Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

My thesis research began with a project in which we were trying to determine the function of embryonic stem cell (ESC)-specific miRNAs. Using luciferase constructs containing miRNA binding sites, luciferase expression was inhibited by endogenous miRNAs in ESCs, and by exogenous miRNAs in HeLa cells. Inhibition of luciferase expression by miRNAs was inhibited in HeLa cells using 2’O-methyl-oligonucleotides. In ESCs, 2’O-methyl-oligonucleotides were only effective in partially inhibiting miR290 function. Partial inhibition of miR290 did not result in any obvious phenotypic changes in mESCs. Later studies using 2’O-methyl-oligonucleotides in ESCs were also unsuccessful. The function of ESC-specific miRNAs has since been studied by re-introducing miRNAs into Dicer -/- cells which cannot make miRNAs. These studies have shown that ESC-specific miRNAs are involved in de novo DNA methylation, self-renewal, and cell-cycle regulation. Newly diagnosed glioblastoma (GBM) patients rarely survive more than two years even after surgery, radiotherapy, and chemotherapy using temozolomide (TMZ) or 1,3-bis(2-chloroethy)-1-nitrosourea (BCNU). Eventual regrowth of the tumor indicates that some tumor cells are resistant to therapy. GBM neurosphere-initiating cells (NICs) are thought to be similar to tumor-initiating cells in vivo, and will form invasive tumors in mice, making neurosphere cultures a good model system for studying GBMs. To test whether GBM NICs were resistant to chemotherapy, we used a neurosphere formation assay to measure the number of proliferating NICs in the presence of TMZ or BCNU. The concentrations of chemotherapy drugs required to inhibit neurosphere formation were much less than those required to inhibit bulk cell proliferation or to induce cell death in our neurosphere cultures. For some cultures, there was a robust recovery of neurosphere formation after chemotherapy treatment which appeared to be DNA damage independent. Some of the cultures that showed significant recovery of neurosphere formation underwent reversible cell cycle arrest, possibly reducing chemotoxicity in these cultures. Collectively, these results indicate that GBM neurosphere cultures can regrow after being treated with clinically relevant doses of chemotherapy drugs. Chemotherapy-treated neurosphere cultures remained viable, and formed tumors when injected into mice. Our experiments show that these in vitro assays may be useful in predicting in vivo responses to chemotherapeutic agents.

The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA expression and human diseases, such as cancer. The contribution of miRNAs to TGF-β-mediated cellular responses remains an open question. Thus, I became interested if miRNAs offered an additional layer of regulation in TGF-β signaling through which this cytokine exerts its pro-metastasis function. To address this inquiry, in the first part of this dissertation I investigated whether miRNAs influenced the ability of TGF-β to induce cellular responses directly involved with carcinoma metastasis, such as epithelial-mesenchymal transition (EMT). Here, I identified two miRNAs, miR-21 and miR-31, that are upregulated during EMT in LIM 1863 organoids, a colon carcinoma model of EMT driven by TGF-β. We performed in vitro studies to characterize the function of miR-21 and miR-31 and found that these two miRNAs positively impact the induction of EMT, migration and invasion by TGF-β. Furthermore, we uncovered TIAM1 (T lymphoma and metastasis gene 1) as a novel target of both miR-21 and miR-31 and show that downregulation of TIAM1 is critical for the pro-migration and pro-invasion activities of miR-21 and miR-31. Together these findings reveal miR-21 and miR-31 as downstream effectors of TGF-β signaling by facilitating EMT, migration and invasion of colon carcinoma cells. How TGF-β regulates miR-21 and miR-31 became important questions and thus the focus of the second part of this thesis. Interestingly, I found that TGF-β and TNF-α synergize to increase miR-21 and miR-31 levels in LIM 1863 organoids and that the synthesis of new factors induced by TGF-β/TNF-α are required for this upregulation. Moreover, I report that regulation of miR-21 by TGF-β/TNF-α occurs at multiple levels of biogenesis. More specifically data provided here show that Smad4 binds to the promoter of miR-21 to upregulate its expression thereby specifying miR-21 as a typical TGF-β target gene. This mechanism is different from one recently observed in smooth muscle cells in which TGF-β did not stimulate miR-21 transcription, but interestingly, Smad4 enhanced the Drosha-mediated processing of the miR-21 precursor. These two mechanisms suggest that TGF-β regulation of miR-21 is contextual and highlight the complexity of TGF-β signaling. As a whole, my findings establish important roles for miR-21 and miR-31 in TGF-β-mediated cellular responses that facilitate the pro-metastasis utility of TGF-β in colon cancer. Also, I describe a novel mechanism by which TGF-β/TNF-α signaling elevates the level of miR-21 and miR-31. Future studies that identify additional targets of miR-21 and miR-31 may offer further insight into the molecular mechanisms underlying cellular regulation by TGF-β. This information will be vital for the design of therapeutic interventions for colon cancer patients.

