David Huntsman

Clear cell ovarian cancer (CCOC) is a molecularly unique subtype of epithelial ovarian cancer for which treatment options are still limited. Patients with late stage CCOC do not respond to gold-standard platinum and taxane based chemotherapeutics, and effective targeted therapies for this cancer are still lacking. Due to its relative rarity, the molecular landscape of this ovarian cancer subtype has not been fully deciphered. In this thesis, I used global screening techniques to elucidate the proteomic and metabolomic landscapes of CCOC, compared to other common epithelial ovarian cancer subtypes. I found that CCOC is a unique entity compared to its other epithelial ovarian cancer counterparts in both its proteomic and metabolomic landscapes. I reported proteomic diversity within CCOC cases presented as subgroups of distinct molecular signatures. Moreover, through integrated proteometabolomic analysis, I identified aberrancies in purine metabolism, cysteine/glutathione metabolism, as well as glucose metabolism. I further demonstrated that available CCOC cell lines reflect the proteomic and metabolomic signatures in CCOC clinical samples. The heterogenous biology seen in clinical samples and cell lines suggests that the comprehensive understanding of this cancer require appropriate in-vitro models to represent its diverse molecular phenotypes. Lastly, our proteomic understanding led to the identification of the lack of an arginine synthesizing enzyme, arginosuccinate synthase, in CCOC and other rare ovarian cancer subtypes including small cell ovarian carcinomas hypercalcemic type. Cancers lacking this enzyme has been shown to be sensitive to a molecular agent depriving extracellular arginine. I show that this therapeutic agent was effective in curbing the growth of arginosuccinate synthase-negative rare ovarian cancers in-vitro and in in-vivo murine models, thus identifying a potential therapy for these very aggressive subtypes. My research provides the largest reported proteomic landscape of CCOC cases, in addition, I report the first metabolic landscape of CCOC and other ovarian cancer subtypes. Both will serve as valuable resources for further research into CCOC biology and therapeutic development. Furthermore, I demonstrate of how proteomic and metabolomic understanding can accelerate therapeutic development in rare ovarian cancers by using arginosuccinate synthase deficiency as an example.

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Adult-type granulosa cell tumours (AGCT) represent 3-5% of ovarian cancers. AGCT is typically diagnosed at an early stage and is treated by surgery. However, tumour recurrence occurs in one-third of patients, and approximately 50% of relapsed patients succumb to their disease. A somatic missense mutation (c.402C>G; pC134W) in FOXL2 was identified in over 95% of AGCTs. Although this FOXL2 mutation enhanced the ability to accurately diagnose challenging cases, there is still a major clinical challenge in identifying patients that are at risk of relapse and how the FOXL2 C134W mutation contributes to tumourigenesis remains to be elucidated. Objectives: 1) Characterize the genomic and transcriptomic features of AGCT and 2) Develop novel model systems to study the FOXL2 C134W mutation in AGCTHypotheses: 1) Secondary mutations and/or transcriptional alterations explain the clinical and biological diversity in AGCT and 2) An appropriate cell context and microenvironment is required to understand the role of FOXL2 C134W in AGCT. Methods: Whole genome and targeted sequencing were performed to identify secondary mutations. RNA-Sequencing was performed to describe the transcriptional landscape. Transduction of FOXL2 C134W mutant and hTERT into primary granulosa cells was investigated for the establishment of a de novo transformation model. Development of tumour-derived cell lines and patient-derived xenografts were explored. Results: AGCT is not a disease that cannot be sub-stratified by genomic features. Besides the AKT1 (c.49G>A; pE17K) hotspot mutation identified at a low frequency (2/130, 1.5%) in AGCT, no actionable mutations were identified. TERT C228T promoter mutations were identified in primary AGCTs (51/229, 22%) and recurrent AGCTs (24/58; 41%) suggesting that these mutations play a role in tumour initiation and progression. Transcriptome analysis did not separate AGCTs based on disease status. The transduction of FOXL2 C134W mutant and ectopic expression of TERT into primary granulosa cells was not sufficient for oncogenic transformation. Immortalization of AGCT-derived patient cells and development of patient-derived xenografts were unsuccessful. Conclusions: AGCT represents a specific clinical entity driven by the FOXL2 C402G mutation. Therapeutic strategies focusing on targeting the FOXL2 C134W protein or its downstream consequences are the most promising approach to developing novel treatments for AGCT.

