The advancement of cancer treatment depends on understanding the biological processes that contribute to disease progression. The spread of tumor cells from the primary site to distant organs is the biggest obstacle to efficacious treatment. The insulin receptor substrate (IRS) proteins IRS1 and IRS2 are cytoplasmic adaptor proteins that organize signaling events downstream of the Insulin receptor (IR) and the Insulin-like growth factor receptor 1 (IGF1R). Both of these receptors have been implicated in cancer progression. The IRS proteins share a significant level of homology and are both capable of recruiting and activating phosphatidylinositol-3 kinase (PI3K). Despite these similarities, signaling through IRS1 and IRS2 leads to distinct tumor cell outcomes in vitro and in vivo. In vitro, IRS1 regulates cell proliferation and growth and IRS2 regulates metabolism, survival and invasion. In vivo, Irs2 is a positive regulator of tumor metastasis, whereas Irs1 does not promote metastasis. The major objective of this thesis work was to further the understanding of the mechanism by which IRS2 signaling regulates tumor progression. To investigate how IRS-1 and IRS-2 regulate distinct tumor cell outcomes, I examined the involvement of the microtubule cytoskeleton in IRS-dependent signaling. I determined that IRS2-mediated AKT activation is dependent upon an intact microtubule cytoskeleton, whereas IRS1-mediated AKT signaling occurs independently of microtubules. As a result, drugs that disrupt microtubules promote apoptosis in cells that signal through IRS2, but cells that signal through IRS1 are resistant to the effects of microtubule disruption. However, AKT inhibition sensitizes IRS1-dependent cells to apoptotic cell death upon microtubule disruption. From a clinical perspective, my studies identify IRS2 as a potential biomarker for the response of breast cancer patients to anti-microtubule drug therapy. To investigate further the mechanism of IRS2 contributions to tumor progression, I employed a mutagenesis approach to identify structural requirements of IRS2 for its function. I established that the ability of IRS2 to activate PI3K is necessary for its regulation of both invasion and tumor initiating cell (TIC) self-renewal. I also identified two independent regions within the IRS2 C-terminus that are required for invasion and self-renewal, respectively. Characterization of the invasion-promoting region identified BMP2-induced protein kinase (BMP2K) as an interacting protein. Suppression of BMP2K expression in mammary tumor cells disrupts IRS2-mediated tumor cell invasion. Taken together, my work advances the understanding of how IRS2 contributes to breast cancer progression and provides a molecular understanding for the development of novel approaches for the treatment of breast cancer and other malignancies that rely upon IRS2.

<p>Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. Previous studies have indicated that many of these changes are dependent on the activity of transcription factors belonging to the AP-1, STAT and Ets families. This study took advantage of transformation-deficient and temperature sensitive mutants of the Rous sarcoma virus to characterize transformation-induced patterns of gene expression of two primary cell types, chicken embryo fibroblasts (CEF) and chicken neuroretina (CNR). In these cells, v-Src alters the expression of up to 6% of the protein coding genes. Comparison of this program with independent breast carcinoma data sets identified a group of 42 v-Src inducible genes associated with reduced disease-free survival. Pathway and ontological analyses of the genes differentially regulated by transformation in CEF and CNR indicated a generalized program of de-differentiation induced by Src.</p> <p>To investigate the role of AP-1 inSrc-mediated transformation, a gene profiling study was conducted to characterize the transcriptomes of v-Src-transformed CEF expressing the <em>Jun</em> dominant-negative allele or the <em>JunD</em> short-hairpin RNA (shRNA). Microarray data analysis indicated a cluster of 18 co-regulated probe-sets activated in v-Src-transformed CEF with repressed AP-1 activity but not activated in normal CEF or CEF transformed only by v-Src. One gene, <em>death-associated protein kinase 1</em> (<em>DAPK1</em>), is a C/EBPβ-regulated mediator of apoptosis in IFN-γ-induced cell death. Inhibition of DAPK1 abrogated cell-death in v-Src-transformed CEF expressing the JunD shRNA and expression of DAPK1 was dependent on C/EBPβ but antagonized by AP-1. Chromatin immunoprecipitation indicated that C/EBPβ, but not JunD, is recruited to the DAPK1 promoter. In conclusion, JunD promotes survival by indirectly antagonizing C/EBPβ-dependent expression of DAPK1.</p> / Doctor of Philosophy (PhD)