Glioma therapy relies on induction of cytotoxicity; however, the current combination of surgery, irradiation (IR) and temozolomide (TMZ) treatment does not result in a long-term cure. Our lab previously demonstrated that a small population of glioma cells enters a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ significantly decreases initial neurosphere formation; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate the culture. This recovery of neurosphere growth recapitulates the inevitable glioma recurrence in the clinic. The focus of our laboratory is to study direct-target therapies that can be combined with TMZ to inhibit neurosphere recovery. The Notch pathway is a promising target because it is involved in cell growth and survival. Here, we demonstrate that blocking the Notch pathway using gamma-secretase inhibitors (GSIs) enhances TMZ treatment. The combination of TMZ and GSI treatments targets the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. Interestingly, recovery is inhibited when the GSI is administered 24 hrs after TMZ treatment, demonstrating a sequence-dependent mechanism. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in NU/NU nude mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. Cultures treated with TMZ + GSI have decreased proliferation, but no increase in cell death. We observed an increase in the number of cells expressing senescence-associated β-galactosidase in vitro and in vivo. This demonstrates that inhibition of the Notch pathway shifts TMZ-treated cells from a transient cell cycle arrest into a permanent senescent state. Senescent cells can stimulate the innate immune system. Here we demonstrate that TMZ + GSI treatment increases phagocytosis in vitro. New therapy combinations, such as TMZ + GSI, are arising in the field of therapy-induced senescence (TIS). Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising target-directed therapy to decrease the rate of brain tumor recurrence by inducing senescence and tumor clearance.

Tumor recurrence after chemotherapy is a major cause of patient morbidity and mortality. Recurrences are thought to be due to small subsets of stem-like cancer cells that are able to survive chemotherapy and drive tumor re-growth. A more complete understanding of stem-like cancer cell regulation is required to develop therapies to better target and eliminate these cells. Slow-cycling stem cells are integral components of adult epithelial tissues and may give rise to cancer stem cell populations that share similar characteristics. These slow-cycling adult stem cells are inherently resistant to traditional forms of chemotherapy and transference of this characteristic may help to explain therapy resistance in cancer stem cell populations. Using a novel application for the proliferation marker CFSE, we have identified populations of slow-cycling cancer cells with tumor initiating capabilities. As predicted, slow-cycling cancer cells exhibit a multi-fold increase in chemotherapy resistance and retain the ability to re-enter the cell cycle. Furthermore, we observed consistent over-expression of the CDK5 activator, p35, in slow-cycling cancer cells. Manipulation of p35 expression in cancer cells affects cell cycle distribution and survival when these cells are treated with traditional forms of chemotherapy. Additionally, we demonstrate that alterations in p35 expression affect BCL2 levels, suggesting a mechanism for the survival phenotype. Combined, our data suggest a model whereby slow-cycling stem-like cancer cells utilize the p35/CDK5 complex to slow cell cycling speed and promote resistance to chemotherapy. Future p35 targeting, in combination with traditional forms of chemotherapy, may help eliminate these cells and reduce tumor recurrence rates, increasing long-term patient survival.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, develops through progression of premalignant pancreatic intraepithelial neoplasias (PanINs). In mouse-models, KRAS-activation in acinar cells induced an acinar-to-ductal metaplasia (ADM), and mutation of the Kras oncogene is believed to initiate PanIN formation. ADM is also promoted by pancreatic injury, which cooperates with activated KRAS to stimulate PanIN and PDAC formation from metaplastic ducts. Our lab, and others, have shown that the downstream PI3K/AKT pathway is important for KRAS-mediated proliferation and survival in vitro and in vivo. Prior studies have demonstrated that full activation of AKT requires both PDK1- mediated phosphorylation of AKTT308 and mTOR complex 2 (mTORC2)-mediated phosphorylation of AKTS473. Given the importance of the PI3K/AKT signaling axis, I hypothesized that mTORC2 is required for KRAS-driven pancreatic tumorigenesis and investigated this relationship in mice by combining pancreasspecific expression of an activated KRASG12D molecule with deletion of the essential mTORC2 subunit RICTOR. In the context of activated KRAS, Rictor-null pancreata developed fewer PanIN lesions; these lesions lacked mTORC2 signaling and their proliferation and progression were impaired. Higher levels of nuclear cyclin dependent kinase inhibitors (CDKIs) were maintained in Rictor-null lesions, and nuclear BMI1, a known regulator of the CDKI Cdkn2a, inversely correlated with their expression.Rictor was not required for KRAS-driven ADM following acute pancreatitis, however the inverse correlation between CDKIs and BMI1 was maintained in this system. Treatment of PDX-Cre;KRASG12D/+;Trp53R172H/+ mice with an mTORC1/2 inhibitor delayed tumor formation, and prolonged the survival of mice with late stage PDAC. Knockdown of Rictor in established PDAC cell lines impaired proliferation and anchorage independent growth supporting a role for mTORC2 in fully transformed cells. These data suggest that mTORC2 cooperates with activated KRAS in the initiation and progression of PanIN lesions and is required for the transformation and maintenance of PDAC. My work illustrates phenotypic differences between pancreatic loss of Rictor and PDK1 in the context of KRAS, broadens our understanding of this signaling node and suggests that mTORC2 may potentially be a viable target for PDAC therapies.