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Mutations in the Capicua (CIC) gene, located on chromosome 19q, were first identified in up to 70% of 1p19q-codeleted, IDH-mutated Oligodendrogliomas (ODGs), a subtype of diffuse low grade glioma (DLGG). Since then, several studies have highlighted that loss or dysregulation of CIC is associated with tumour progression, worse prognosis, and treatment resistance in multiple cancer types. One CIC binding partner, Ataxin-1-Like (ATXN1L), has been implicated as an important regulator of CIC function in murine development, but little is known in the context of cancer. In this thesis, we characterize the functional interaction between CIC and ATXN1L and their relationship in regulating pathways involved in tumourigenesis.The function of ATXN1L in relation to CIC was investigated by assessing and comparing the transcriptomic consequences of ATXN1L knockout (KO) and CIC KO using isogenic cell lines. Loss of either CIC or ATXN1L led to concordant dysregulation of gene sets in each context which converged upon several pathways involved in differentiation and activation of the mitogen activated protein kinase (MAPK) pathway as a result of decreased CIC-DNA binding. Further analysis of cancers harboring deletions in CIC and ATXN1L, namely, stomach adenocarcinoma, prostate adenocarcinoma, and astrocytoma, resulted in convergent dysregulation of several pathways involved in cell proliferation and growth.In addition to decreased CIC-DNA binding, loss of ATXN1L resulted in increased CIC protein instability as a result of proteasomal degradation which was found to be independent of ERK activity, the canonical CIC degradation pathway. Instead, loss of ATXN1L was found to promote CIC instability through ubiquitination by the E3-ligase TRIM25, a novel CIC interactor. Transcriptomic analyses of breast carcinomas with TRIM25 amplification and liver hepatocellular carcinomas with high TRIM25 expression revealed dysregulation of genes and pathways reminiscent of CIC loss, supporting its role in the post-translational regulation of CIC.The results of these studies illuminate our understanding and the intricacy of mechanisms which regulate normal CIC function in cancer; and further supports the role of CIC as a potent tumour suppressor in a multitude of cancer types.

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Genetic variation makes important and often uncharacterized contributions to both rare syndromes and more common complex diseases. Around 8% of all cancers are caused by high- and moderate-penetrance deleterious germline variants that are present in an individual from birth and predispose to specific cancer types throughout life. Such cancer predisposition genes associated with moderate to high lifetime cancer risks, conventionally defined as greater than twofold and fivefold increases, respectively, are involved in various biological pathways required for regulating cellular proliferation, maintaining genome integrity, and mediating inter- and intracellular signaling. Clinical use of multigene next-generation sequencing panels has improved molecular diagnosis of cancer predisposition syndromes, demonstrating both genetic heterogeneity and phenotypic variability amongst carriers. However, many individuals with a strong personal or family cancer history receive uninformative results from clinical genetic testing that may lead to increased health anxiety and missed opportunities for increased cancer screening, cancer prevention or use of targeted therapies. Therefore, the main objective of my dissertation was to characterize the biological significance, functional impact, and heterogeneity of genetic variation underlying high-penetrance cancer predisposition syndromes to improve rates of genetic diagnosis. Using genome and transcriptome sequencing, I explored molecular characteristics associated with inactivation of high-penetrance cancer predisposition genes across advanced cancers and in an organoid model system. Tissue-specific molecular signatures provided insights into the aetiology of site-specific tumour development in carriers, allowing opportunities for carrier ascertainment and differential genetic diagnosis of suspected hereditary cancer families. Based on findings that structural variants account for 10% of causal variants, I investigated the utility of long-read sequencing in the clinical interpretation of germline structural variants that were undetected or unresolved through next-generation sequencing. Short- and long-read genome sequencing improved genetic diagnosis in known and suspected carriers for autosomal dominant cancer syndromes, demonstrating incomplete penetrance and phenotypic heterogeneity in population-based and disease-specific cancer cohorts. The research presented here thus supports a broader understanding of the contributions of germline variation to cancer susceptibility and disease progression, ultimately informing guidelines for screening, variant interpretation, and clinical management.

