E2F1 is a transcription factor which paradoxically has major influence on both apoptosis and cell cycle progression. One of the most important questions in E2F1 biology therefore is the mechanism underlying regulation of these opposing physiological outcomes. Post-translational modifications (PTM) provide proteins with an additional layer of complexity, potentially altering interactions with partner DNA and protein. The importance of arginine methylation has recently been implicated in modulating the activity of the tumour suppression pathway proteins, p53 and E2F1. Previous studies have established that the methyltransferase, PRMT5, is responsible for the symmetrical dimethylation of E2F1, which inhibits its pro-apoptotic activity. In this thesis, E2F1 was found to be a substrate of PRMT1, which catalysed asymmetrical dimethylation of E2F1 at arginine 109. In addition, a positive correlation was found between the percentage of apoptotic cells and levels of PRMT1. Conversely, an increase in cancer cell colony formation was shown when the site of PRMT1 methylation on E2F1 was changed from arginine to lysine at position 109. These findings suggested a growth inhibition effect by PRMT1 methylation on E2F1. At the transcriptional level, depletion of PRMT1 increased E2F1 binding to the promoter region of Cdc6, a cell cycle regulator, and decreased binding to the promoter region of Apaf1, which has a pro- apoptotic role. Genome-wide ChIP-sequencing technology was undertaken and results further clarified that the depletion of PRMT1 preferably enriched E2F1 binding to promoters of positive regulators of cell proliferation and promoters of the cell cycle. Collectively, the findings of this thesis suggested that the opposing roles E2F1 demonstrated in promoting both cell proliferation and apoptosis was due to different types of arginine methylation which trigger E2F1 binding to different promoters. Lastly, arginine methylation was shown to influence protein-protein interactions. PRMT5 induction resulted in the identification by mass spectrometry of β-catenin as an E2F1 interacting partner. As the Wnt/β-catenin signalling pathway is broadly recognised as having pro- cell proliferation activity, this finding is consistent with previous reports that suggest the oncogenic role PRMT5 methylation has on E2F1.

<p> Breast cancer is the second leading cause of cancer death and the leading cause of premature death of women in the United States (US). It was estimated that 231,840 women were expected to develop breast cancer in the US in 2015 and approximately 40,290 women were estimated to die of the disease. Even though most breast cancers are sporadic, 5-10% of women are at an increased risk for developing breast cancer due to a hereditary risk. Too few healthcare providers are identifying women with family histories suggestive of hereditary cancer syndromes. An efficient way to identify high risk women in the primary care setting is through an easy to understand, self-administered family history risk assessment tool. The Pedigree Assessment Tool (PAT) family history questionnaire was offered to women age 18 and over at a primary care clinic in northern Louisiana. A PAT score of 8 or above prompted a cancer family history discussion by the physician or nurse practitioner and was followed by a genetic counseling referral. A total of 428 women completed the risk assessment tool during a 4 month period, 32 were high risk as evidenced by scoring 8 or higher on the PAT. Fourteen women were referred for genetic counseling. Twelve declined testing due to lack of insurance coverage, previous completion of genetic testing or felt the information would not improve their health. Six of the thirteen women completed genetic counseling and genetic testing. Lack of insurance coverage was identified as a major barrier to genetic counseling referrals. Utilization of the PAT identifies high risk women who would benefit from a genetic counseling referral. Genetic testing provides information that allows the patient and primary care provider to make informed decisions regarding surveillance protocols or prophylactic surgeries to diagnose cancer at an early stage or prevent cancer from developing.</p>

