767 research outputs found
Adult-type granulosa cell tumour of the ovary : a FOXL2-centric disease
No full textAdult-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 AGCT
Hypotheses: 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.Medicine, Faculty ofMedical Genetics, Department ofGraduat
The pulmonary metastasis assay (PUMA) : a tool to study novel therapies for ovarian cancer
No full textClear Cell Ovarian Cancer (CCOC) is a chemoresistant subtype of ovarian cancer, accounting for ~10% of epithelial ovarian carcinomas (EOC) in North America and up to 30% in Japan. Unlike in the more common high-grade serous ovarian cancers, hematogenous spread and extraperitoneal metastasis are prevalent in CCOC but have been understudied due to a lack of suitable models.
High expression of Cystathionine Gamma-Lyase (CTH) and mutations in AT-Rich Interactive Domain-Containing Protein 1A (ARID1A) are common in CCOC. CTH, a key enzyme in the transsulfuration pathway, is a marker for CCOC. Inactivating CTH reduces CCOC’s metastatic potential and improves chemotherapy responsiveness. Loss of ARID1A occurs in ~65% of CCOC cases, inducing ROS accumulation and reliance on oxidative phosphorylation (OXPHOS) for energy. EO3001, a synthetic drug, selectively transports extracellular Cu (II) to mitochondria, inducing ROS and triggering cuproptosis. ARID1A-deficient cells show increased sensitivity to EO3001.
To investigate the effectiveness of EO3001 and CTH inhibitors in CCOC, we used the Pulmonary Metastasis Assay (PuMA). This model enables metastases to be seeded via tail vein injections and studied either immediately or after full establishment, providing a more complex microenvironment than traditional 2D/3D models.
We generated isogenic CCOC cell lines (-/+ CTH loss & -/+ ARID1A loss) using CRISPR-Cas9. EO3001 was obtained through collaboration, while a CTH inhibitor library was screened by our collaborators; 3 candidates showed promising effects and were further tested. We evaluated EO3001's therapeutic impact on tumorigenic potential and cell growth in vitro and ex vivo. Both early- and late-harvest PuMA were optimized to assess CTH inhibitors and EO3001.
In both assays, all 3 CTH inhibitors showed significant inhibitory effects on CCOC, offering a novel therapeutic strategy not only for CCOC but also other cancers expressing CTH. Differences in EO3001 response were noted between CCOC -/+ ARID1A cell lines, with hypoxia reducing EO3001's effects, likely due to changes in glycolysis and OXPHOS. Exploiting OXPHOS dependence in ARID1A-deficient CCOC, EO3001 could be a promising therapeutic approach.
These findings highlight the PuMA assay’s potential for testing novel treatments for metastatic CCOC.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat
Human fallopian tube organoid models of high grade serous ovarian cancer initiation
No full textOvarian cancer is the sixth most lethal cancer in women. Out of all ovarian cancers – the high grade serous (HGSC) subtype is the most common and deadliest. This cancer originates from fallopian tube secretory cells and has several putative precursor lesions that have been described in the fallopian tube. These precursors show a spectrum of morphology from normal to abnormal and have been identified in patients with and without cancer. Genetically, HGSC is characterized by widespread genomic instability and ubiquitous TP53 mutation. A variety of model systems have attempted to interrogate how carcinogenesis occurs in the fallopian tube, but few examine the effect of TP53 mutation alone, and virtually all use other species to model human disease. In this thesis I describe the development, validation, and characterization of normal human fallopian tube derived organoids that have had exogenous mutations in TP53 introduced by the targeted cutting of the Cas9 nuclease. Analysis of these organoids was undertaken by live-cell imaging, histochemistry, and single-cell genomic sequencing. Relative to controls, mutant organoids showed normal morphology and growth but abnormal p53 immunohistochemistry. Single-cell sequencing further validated the strength of this model system and revealed comparable genomic stability between TP53 mutants and controls. These findings make sense in light of p53 signature precursor lesions’ normal morphology and low proliferative index; and support the idea that TP53 mutation alone is not sufficient to cause carcinogenesis in the fallopian tube. Reagents and theoretical approaches to interrogating the effects of TP53 missense mutations and homologous recombination deficiency pathway members have also been designed. This research supports genetic perturbation of patient-derived normal organoids as a robust and promising avenue of research.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Fibroblast activation protein and the progression of low-grade serous ovarian cancer