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Germline CDH1 mutation carriers are at risk for early-onset diffuse gastric cancer and female carriers have an additional risk of lobular breast cancer. Reliable estimates of cancer risk are essential for genetic counselling and clinical management of mutation carriers. Prophylactic total gastrectomy (PTG) to eliminate the gastric cancer risk is an option for mutation carriers. Current information on post-surgical outcomes and quality of life is limited. The objectives of this research were 1) To improve the evidentiary basis of genetic counselling by deriving reliable estimates of cancer risk in CDH1 mutation carriers; 2) To catalogue a comprehensive list of all novel and previously reported germline mutations to date; and 3) To provide data on post-surgical clinical outcomes and to describe the impact of the surgery on participants’ quality of life. Methods: Penetrance was derived from 67 mutation-positive families comprising 4031 individuals (350 affected with gastric cancer and 99 with breast cancer). Participants were recruited through multiple sources for clinical outcomes and quality of life study. Hospital records provided information on clinical outcomes. All participants were asked to complete validated questionnaires measuring generic and condition specific QOL (PROMIS, EORTC and SF 36v.II) at a single point. Results: By age 75 years, the cumulative incidence of gastric cancer was 70% (95% confidence interval [CI], 40%-94%) for males and 56% (95% CI, 27%-90%) for females. The risk of breast cancer for females was 42% (95% CI, 23%-68%) by 75 years. The mutational landscape of CDH1 did not reveal mutational hotspots but several shared mutations are seen in unrelated families. The 53 participants who had undergone PTG reported frequent symptoms of fatigue (59%), abdominal pain (55%), and diarrhea (45%). Cognitive, role and social function plus the symptoms anxiety, pain, taste, dyspnea and diarrhea were significant predictor variables for quality of life (p
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Endometrial carcinoma is the most common gynaecological cancer in developed countries. The current endometrial pathologic classification system lacks reproducibility, which has hampered the development of new treatments for these cancers. The PP2A phosphatase complexes are responsible for regulating many cellular pathways, and may play a role in the deregulation of endometrial cancer-associated pathways. In this thesis, the role of PPP2R1A mutations in the subtype-specific classification of gynaecological tumours was investigated. Additionally, mutational profiles will be used to improve the classification of the subtypes of endometrial carcinomas. Lastly, the functional effect of mutant PPP2R1A on PP2A-subunit protein interactions will be determined, in the context of endometrial cancer cell lines. Next-generation and Sanger sequencing was used to determine the presence of mutations in endometrial and ovarian carcinomas. PPP2R1A isogenic endometrial-specific cell lines were generated using somatic cell gene knockout by homologous recombination. Co-immunoprecipitation and mass spectrometry was used to determine effects of the PPP2R1A W257L mutation on its ability to interact with PP2A subunits. Subtype-specific somatic PPP2R1A mutations were identified in endometrial serous carcinomas. Low-grade endometrial endometrioid carcinomas were defined by mutations in the genes: ARID1A, PTEN, PIK3CA, CTNNB1, and KRAS, whereas high-grade endometrioid also harbor TP53 mutations. Endometrial serous carcinomas harbor mutations in PPP2R1A, FBXW7, PIK3CA and TP53. Consequently, the molecular profiles proved useful in assisting classification of tumours with overlapping morphological features that cause irreproducibility in diagnoses. Proteomic analysis of isogenic cell lines determined that the PPP2R1A W257L mutation disrupts interaction with PPP2R5C and PPP2R5D subunits. In addition, PPP2R1A mutated protein caused an increased interaction with the endogenous PP2A inhibitor SET/I2PP2A. The integration of mutational profiles and other genomic features will be used to improve clinical and pathological classification in endometrial tumours that are difficult to diagnose. PPP2R1A mutations are likely playing a role in the transformation of gynaecological carcinoma, by disrupting PP2A subunit interactions with tumour suppressor functions. Increased interaction of mutant PPP2R1A with SET/I2PP2A adds another layer of complexity to the tumour suppressive role of PP2A. In the future, targeting the PP2A complex with novel therapeutics could provide an alternative method for treating gyneacological cancers with poor outcomes.

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Hereditary cancer syndromes predispose to early-onset or multiple cancers in a person or family, follow Mendelian inheritance patterns and demonstrate stereotyped patterns of tumor development. Genotype-phenotype correlations direct clinical genetic testing and provide guidance for hereditary cancer management. This thesis began by examining the association of lobular breast cancer with germline mutations in CDH1, the gene encoding the epithelial cell-cell adhesion molecule E-cadherin and tested whether CDH1 represented a high-frequency breast cancer susceptibility gene, apart from its association with hereditary diffuse gastric cancer. In addition it examined other genotype-phenotype correlations including the association between granular cell tumors and a multiple congenital anomaly syndrome, the specific correlation between a recurrent somatic mutation in a transcription factor and adult-type granulosa cell tumors and the strong association of germline BRCA1 and BRCA2 mutations with high-grade serous epithelial ovarian cancer. These candidate gene analyses were performed using low-throughput molecular technologies. With the advent of cheaper DNA-sequencing capabilities, the application of these new technologies to novel Mendelian disease gene discovery and hereditary cancer management became the subsequent focus of the thesis. Objectives: To determine the frequency of germline CDH1 mutations in women with lobular breast cancer unselected for familial gastric cancer; to define the associations between several alternative genotype-phenotype correlations; and, to apply next-generation sequencing to determine the basis of a Mendelian disorder, in order to determine its utility as a potential familial cancer gene discovery and clinical tool. Selected Methods: Single amplicon mutation screening and sequence analysis of a large cohort of women with early-onset or familial lobular breast cancer. Next-generation sequencing analysis of a family with multiple individuals cosegregating spondyloepiphyseal dysplasia and retinitis pigmentosa. Results: Without a selective history of diffuse gastric cancer, potentially pathogenic germline mutations in CDH1 occured in women with early-onset or hereditary lobular breast cancer in less than two percent of individuals. Diagnosis of a Mucolipidosis type III gamma was possible using new sequencing technologies. Conclusion: There is utility in understanding genotype-phenotype correlations in order to direct genetic testing and novel gene identification. Next-generation sequencing technologies can succesfully be applied to Mendelian disorders with clear phenotypes for gene discovery.

