CHEK1 inhibitors are currently in clinical trials for their ability to abrogate chemotherapy-induced CHEK1 activation and S phase arrest resulting in cancer cell apoptosis. No studies have yet identified ovarian cancers that could benefit from CHEK1-targeting therapy. I hypothesised that knowledge of CHEK1 and CHEK2 signalling in the DNA damage response can assist in identifying potential biomarkers for platinum responsiveness and CHEK-targeting therapy in ovarian cancer. In vitro studies investigated the CHEK1/2 inhibitor AZD7762 (AZD) and cisplatin (CP) in same patient-derived platinum-sensitive/resistant high-grade serous ovarian cancer cell lines (PEO1/PEO4 and PEO14/PEO23). Cytotoxicity assays confirmed higher CP IC50’s for PEO4 and PEO23 relative to PEO1 and PEO14 cell lines, respectively. AZD was more toxic to PEO1 cells and an additive effect of AZD with CP relative to CP alone was seen. A nontoxic AZD treatment to PEO4 cells sensitised the cells to CP when applied in combination. PEO14 and PEO23 cells had similar cytotoxicity profiles for combination treatments. BRDU DNA synthesis assays and cell cycle analysis revealed increased BRDU incorporation and accumulation in S phase when all cell lines were treated with CP. AZD treatment had a similar effect in PEO14 and PEO23 cells and increased the sub-G1 population, a marker of apoptotic DNA fragmentation, relative to control. Drug combination had no major effect on cell cycle distributions of both PEO14 and PEO23 cells relative to single agents but resulted in BRDU incorporation levels below CP and control levels for PEO14 cells. In PEO1 and PEO4 cells, AZD did not affect the cell cycle or DNA synthesis levels relative to control. Drug combination did not alter the cell cycle relative to CP treatment for PEO1 cells but decreased S phase and increased G2/M and sub-G1 populations in PEO4 cells. This was coupled with a decrease of CP-induced BRDU levels in PEO4 control levels. Apoptotic PARP cleavage/total PARP occurred early in CP treated PEO1 and PEO14 cells. A surrogate CHEK1/2 activity marker, p-CDC2 (Y15), decreased in all lines treated with AZD relative to control. Within PEO1 and PEO4 cells, greatest PARP cleavage was observed with combination treatment and coincided with high p-H2AX (S139), a DNA damage marker. p-CHEK1 (S317) and p-CHEK2 (T68), both ATR and ATM phosphorylation sites during DNA damage, increased for lone drug treatment and, to a greater extent, the combination drug treatments. PARP cleavage occurs across all treatments in PEO1 cells while it only occurs in the combination treatment for PEO4 cells. The latter coincides with a decrease in p-CHEK1 (S296) a CHEK1 autophosphorylation site, p-TP53 (S15), and p-BRCA1 (S1524), a homologous recombination marker, relative to the CP treated sample. In PEO14 and PEO23 cells, lone AZD and combination treatments had similar cleaved PARP/total PARP levels compared to the PEO14 CP treated cells. This was coupled with increased p-H2AX (S139), decreased CHEK1, and decreased CHEK2 autophosphorylation p-CHEK2 (S516). A human ovarian cancer xenograft model identified increases in p-H2AX (S139), CHEK1, p-CHEK1 (S317), p-CHEK2 (T68), and p-BRCA1 (S1524) in the carboplatin responsive cancers. In the paired pre- and post-chemotherapy human ovarian cancer samples, p-CHEK1 (S317) was elevated in post-chemotherapy responsive samples. In the first cohort, high p-CHEK1 (S317) was an independent poor overall survival biomarker and correlated with high p-H2AX (S139), MYC, p-CHEK1 (S296), p-CHEK2 (T68), p-CHEK2 (S516), and p-TP53 (S15). p-CHEK1 (S317) was associated with poor overall survival in serous ovarian cancers within the second pre-treatment ovarian cancer cohort. In conclusion, AZD can induce apoptosis in CP resistant cancer cells by synergising with CP to abrogate the S phase checkpoint, increase DNA damage, and inhibit CHEK1, and BRCA1 function. As a single agent, AZD can induce apoptosis by decreasing CHEK1 levels and CHEK2 activity. p- CHEK1 (S317) is a platinum responsive / poor prognostic biomarker.