<p>In the recent years considerable progress has been made to understand how the protein Bcl-2 regulates apoptosis at the mitochondria. By comparison, the cell death mechanisms at the endoplasmic reticulum remain unclear. In response to the agents that cause endoplasmic reticulum stress in breast cancer cells, the cell-cell adhesion molecule E-cadherin is modified by two independent modifications comprising pro-region retention and O-glycosylation. Both the modifications on E-cadherin inhibit its cell surface transport and the resultant loss of E-cadherin on the plasma membrane sensitizes cells to apoptosis. During this process binding of E-cadherin to type I gamma phosphatidylinositol phosphate kinase (PIPKIγ), a protein required for E-cadherin trafficking to the plasma membrane is prevented by O-glycosylation. E-cadherin deletion mutants that cannot be O-GlcNAcylated continue to bind PIPKIγ, traffick to the cell surface and delay apoptosis, confirming the biological significance of the modifications and PIPKIγ binding in the cell death regulation. These results also led me to determine whether there is a cell death pathway in which commitment to cell death is mediated by proteins primarily located at the endoplasmic reticulum. The studies show that the growth of estrogen receptor-positive breast cancer cells in charcoal stripped bovine serum leads to a form of programmed cell death which is protected by Bcl-2 exclusively localized at the endoplasmic reticulum instead of the mitochondria. Interestingly, the BH3 mimetic ABT-737 can abolish the protection mediated by Bcl-2 localized at the endoplasmic reticulum. Taken together, these studies suggest the novel role of the endoplasmic reticulum in programmed cell death through the identification and elucidation of the mechanisms that regulate the cell death pathway at this organelle.</p> / Doctor of Philosophy (PhD)

The development of several organ systems through modeling and shaping of the tissue structure occurs from signaling through axon guidance molecules. The Slit family of ligands has been shown to regulate branching morphogenesis in mammary gland duct development and loss of Slit gene expression during this time leads to the formation of hyperplastic, disorganized lesions suggesting a potential role for Slits in cancer formation. Characterization of human pancreatic ductal adenocarcinoma cell lines showed a loss of SLIT2 expression in cells that contain activated Kras. Loss of SLIT2 expression was associated with DNA methylation of CpG sites within the SLIT2 core promoter and chromatin enrichment of repressive histone modifications at the SLIT2 transcriptional start site. Additionally, treatment of pancreatic ductal adenocarcinoma cell lines with demethylating agent 5-aza-2'-deoxycytidine led to SLIT2 re-expression while treatment with histone deacetylase inhibitor Trichostatin A did not. Mir-218-1 is an intronic microRNA encoded within intron 15 of the SLIT2 gene. Expression of mir-218-1 does not correlate with SLIT2 mRNA expression suggesting that it is transcribed from a promoter independent of the SLIT2 gene promoter. Pancreatic ductal adenocarcinoma cell lines showed a peak of H3K4me3 chromatin enrichment localized to a 1kb region within intron 4 of the SLIT2 gene denoting a candidate alternative promoter for mir-218-1. A concordant peak of H4ac chromatin enrichment overlapped the peak of H3K4me3 enrichment and transcriptional activity was measured from the 1kb region in all pancreatic ductal adenocarcinoma cell lines. A NF-κB binding site was also predicted to exist within the 1kb region. Transfection with two independent siRNAs to NF-κB led to an increase in both pre-mir-218-1 and mature mir-218-1 while treatment with an inhibitor to IκB kinase led to an increase in pre-mir-218-1 expression. Additionally, the p65 subunit of NF-κB was found to bind to the candidate mir-218-1 alternative promoter in pancreatic ductal adenocarcinoma cell lines that do not contain DNA CpG methylation at the predicted NF-κB binding site. It was discovered that miR-218 is a modulator of ARF6 expression suggesting a role in the inhibition of pancreatic ductal adenocarcinoma cell invasion through modulation of the actin cytoskeleton. Overexpression with a miR-218 precursor showed that miR-218 is an inhibitor of pancreatic ductal adenocarcinoma cell invasion in two dimensions. Additionally, it was found that while miR-218 does not have an affect on the ability of pancreatic ductal adenocarcinoma cells to form functional invadopodia, miR-218 is an inhibitor of the extracellular matrix degradation properties of mature invadopodia. Interestingly, the effect of miR-218 on pancreatic ductal adenocarcinoma cell invasion or extracellular matrix degradation is not reliant on the cell's dependency on Kras signaling for growth and survival. Collectively, these observations indicate that understanding the transcriptional regulation of SLIT2 and mir-218-1 expression as well as their signaling properties may provide a step toward the development of diagnostic tests and therapeutic treatments for patients with invasive or metastatic pancreatic ductal adenocarcinoma.