No full textEpithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Among the five histologically distinct types of EOC, low-grade serous ovarian carcinoma (LGSC) is among the rarest with an incidence of 5%, compared to the high-grade serous ovarian carcinoma (HGSC) standing on an incidence of 70%. The low incidence of LGSC has contributed to it being underrepresented in EOC research endeavours. The current therapeutic options provided to LGSC patients are chemotherapy, either paclitaxel, carboplatin, or a combination. Yet, the disease is refractory to these treatments. As conventional treatment is not effective at prolonging survival of LGSC patients, alternative approached must be explored. Here, we investigate the proteome of LGSC, its precursor lesion serous borderline tumour (SBT) to identify novel prognostic and therapeutic targets against LGSC. Fibroblast Activation Protein (FAP) was identified as the most differentially abundant protein in LGSC relative to SBT. FAP is widely expressed and recognized protein in epithelial cancers, marking the presence of a cancer-associated fibroblasts (CAFs). FAP+ CAFs have been associated with the T regulatory cells, a population of immune cells that foster an immunosuppressive microenvironment, in breast cancer. That correlation was also observed in LGSC through scoring of FOXP3+ cells via immunohistochemistry. Our proteomic cohort reveals that FAP is present in the stroma of LGSC and is correlated with T regulatory cells, presenting the potential to translate treatment approaches that target FAP to this cancer.Science, Faculty ofGraduat
Murine models of DICER1 syndrome-associated cancers : development and characterization
No full textDICER1 syndrome is a rare genetic disorder that predisposes individuals to hereditary cancers, with tumours arising in organs such as the lung, gynecologic tract, thyroid, kidney, and brain. This syndrome is driven by germline loss-of-function mutations in the DICER1 gene, typically inherited in an autosomal dominant manner. Tumours acquire somatic mutations affecting the RNase IIIb domain on the alternate allele, resulting in compound heterozygosity. These mutations impair DICER1's ability to cleave precursor miRNAs, disrupting microRNA biogenesis, an essential process for gene expression regulation. While the role of DICER1 mutations in tumour initiation is well-established, the mechanisms underlying tumour progression remain poorly understood. To address this gap, a genetically engineered mouse model harbouring a specific Dicer1 RNase IIIb mutation was developed. Tumours in this model displayed histological and molecular characteristics resembling those of human DICER1-associated cancers, including renal cystic nephroma, Wilms tumours, and anaplastic sarcoma of the kidney. Exome sequencing indicated that tumour progression may be influenced by accumulating secondary mutations, such as alterations in Trp53 and Kras. Syngeneic models and cell lines derived from these tumours were established, offering a robust platform to evaluate therapeutic strategies. This study demonstrates that the Dicer1 RNase IIIb mouse model faithfully recapitulates DICER1-associated tumourigenesis and progression. By enabling the investigation of tumour biology and therapeutic testing, this model provides a foundation for identifying targeted treatments for DICER1 syndrome-associated cancers.Medicine, Faculty ofMedical Genetics, Department ofGraduat
The proteomic and metabolomic characterization of clear cell ovarian cancer : towards better management strategies
No full textClear 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.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat
Investigation into the early pathogenesis of lynch syndrome associated endometrial cancer
No full textEndometrial cancer is the most common gynecologic malignancy in Canada with increasing rates of both incidence and mortality. Historically, histopathological subtyping of endometrial cancer was employed but was determined to be less than accurate at patient risk stratification. Recent advances in molecular subtyping have revealed mismatch repair deficient endometrial cancer as the second most deadly subtype. Mismatch repair deficient endometrial cancers may be acquired through somatic or germline aberrations to the four mismatch DNA repair genes: MLH1, PMS2, MSH2, and MSH6. Germline mutations to mismatch DNA repair genes results in Lynch Syndrome, an autosomal dominant hereditary cancer predisposition syndrome which increases the risk of endometrial cancer in affected individuals, however, the pathogenesis of Lynch Syndrome associated endometrial cancer has yet to be fully elucidated. To investigate the early pathogenic events following the introduction of MLH1 and PMS2 deficiencies, both in vivo and ex vivo models were utilized. A cohort (n = 6) of archival FFPE tissues, taken from prophylactic hysterectomies performed for Lynch Syndrome patients with germline MLH1 and PMS2 mutations, were immunohistochemically profiled for mismatch repair deficient endometrial glands. MLH1 deficient benign endometrial glands were identified in four cases with a novel report of zonal MLH1 deficient benign endometrial glands. In one case with zonal MLH1 deficient endometrial glands, multiplex immunohistochemical staining based panel was used to quantify endometrial epithelial cell differentiation by examination of secretory, ciliated, and proliferative cell types in both MLH1 proficient and deficient endometrial glands where no significant difference in cell type proportion was observed. MLH1 and PMS2 deficient endometrial organoid models were established using benign hysterectomy tissues and CRISPR-Cas9 mediated gene editing. Single cell RNA sequencing of MLH1 and PMS2 deficient endometrial organoids revealed no significant alterations in MLH1 deficient organoid cells and slight alterations to epithelial mesenchymal transition genes in PMS2 deficient organoids. Altogether, this data suggests that mismatch repair deficiency does not result in immediate changes to gene expression or cell differentiation in endometrial glands and that progression towards endometrial cancer is reliant on the accumulation of mutations.Medicine, Faculty ofGraduat
