled in vitro using primary B cells and lymphoblastoid cell lines (LCLs). However, epithelial cell infection models have proven much more difficult to develop. Given these difficulties, EBV infection and outgrowth in epithelial cells is comparatively understudied and the process of tumorigenesis in vivo is poorly understood. In this dissertation, I developed methods to generate EBV infected epithelial cell lines derived from both gastric cancer and lung adenocarcinoma using diverse strains of EBV. I used these models to complete a CRISPR/Cas9 whole genome knockout screen to identify cellular restriction factors of infection and outgrowth. Together, these data will provide novel insights into the process of EBV infection and the dynamic interplay between virus and host during tumorigenesis. Furthermore, I have used these models to explore unique therapeutic approaches for EBV+ epithelial cancers. Specifically, I have characterized the lytic reactivation potential to histone deacetylase (HDAC) inhibitors and generated preclinical data supporting the use of HDAC inhibitors and the anti-viral ganciclovir for treatment of EBV+ epithelial tumors. Lastly, I have identified modulators of the response to a PI3Ka inhibitor in PIK3CA mutant gastric cancers. I found that loss of NEDD9 or inhibition of BCL-XL rendered cells hyper-sensitive to the PI3Ka inhibitor BYL719. Additionally, I found that loss of CBFB conferred resistance to BYL719 through up- regulation of the protein kinase PIM1 and defined the clinical utility of our data in the context of PI3K inhibition more broadly. The work outlined in this dissertation contributes to the study of EBV infection and tumorigenesis in the stomach as well as provides mechanistic insights into novel therapeutic approaches for EBV+ epithelial cancers and PIK3CA mutant gastric cancers.

Chlamydia trachomatis is a bacterial pathogen with a large socioeconomic impact: it is the leading cause of preventable blindness worldwide, and the most prevalent sexually transmitted infection in the United States. Despite its importance, relatively little is known about the molecular mechanisms that Chlamydia employs to invade epithelial cells, manipulate the secretory pathway, evade innate immune responses and acquire nutrients from its host. Chlamydia, like many other intracellular pathogens, is known to use a type III secretion mechanism to deliver bacterial effector proteins directly to the host cell cytoplasm. These effectors are thought to be the principle actors involved in co-opting host cell functions. TepP is an effector protein that is pre-loaded into infectious Chlamydia particles, and that is secreted early during infection, but whose function is unknown. We took large-scale, unbiased approaches to identify genes whose transcription is modified during the course of infection in a TepP-dependent manner (microarrays), and proteins that interact with TepP and and/or whose phosphorylation is altered by the absence of TepP (proteomics). We used biochemical techniques, cell biology, and molecular techniques to validate interactions identified using large-scale methods, and to further probe the molecular mechanism underlying these connections. In sum, we have determined that TepP contributes to four major phenotypes: changes in the host cell cytoskeleton, modification of the host cell phosphoproteome, bacterial replication, and interferon-dependent gene activation. We have additionally determined that TepP interacts with the Crk family of host cell adaptor proteins, and the class 1 phosphoinositol-3-kinase (PI3K). Cell lines where the levels or activity of TepP interacting partners were modified by deletion, knockdown, or inhibitors, showed that these host proteins are important for the growth of Chlamydia during infection, but are not required for all TepP-dependent phenotypes. TepP not only interacts with PI3K but also induces its activation during infection. Finally, we have determined that the requirements for phosphorylation of TepP are complex, but that the Src kinases are largely responsible for its phosphorylation. Additionally, Src kinases are required for some TepP-dependent phenotypes, but are not required for the recruitment of TepP-interacting proteins during infection.

The homeostasis of naïve T lymphocytes is maintained by several mechanisms involving basal TCR and cytokine signaling, and nutrient factors. One of the common net results of these input signals is the production and stabilization of anti-apoptotic Bcl-2 family members. A second result of these processes is the induction of autophagy, an intracellular, catabolic, lysosomal targeting pathway. Autophagy induction in most systems involves the class III phosphatidylinositol-3 kinase (PI3K), Vps34, to produce phosphatidylinositol-3-phosphate (PI(3)P). To test this in T lymphocytes, I generated mice specifically lacking Vps34 in T cells (Vps34f/fLck-cre mice). However, Vps34-deficient T lymphocytes have normal levels of basal autophagy, and upregulate autophagy normally in response to cytokine or nutrient withdrawal, or TCR stimulation. Therefore I conclude that Vps34 activity is not required for autophagy induction in T lymphocytes. T lymphocytes lacking Vps34 do have enhanced rates of apoptosis, but this is due to defects in intracellular trafficking, specifically of the Interleukin-7 receptor alpha subunit (IL-7Rα). Additionally, multivesicular body (MVB) maturation is impaired in T cells lacking Vps34 such that extracellular ligands are not efficiently targeted to the lysosome.

Autophagy induction in Vps34-deficient T lymphocytes is still sensitive to pan-PI3K inhibitors, such as wortmannin and 3-methyladenine (3MA). Therefore, I hypothesized that other classes of PI3K are necessary to induce autophagy in T lymphocytes through the production of PI(3)P. Autophagy induction is sensitive to specific class I PI3K (PI3KI) inhibitors, such as PIK75. Additionally, T cells lacking the p85 regulatory subunit of PI3KI also have severe defects in T cell receptor (TCR) mediated autophagy induction. PI3KI activity results in the production of PI(3,4,5)P3, though, and not PI(3)P. Because of this specificity, I hypothesize that additional inositol polyphosphatases (Inpp) are required for autophagy induction downstream of PI3KI activity. Indeed, utilizing both inhibitors of pharmacological inhibition and siRNA-mediated knockdown of two classes of phosphatidylinositol phosphatases, inositol polyphosphate-4-phosphatase (Inpp4) and SH2 containing inositol phosphatase (SHIP), had dramatic impacts on autophagy induction. Furthermore, exogenous addition of PI(3,4)P2, a hypothesized intermediate in this pathway, positively regulates autophagy induction and leads to enhanced progression of autophagy. These observations indicate that PI3KI activity, linked to Inpp activity, are necessary and positive regulators of autophagy through the production of PI(3)P.