Integrin α6β4 is upregulated in pancreatic carcinoma, where signaling promotes metastatic properties, in part by altering the transcriptome. Such alterations can be accomplished through DNA demethylation of specific promoters, as seen with the pro-metastatic gene S100A4. I found that signaling from integrin α6β4 dramatically upregulates expression of amphiregulin (AREG) and epiregulin (EREG), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if AREG and EREG are regulated by DNA methylation, pancreatic cancer cells with low AREG and EREG expression were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR), resulting in stable overexpression of AREG and EREG, and this induction required signaling from integrin α6β4. Similarly, treatment of cells with high integrin α6β4 with the methyl donor S-adenosylmethionine inhibited gene expression of AREG and EREG. Whole genome bisulfite sequencing on pancreatic cancer cells reveled hypomethylation of the promoter regions of AREG and EREG when integrin α6β4 is high, and these regions correspond to H3K27Ac, indicative of enhancer location. Interestingly, I also observed genome-wide DNA demethylation, and a large proportion of altered CpGs correspond to potential enhancers. It is currently accepted that active DNA demethylation occurs via DNA repair. I tested this hypothesis by treating cells with Gemcitabine, which inhibits multiple components of DNA repair, including DNA demethylation mediated by GADD45A. Gemcitabine treatment resulted in marked reduction in AREG and EREG expression. To further test the involvement of GADD45A, I used RNAi-mediated knockdown or cDNA overexpression to alter GADD45A levels. In both instances, AREG and EREG expression positively correlated with GADD45A, particularly when integrin α6β4 is high, indicating that GADD45A is a rate-limiting step in AREG and EREG overexpression. Similarly, using stable shRNA, I show that Thymine DNA Glycosylase (TDG), and TET1 known modulators of DNA demethylation, are required for AREG and EREG expression in integrin α6β4 high cells, and nuclear localization of TDG is much higher in cells with high integrin α6β4. Using a specific inhibitor I found that AREG and EREG expression is dependent on Parp-1. Finally, I determined that integrin α6β4 signaling enhances cells ability to respond to and survive in the presence of DNA damage, and that active DNA repair is required for integrin α6β4 mediated DNA demethylation. Taken together, these data indicate that DNA repair is required to maintain overexpression of AREG and EREG in response to signaling from integrin α6β4 and that integrin α6β4 promotes this overexpression by enhancing DNA repair.

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.

The field of prostate cancer research is in need of biological markers that predict which cancers do not need treatment, those that can be treated successfully with a localized treatment and more specific cases in which patients are likely to have an aggressive form of cancer that will require more aggressive surgical and chemotherapeutic treatments. ZIC2 is one of five members of a family of proteins that play critical roles in neural crest and mesoderm growth in normal embryonic brain development and in the adult cerebellum of vertebrates. Found throughout the animal kingdom, ZIC1-5 genes encode five distinct ZIC proteins containing five highly conserved C2H2-type zinc finger motifs whose structural integrity is important in carrying out its function as a transcription factor. We hypothesize that ZIC2 has functional significance at the molecular and cellular levels in the initiation of prostate adenocarcinoma (PRAD) and the progression to metastatic and/or castration resistant prostate cancer (CRPC). Bioinformatic predictions suggest that the function of ZIC2 is regulated by post-translational modifications, such as phosphorylation, ubiquitination and sumoylation. This proposal further outlines the research hypothesis for investigating the role of ZIC2 in prostate cancer progression and the effects of the post-translational modification, ubiquitination, on the loss or gain of function of ZIC2.

Breast cancer is the second most commonly diagnosed cancer among American women and is responsible for the second highest number of cancer-related deaths. Targeted therapeutic agents sildenafil, a phosphodiesterase type 5 inhibitor, and celecoxib, a cyclooxygenase-2 inhibitor, have been used individually in conjunction with other chemotherapeutic agents to enhance cell killing in a variety of cancers. Sildenafil when combined with traditional chemotherapeutic drugs, such as the taxanes and anthracyclines, or celecoxib combined with traditional hormone therapies have been used to increase cytotoxicity and cell killing. The data presented here demonstrates that the novel combination of sildenafil and celecoxib work together to enhance cell killing in both receptor positive and triple negative breast cancer through the induction of autophagy, ER stress, as well as both intrinsic and extrinsic apoptosis.

This study shows for the first time the effect that L-(+)-lactic acid has on mast cell activation. Lactic acid is a byproduct of anaerobic glycolysis and is associated with inflammatory environments such as wounds, tumors and, asthma. In this study, pre-treatment with lactic acid altered cytokine production by bone marrow-derived mast cells (BMMC). Specifically, lactic acid enhanced cytokine secretion following IgE cross-linking, but decreased IL-33 mediated cytokine production. These effects were altered by genetic background, since C57BL/6 mast cells demonstrated the aforementioned result, but lactic acid had no effect on IgE-mediated cytokine production in 129/SvJ mast cells. The affected cytokines included IL-6, TNF, MCP-1, MIP-1α, IL-13, and VEGF. Lactic acid pretreatment promoted a G0/G1 cell cycle arrest. Investigation into the IL-33 signaling pathway showed lactic acid decreased TAK1 and JNK phosphorylation, while increasing phosphorylated AKT levels. Blocking JNK and TAK1 with a small molecule inhibitor mimicked the effects of lactic acid. Interestingly, lactic acid significantly increased IL-33 mediated VEGF. An in vitro angiogenesis assay confirmed that mast cells were pro-angiogenic in a lactic acid-rich environment. Taken together, these data show that lactic acid impacts mast cell function, possibly promoting a pro-angiogenic, anti-inflammatory phenotype.

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS’s cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2-and peroxynitrite (ONOO-). Previous work in our and other’s labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein we show that recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We also see increased tumor cell death measured by in vitro clonogenic assay, as well as decreased metabolic uptake in Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) induced colorectal cancer in vivo measured by [18F]-fluorodeoxyglucose ([18F]-FDG) positron emitted topography (PET) imaging. We believe by recoupling NOS both in vivo and in vitro we are modulating Wnt signaling via Akt and GSK-3β. Lastly, we conducted studies to determine a mechanistic explanation of how tumor cells maintain a decreased BH4:BH2 ratio.