The skeleton is the most common site of prostate cancer bone metastasis, and at present, there are no curable treatments for these patients. To further understand what stimulates tumor cell growth in the bone microenvironment and to find suitable therapies, reliable model systems are needed. For this purpose, we have developed an in vitro co-culture system that can be used to study interactions between tumor cells and murine calvarial bones. To validate the model, we measured the release of collagen fragments and monitored changes in expression levels of genes normally expressed during active bone remodeling. One of the major reasons why prostate cancer cells colonize bone is the abundance of tumor-stimulating factors, such as insulin-like growth factors (IGFs), present in this milieu. We found that the IGF-1 receptor (IGF-1R) was one of the most highly activated receptor tyrosine kinases in tumor cell lines stimulated with bone conditioned media. Since IGF-1 is known to be a strong survival factor for tumor cells, we hypothesized, that concurrent inhibition of IGF-1R signaling can enhance the effects of apoptosis-inducing therapies, such as castration. We used our co-culture model to target human prostate cancer cell lines, PC-3 and 22Rv1, with simvastatin (an inhibitor of the mevalonate pathway and an inducer of apoptosis), in combination with anti-IGF-1R therapy. Tumor cell viability declined with either one of the therapies used alone, and the effect was even more pronounced with the combined treatment. The hypothesis was also tested in rats that had been inoculated with rat prostate cancer cells, Dunning R3327-G, into the tibial bone, and treated with either anti-IGF-1R therapy, castration, or a combination of both therapies. Immunohistochemistry was used to evaluate therapeutic effects on tumor cell proliferation and apoptosis, as well as tumor cell effects on bone remodeling. The tumor cells were found to induce an osteoblastic response, both in vivo in rats, and in vitro using the co-culture model. Interestingly, the therapeutic response differed depending on whether tumor cells were located within the bone marrow cavity or if they had leaked out into the knee joint cavity, highlighting the role of the microenvironment on metastatic growth and therapeutic response. Therapies targeting the IGF-1R have been tested in clinical trials, unfortunately with disappointing results. By immunohistochemical evaluation of bone metastases from patients with castration-resistant prostate cancer, we found a large variance in IGF-1R staining within this group of patients. Hence, we postulate that the effects of anti-IGF-1R therapies could be more beneficial in patients with high tumoral IGF-1R-activity than in IGF-1R negative cases. We also believe that side effects, such as hyperglycemia, associated with anti-IGF-1R therapy, could be reduced if this treatment is administered only to selected patients and for shorter time periods. In a separate study, using whole-genome expression data from bone metastases obtained from prostate cancer patients, we present evidence that a high activity of osteoblasts is coupled to a high activity of osteoclast. Moreover, we found that high bone remodeling activity is inversely related to tumor cell androgen receptor (AR) activity. The results from this study may be of importance when selecting therapy for patients with bone metastatic cancer, especially when bone-targeting therapies are considered, and could aid in the search for novel therapeutic targets. In summary, we present an in vitro model for studies of the bidirectional interplay between prostate cancer cells and the bone microenvironment. We also demonstrate the importance of IGF-1 in prostate cancer bone metastases and suggest that inhibition of IGF-1R signaling can be used to treat prostate cancer as well as to enhance effects of other treatments such as androgen deprivation therapy. Furthermore, we emphasize the possibility of molecular tumor characterization when designing treatment plans for individual patients, thereby maximizing the therapeutic effects.

Master of Science / Department of Kinesiology / Bradley J. Behnke / Given the critical role of tumor O₂ delivery on patient prognosis and the rise in preclinical exercise-oncology studies, we investigated tumor and host-tissue blood flow at rest and during exercise as well as vascular reactivity using a rat prostate cancer model grown in two transplantation sites. Methods. In male COP/CrCrl rats, blood flow (via radiolabeled microspheres) to prostate tumors (R3327-MatLyLu cells injected in the left flank (ectopic) or ventral prostate (orthotopic)) and host-tissue was measured at rest and during a bout of mild-intensity exercise. Alpha-adrenergic vasoconstriction to norepinephrine (NE: 10⁻⁹ to 10⁻⁴ M) was determined in arterioles perforating the tumors and host-tissue. To determine host-tissue exercise hyperemia in healthy tissue, a sham-operated group was included. Results. Blood flow was lower at rest and during exercise in ectopic tumors and host-tissue (subcutaneous adipose) versus the orthotopic tumor and host-tissue (prostate). During exercise, blood flow to the ectopic tumor significantly decreased by 25 ± 5%, whereas flow to the orthotopic tumor increased by 181 ± 30%. Maximal vasoconstriction to NE was not different between arterioles from either tumor location. However, there was a significantly higher peak vasoconstriction to NE in subcutaneous adipose arterioles (92 ± 7%) versus prostate arterioles (55 ± 7%). Establishment of the tumor did not alter host-tissue blood flow from either location at rest or during exercise. Conclusion. These data demonstrate blood flow in tumors is dependent on host-tissue hemodynamics and that the location of the tumor may critically affect how exercise impacts the tumor microenvironment and treatment outcomes.