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Clear cell carcinomas (CCCs) are a subtype of ovarian cancer that is understudied, and whichdoes not respond well to conventional therapeutic strategies. There is a desperate need toclarify the genetic mechanisms of CCC to allow for the development of subtype specifictherapeutics. To determine genetic changes responsible for the development of CCC, wholetranscriptomes of 18 ovarian CCCs and one ovarian CCC cell line were sequenced. Somaticmutations were found in ARID1A in 6 samples. ARID1A encodes BAF250a, a key componentof the SWI/SNF chromatin remodeling complex. ARID1A was sequenced in an additional210 ovarian carcinomas and a second CCC cell line, and BAF250a expression was measuredby immunohistochemistry (IHC) in an additional 455 ovarian carcinomas and over 3000 nonovarianmalignancies. Overall, ARID1A mutations were seen in 46% of CCCs and 30% ofendometrioid carcinomas (EC) implicating ARID1A as a tumour suppressor frequentlydisrupted in CCC and EC. Loss of the BAF250a protein correlated strongly with the presenceof ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expressionwere evident in the tumour and contiguous atypical endometriosis, implicating ARID1A lossas an early event in tumourigenesis. Screening 3000 cases of different malignancies by IHCshowed loss of BAF250a was is most frequent in cancers of endometrial origin. Reversephase protein array (RPPA) for tumour samples with known ARID1A mutation statusrevealed a notable change pAKT-Thr³⁰⁸ (FDR
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Kisspeptins and their receptor, GPR54, mediate sex hormone release through stimulation of the hypothalamic-pituitary-gonadal axis and have been implicated as metastasis suppressors. Expression of kisspeptin and GPR54 has been associated with less invasive cancers as determined by RNA expression, and a multitude of in vitro studies has consistently shown that overexpression of either ligand or receptor in malignant cell lines results in a less invasive phenotype. We hypothesized that expression of GPR54/kisspeptin in epithelial malignancies is predictive of disease outcome and altering endogenous GPR54 signalling in malignant breast and ovarian epithelial cells could alter their metastatic properties. We have determined by immunohistochemistry that kisspeptin and GPR54 are independent favourable prognostic markers for ovarian carcinoma and are specific for the clear cell cancer subtype; the least characterized of the subtypes. Additionally, loss of GPR54 is associated with poor prognosis in node positive breast cancer patients and is also lost in prostate cancer and testicular germ cell nonseminomas as compared to more benign disease. Moreover, secreted kisspeptin is elevated above physiological levels in the plasma of women with gynaecological cancers, including ovarian cancer. We evaluated GPR54 expression across a panel of breast and ovarian cancer cell lines to create an in vitro model system with which to knockdown GPR54 expression using RNA interference. However, we discovered that endogenous GPR54 was internalized rather than localized to the plasma membrane of these cancer cell lines. Consequently, internal GPR54 was unable to signal through its canonical Gαq pathway. To discover novel genes involved in kisspeptin-GPR54 signalling, we assessed gene expression differences between the Gpr54 and Kiss1 knockout mice as compared to wildtype mice. Our novel candidate list provides insight into physiological signalling in the hypothalamus that can then be applied to epithelial anti-metastatic signalling. Our results also support the sex hormone negative feedback effect on kisspeptin expression as reported in the current literature.In summary, we have confirmed kisspeptin and GPR54 as favourable prognostic markers, are the first to report the intracellular localization of GPR54 in endogenously expressing cancer cell lines, and we have introduced a list of novel genes involved in signalling.

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