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.

In cancer, aberrant activation of developmental signaling pathways such as the Hippo Pathway has been shown to drive proliferation and invasion of cancer cells. Therefore, understanding the normal function of the Hippo Pathway during embryonic development can provide critical insight into how aberrant activity contributes to tumorigenesis. This dissertation explores the role of the Hippo Pathway members YAP and TAZ in gastrointestinal (GI) development and tumorigenesis. I use mouse genetics to systematically dissect the roles of YAP/TAZ in the endoderm-derived gastrointestinal epithelia and mesoderm-derived gastrointestinal mesenchyme during mammalian development. In the GI epithelium, I demonstrate that YAP/TAZ are dispensable for development and homeostasis. However, YAP/TAZ are required for Wnt pathway-driven tumorigenesis. I find that YAP/TAZ are direct transcriptional targets of Wnt/TCF4 signaling. In the GI mesenchyme, I describe a previously unknown requirement for YAP/TAZ activity during mammalian GI development. YAP/TAZ are involved in normal GI mesenchymal differentiation and function as transcriptional co-repressors in a progenitor cell population. In this way, YAP/TAZ act as molecular gatekeepers prior to Hedgehog-mediated differentiation into smooth muscle cells. This work unveils a previously unknown requirement for Hippo pathway signaling in the mammalian GI tract and a novel mechanism wherein YAP/TAZ function as transcriptional co-repressors to maintain a mesenchymal progenitor cell population.

The development and survival of multicellular organisms depends upon the ability of cells to move. Embryogenesis, immune surveillance, wound healing, and metastatic disease are all processes that necessitate effective cellular locomotion. Central to the process of cell motility is the family of integrins, transmembrane cell surface receptors that mediate stable adhesions between cells and their extracellular environment. Many human diseases are associated with aberrant integrin function. Carcinoma cells in particular can hijack integrins, harnessing their mechanical and signaling potential to propagate cell invasion and metastatic disease, one example being integrin α6β4. This integrin, often referred to simply as β4, is defined as an adhesion receptor for the laminin family of extracellular matrix proteins. The role of integrin β4 in potentiating carcinoma invasion is well established, during which it serves both a mechanical and signaling function. miRNAs are short non-coding RNAs that regulate gene expression posttranscriptionally, and data describing the role of extracellular stimuli in governing their expression patterns are sparse. This observation coupled to the increasingly significant role of miRNAs in tumorigenesis prompted us to examine their function as downstream effectors of β4, an integrin closely linked to aggressive disease in breast carcinoma. The work presented in this dissertation documents the first example that integrin expression correlates with specific miRNA patterns. Moreover, integrin β4 status in vitro and in vivo is associated with decreased expression of distinct miRNA families in breast cancer, namely miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, with purported roles in cell motility. Another miRNA, miR-29a, is significantly downregulated in response to de novo expression of β4 in a breast carcinoma cell line, and β4-mediated repression of the miRNA is required for invasion. Another major conclusion of this study is that β4 integrin expression and ligation can regulate the expression of SPARC in breast carcinoma cells. These data reveal distinct mechanisms by which β4 promotes SPARC expression, involving both a miR-29a-mediated process and a TOR-dependent translational mechanism. Our observations establish a link between miRNA expression patterns and cell motility downstream of β4 in the context of breast cancer, and uncover a novel effector of β4-mediated invasion.

Apoptosis-induced compensatory proliferation (AiP) is a mechanism that maintains tissue homeostasis after stress-induced cell death. During AiP, apoptotic cells induce proliferation of the neighboring surviving cells to compensate for tissue loss. AiP is important for wound healing and tissue regeneration in several model organisms. Additionally, AiP is an important feature of tumorigenesis and tumor relapse as it contributes to tumor repopulation following radiation or chemotherapy. Using an overgrowth tumor model (“undead tissue”) in Drosophila melanogaster, we determined that the initiator caspase Dronc promotes generation of extracellular Reactive Oxygen Species (ROS), which drive activation of the stress kinase JNK and downstream mitogens to promote AiP. We also observed increased numbers of Drosophila macrophages, termed hemocytes, which are attracted to undead tissue. However, the specific mechanisms by which macrophages are recruited to undead tissue are still unclear. Here, we report that the tissue microenvironment of the overgrown undead tissue directs macrophage recruitment during AiP. We demonstrate that ROS, JNK, and the matrix metalloproteinase Mmp2 are important for recruiting macrophages. Mechanistically, undead tissue-produced ROS and active JNK damage the basement membrane (BM) surrounding the undead tissue, by upregulating the expression and activity of Mmp2. The damaged BM then recruits macrophages to the undead tissue. Taken together, we propose a model in which the ROS-JNK-Mmp2 signaling axis damages the BM of undead tissue, resulting in changes in the tissue microenvironment that recruit macrophages to the area of damage to promote AiP and overgrowth.