Sarcomas are cancers of connective tissues, such as bone, adipose and cartilage, and are thought to arise from the aberrant development of the mesenchyme. As such, mesenchymal stem cells are thought to be the cell of origin for sarcomas. Genetic or epigenetic lesions at particular points during the differentiation of a mesenchymal stem cell into its terminal mesenchymal cell type are able to give rise to specific subtypes of sarcomas. Recently, a number of reports have identified elevated expression of the human Piwi homolog--called Hiwi--in a variety of human cancers, including gastric cancer, pancreatic cancer, gliomas and, most relevant for this dissertation, sarcomas. In sarcomas, Hiwi is highly expressed and elevated Hiwi prognosticates shorter patient survival. Hiwi is the human homolog of the Piwi family of proteins, which are members of the Paz-Piwi Doman (PPD) family. During normal development, Piwis are thought to maintain stem cells of the germline, and indeed their expression is limited to early development and to the adult germline. Piwis are thought to maintain stem cells in the germline with small RNA partners, called piwi-interacting RNAs (piRNAs). More specifically, Piwi/piRNA complexes in the germline are thought to maintain transposon silencing, and thus ensure genomic stability. A detailed mechanism by with Piwis suppress transposon migration in the germline remains an area of active investigation, but is thought to occur via DNA methylation of transposon regions. In this way, Piwis are critical for maintenance of genomic integrity of germline stem cells during normal development. Thus, the finding that Piwis are elevated in human cancers is directly in conflict with its known role in ensuring genomic stability during development. Piwi homologs are critical for maintenance of germline stem cells during development but aberrant Hiwi expression has also been identified in all cancers examined, including in sarcomas. A potential connection between mesenchymal stem cells, sarcomas and Hiwi remains unexplored. Moreover, the role of Hiwi in sarcomas is unknown. In the studies presented here, we demonstrate that over-expressing Hiwi in mesenchymal stem cells inhibits their differentiation in vitro and generates sarcomas in vivo. Secondly, transgenic mice expressing Hiwi (mesodermally-restricted) develop sarcomas. Conversely, inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. These data reveal that Hiwi is directly tumorigenic. We have also identified the presence of piRNAs in our Hiwi-expressing models. We further show that DNA methylation correlates with Hiwi expression and that cyclin-dependent kinase inhibitor (CDKI) tumor suppressor genes are silenced upon Hiwi over-expression. Moreover, Hiwi's tumorigenic effects are reversible using DNA de-methylating agents. These studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.

Familial cases of breast and ovarian cancer are often attributed to germline mutations of the BRCA1 tumor suppressor gene. Although the mechanisms of BRCA1 tumor suppression are poorly understood, its protein product has been implicated in multiple aspects of the DNA damage response. As such, BRCA1 may suppress tumor development through its role in the maintenance of genomic integrity. The C-terminus of BRCA1 contains two tandem BRCT motifs that form a single phosphoprotein binding motif that mediates the interaction of BRCA1 with at least three distinct phospho-ligands; Abraxas, BACH1, and CtIP. We recently reported that the tumor suppression activity of BRCA1 is dependent on the phospho-recognition function of its BRCT motifs. Of the three known phospho-ligands, the CtIP repair protein is intriguing because its interaction with BRCA1 is ablated by tumor-associated missense mutations in the BRCT domain. Accordingly, CtIP may be a critical mediator of the genome maintenance and tumor suppression functions of BRCA1. Here we evaluate the role of CtIP in these BRCA1-dependent processes using murine cells expressing Ctip polypeptides (Ctip-S326A) that fail to interact with Brca1. Surprisingly, we demonstrate that the BRCA1-CtIP interaction is dispensable for mammalian cell viability, critical aspects of BRCA1 function in genome stability, and BRCA1-mediated tumor suppression. Given that CtIP plays a diverse role in maintaining genome integrity, we also assessed whether CtIP has functions relevant to tumor suppression independent of its interaction with BRCA1. To test this hypothesis, we generated mice carrying a conditional-null Ctip (CtipCo) allele and used Cre recombination to inactivate the gene specifically in mammary epithelial cells. Furthermore, since we recently demonstrated that the genome maintenance and tumor suppression functions of BRCA1 do not depend on its individual interaction with the BRCT phospho-ligands Abraxas, BACH1, or CtIP, we examined whether the interaction of BRCA1 with two or more of these phospho-ligands mediates these functions.

Prostate cancer is a common malignancy with nearly one million annual diagnoses worldwide. Among a subset of patients, primary disease eventually progresses to disseminated castration resistant prostate cancer (CRPC). In recent years, treatment modalities that improve survival in CRPC have emerged including taxane chemotherapy and second generation androgen signaling inhibitors, among others. Indeed, today the first line chemotherapeutic docetaxel as well as the second line agent cabazitaxel are mainstays of treatment. However, CRPC inexorably progresses to a chemotherapy resistant state that ultimately precedes lethality. Elucidating the molecular determinants of aggressiveness in chemotherapy resistant CRPC may therefore stimulate new therapeutic strategies that improve clinical outcomes. We used laboratory models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Whole genome expression profiling, clinical validation and genetic screening approaches revealed that GATA2 regulates a signature of cancer progression associated genes. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a significant mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this regulatory axis prompted a combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in chemotherapy resistant prostate cancer and identify a therapeutic opportunity in this challenging disease.