Investigating molecular mechanisms of dedifferentiation and mutations in dedifferentiated endometrial carcinoma
No full textDedifferentiated endometrial carcinoma (DDEC) is an aggressive endometrial cancer composed of two components: a well differentiated component and an undifferentiated component thought to arise clonally from the well differentiated component through dedifferentiation. In some cases, these tumours are presented as pure undifferentiated endometrial carcinomas (UECs) likely due to outgrowth of their undifferentiated components. A majority of DDECs and UECs are known to have defects in mismatch repair (MMR). They are also frequently deficient in components of SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes, a family of chromatin remodelling complexes with roles in differentiation, and p53, both of which are known tumour suppressors. In this thesis, I assembled a cohort containing 88 DDEC/UEC cases, the largest cohort to date. The protein expression levels of SWI/SNF components, MMR, and p53 were evaluated by immunohistochemistry (IHC). Multiple SWI/SNF members were studied, including SMARCA4, ARID1A, ARID1B, SMARCB1, and SMARCA2 to determine the extent of the abnormality of SWI/SNF components and p53 in DDEC/UEC. The prognostic value of these mutations was also assessed. I found that SMARCA4/A2 dual loss, ARID1A/1B dual loss, and p53 abnormality occurred in 10/88 (11%), 25/88 (28%) and 53/87 (40%) cases, respectively. These three abnormality groups were not statistically associated in DDEC/UEC, representing distinct subgroups of DDEC/UECs. In addition, these defects occurred more frequently in the undifferentiated component, suggesting that they have roles in histologic dedifferentiation. However, neither SWI/SNF, MMR, nor p53 abnormality were found to be prognostic of survival.
Lastly, the effect of ARID1B re-expression on the transcriptomes of ARID1A/1B dual deficient undifferentiated endometrial cancer cell lines was analyzed, to identify molecular pathways by which ARID1B loss may drive dedifferentiation. ARID1B re-expression did not consistently result in the upregulation of gene sets associated with differentiation across the two cell lines examined, indicating that the loss of ARID1B in an ARID1A-deficient context may not be sufficient to drive dedifferentiation in vitro.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat
The magnitude of androgen receptor positivity in breast cancer is critical for reliable prediction of disease outcome
No full textPublished first March 7, 2018.Abstract not availableCarmela Ricciardelli, Tina Bianco-Miotto, Shalini Jindal, Lisa M. Butler, Samuel Leung, Catriona M. McNeil, Sandra A. O'Toole, Esmaeil Ebrahimie, Ewan K.A. Millar, Andrew J. Sakko, Alexandra I. Ruiz, Sarah L. Vowler, David G. Huntsman, Stephen N. Birrell, Robert L. Sutherland, Carlo Palmieri, Theresa E. Hickey, and Wayne D. Tille
Case study of California Proposition 34
No full textThe problem with the death penalty statute in California is the cost impact it has on taxpayers.The cost impact was one of the goals for Proposition 34 to address. If Proposition 34 would have passed, taxpayers could have saved about $1 billion every five or six years (Williams, 2013). The purpose of this study is to analyze and explain why California Proposition 34 failed. The author utilizes Stone’s (2002) research of policy paradox to explain why some policies fail. The author’s analysis of Stone’s research reveals that public interest and influence are potent forces in a policy decision. The research also indicated that influence has to be effective in reaching out to people. The author feels that the two campaigns were effective in utilizing measures in influencing the people they reached. The two campaigns were effective in campaigning to carry out their purpose and goals. However, the Californians for Justice and Public Safety were the winner of the 2012 Proposition 34 election which was designed to protect California and preserve California’s death penalty law. The author believes that the “no on 34” campaign worked on framing that the “yes on 34” campaign was about saving money and that it did not protect justice for the people of California. This counterargument worked because it led voters to believe that Proposition 34 was an injustice. In addition, the author believes this argument led to the “no on 34” campaign victory in the 2012 Proposition 34 election. As a result, the Californians for Justice and Public Safety was the more influential campaign which led them to win the 2012 Proposition 34 election. Recommendations consists of reaching out to Governor Jerry Brown and that in future attempts to abolish the death penalty, supporters should try to convince the sitting governor to sign a bill to abolish capital punishment, and the “yes on 34” campaign should focus on justice arguments rather than economics by citing facts that justice would be carried out on their ballot initiative
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