Cancer research, like many areas of science, is adapting to a new era characterized by increasing quantity, quality, and diversity of observational data. An example of the advances, and the resulting challenges, is represented by The Cancer Genome Atlas, an enormous public effort that has provided genomic profiles of hundreds of tumors of each of the most common solid cancer types. Alongside this resource is a host of other data and knowledge, including gene interaction databases, Mendelian disease causal variants, and electronic health records spanning many millions of patients. Thus, a current challenge is how best to integrate these data to discover mechanisms of oncogenesis and cancer progression. Ultimately, this could enable genomics-based prediction of an individual patient's outcome and targeted therapies, a goal termed precision medicine. In this thesis, I develop novel approaches that examine patterns in populations of cancer patients to identify key genetic changes and suggest likely roles of these driver genes in the diseases. In the first section I show how genomics can lead to the identification of driver alterations in melanoma. The most recurrent genetic mutations are often in important cancer driver genes: in a newly sequenced melanoma cohort, recurrent inactivating mutations point to an exciting new melanoma candidate tumor suppressor, FBXW7, with therapeutic implications. But each tumor is unique, underlining the fact that recurrence will never capture all relevant mutations responsible for the disease. Tumors are a result of random events that must collaborate to endow a cell with all of the invasive and immortal properties of a cancer. Some combinations of events are lethal to a developing tumor, while other combinations are simply not preferentially selected. In order to discover these complex patterns, I develop a method based on the joint entropy of a set of genes, called GAMToC. Using GAMToC, I identify sets of recurrently altered genes with a strongly non-random joint pattern of co-occurrence and mutual exclusivity. Then, I extend this method as a means of identifying novel genes with a role in cancer, by virtue of their non-random pattern of alteration. Insights into the roles of these novel drivers can come from their most strongly co-selected partners. In the final section of the main text, I develop the use of cancer comorbidity, or increased cancer risk, as a novel data source for understanding cancer. The recent availability of clinical records spanning a large percentage of the American population has enabled discovery of many cancer comorbidities. Although most cancers arise as a result of somatic mutations accumulating over a patient's lifespan, mutations present at birth could predispose some rare populations to increased cancer risk. Mendelian disease phenotype provides strong insight into the genotype of an afflicted individual. Thus, if Mendelian diseases with cancer comorbidity can be shown to have specific defects in processes that are important in the development of that cancer, statistical comorbidity could provide a new a resource for prioritizing Mendelian disease genes as novel cancer related genes. For this purpose, I integrate clinical comorbidity, Mendelian disease causal variants, and somatic genomic profiles of thousands of cancers. I demonstrate that comorbidity indeed is associated with significant genetic similarity between Mendelian diseases and the cancers these patients are predisposed to, suggesting highly interesting and plausible new candidate cancer genes. While cancer may be the result of a series of selected random events, patterns of incidence across large populations, as measured by genomics or by other phenotypes, contain much non-random signal yet to be mined.

No description available.

abstract: Medulloblastoma is the most common malignant pediatric brain cancer and is classified into four different subgroups based on genetic profiling: sonic hedgehog (SHH), WNT, Group 3 and 4. Changes in gene expression often alter the progression and development of cancers. One way to control gene expression is through the acetylation and deacetylation of histones. More specifically in medulloblastoma SHH and Group 3, there is an increased deacetylation, and histone deacetylase inhibitors (HDACi) can be used to target this change. Not only can HDACi target increases in deacetylation, they are also known to induce cell cycle arrest and apoptosis. The combination of these factors has made HDACi a promising cancer therapeutic. Panobinostat, a hydrophobic, small molecule HDACi was recently identified as a potent molecule of interest for the treatment of medulloblastoma. Furthermore, panobinostat has already been FDA approved for treatment in multiple myeloma and is being explored in clinical trials against various solid tumors. The laboratory is interested in developing strategies to encapsulate panobinostat within nanoparticles composed of the biodegradable and biocompatible polymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG). Nanoparticles are formed by single emulsion, a process in which hydrophobic drugs can be trapped within the hydrophobic nanoparticle core. The goal was to determine if the molecular weight of the hydrophobic portion of the polymer, PLA, has an impact on loading of panobinostat in PLA-PEG nanoparticles. Nanoparticles formulated with PLA of varying molecular weight were characterized for loading, size, zeta potential, controlled release, and in vivo tolerability. The results of this work demonstrate that panobinostat loaded nanoparticles are optimally formulated with a 20:5kDa PLA-PEG, enabling loading of ~3.2 % w/w panobinostat within nanoparticles possessing an average diameter of 102 nm and surface charge of -8.04 mV. Panobinostat was released from nanoparticles in a potentially biphasic fashion over 72 hours. Nanoparticles were well tolerated by intrathecal injection, although a cell culture assay suggesting reduced bioactivity of encapsulated drug warrants further study. These experiments demonstrate that the molecular weight of PLA influences loading of panobinostat into PLA-PEG nanoparticles and provide basic characterization of nanoparticle properties to enable future in vivo evaluation. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019