The majority of women with ovarian cancer (OC) have advanced disease at diagnosis and 5-year survival rates of less than 25%. Women with stage I disease have significantly better 5-year survival rates of over 90%. Recent large studies using CA 125 and transvaginal ultrasound have failed to improve mortality in a screened population. There is therefore a pressing need for new diagnostic biomarkers in OC. The primary aim of my project, as a first step in developing a diagnostic circulating tumour DNA (ctDNA) biomarker for high grade serous ovarian cancer (HGSOC), was to investigate low-cost high-throughput next generation sequencing assays in plasma samples collected from women with newly diagnosed OC. The secondary aim was to apply these methods to other non-invasive samples including cervical liquid based cytology samples that might contribute to earlier diagnosis or screening for women with OC. ctDNA was detected in 30-49% of women with newly diagnosed OC from the UKOPS (n=54) and CTCR-OV04 (n=156) cohorts using targeted sequencing. Using the trimmed median absolute deviation (t-MAD) score, a quantitative measure of genome wide copy number aberration generated from shallow whole genome sequencing (sWGS) data, ctDNA was detected in 39-41% of the women with newly diagnosed disease. To improve sensitivity of ctDNA detection I developed an optimised method for targeted sequencing that has the potential to lower the limit of detection of ctDNA in HGSOC by 100 fold. I have also shown that the size profile of HGSOC ctDNA fragments is different to that of wildtype DNA fragments and shown that selecting for DNA fragments between 90-150 bp can increase rates of ctDNA detection in HGSOC. ctDNA detection increased to 53-67% of women with newly diagnosed OC using the size selected t-MAD score. I have evaluated the utility of cervical sampling for earlier diagnosis of OC by testing and optimising DNA extraction, library preparation and sequencing methods. I have detected tumour DNA in routine cervical cytology samples collected from women subsequently diagnosed with cervical and endometrial cancers. In summary I have developed methods for ctDNA detection in women with newly diagnosed HGSOC that can be applied and refined in larger prospective studies of women undergoing follow-up for treated HGSOC, women with symptoms suggestive of OC and women at high risk of OC.

Despite the high mortality rate of ovarian cancer, there are few selective biomarkers that detect its progression and none have become successful targets for therapy. A complex microenvironment that promotes angiogenesis, reduces immune responses and alters the integrity of the surrounding matrix is involved through the biology of ovarian cancer. Previous studies done by our lab and collaborators indicated that extracellular threonyl-tRNA synthetase (TARS) is a pro-angiogenic mediator of the ovarian tumor microenvironment, which is secreted in response to inflammatory signals, and actively promotes angiogenesis. In order to better understand the mechanisms underlying the angiogenic effects of TARS in ovarian cancer, it is essential to identify whether it directly affects ovarian tumor growth and invasion. Preliminary evidence indicated that TARS is secreted from ovarian cancer cells in response to TNF-α and TARS exhibits extracellular angiogenic activity. In previous studies, TARS was shown to significantly increase migration of HUVECs in a transwell assay to an extent that was similar to VEGF. The purpose of this project was to establish a role for TARS in tumor progression and its potential as a diagnostic marker using an animal model of ovarian cancer. The hypothesis tested is that TARS plays a key role in the angiogenic and invasive potential of ovarian cancer, and TARS inhibition will reduce the angiogenic effect of tumor cells which is reflected by measurement of intratumor microvessel density (MVD). The study tested the effect of BC194-mediated TARS inhibition on the development of ovarian tumors in ID8 mouse model. We found a positive correlation between TARS expression and ovarian cancer progression, and TARS inhibition with BC194 reduce the progression of ovarian cancer. These data suggest that TARS has an important role in the tumor microenvironment and that TARS inhibition should be further investigated as a therapy for ovarian and other angiogenic cancers.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy in Western countries. A better understanding of the etiology and risk factors associated with this disease is crucial for the development of early detection protocols as well as more effective therapies. Epidemiological data has shown that the risks of EOC are highest among peri- or post- menopause women, while increased parity or the use of oral contraceptives is preventive. These data suggest that alterations in reproductive factors are associated with ovarian cancer risks; however, the molecular mechanisms underlying such a link remain to be understood. For decades, EOC was believed to arise from the epithelium that surrounds the ovarian surface, yet this concept fails to explain the morphological resemblance of ovarian epithelial neoplasms with the epithelial cells of the Müllerian-derived female reproductive tract. Alternative ideas have argued that EOC may originate from extra- or para-ovarian tissues such as the fallopian tube and ovarii rete. Studies of the origin of EOC will provide a better understanding of the disease and advance the protocols for early diagnosis. The aims for this thesis are to establish in vivo ovarian tumor models based on the germ cell deficient Wv/Wv mice that mimicking menopausal physiology. The Wv mice harbor a point mutation in c-Kit, which reduces its tyrosine kinase activity to about 1%, resulting in a premature loss of ovarian germ cells and follicles that recapitulates the initiation of menopause in human. We have developed ovarian tumor models by deleting the tumor suppressor genes p53 or p27kip1 in Wv/Wv mice. We found that both Wv/Wv:p27+/- and Wv/Wv :p27 -/- mice developed ovarian epithelial tumors, which consist of papillary structures lined by hyperchromatic neoplastic cells. Positive Cytokeratin 8 (CK8) staining indicated the epithelial origin of these tumors. In vitro primary cultures of mouse ovarian surface epithelial (MOSE) cells from wildtype, p27+/- and p27 -/- mice further confirmed the growth advantage caused by p27 deficiency. However, neither p27 +/- nor p27 -/- MOSE cells were transformed in vitro, probably due to the compensatory increase of cyclin dependent kinase inhibitor (CKI) proteins including p21, p16, p19. When p53 was deleted unilaterally in the ovarian surface epithelial cells of Wv/Wv:p53 loxP/loxP mice by single administration of Adenovirus containing Cre activity (Ad-Cre), ovarian tumors developed after long latency. The ovarian tumors were significantly enlarged when compared with the uninfected ovary from the same mouse. However, most of the lesions in Wv:p53 conditional knockout tumors was negative for epithelial and follicular markers. In vitro deletion of p53 in MOSE cells significantly increased the proliferation and passage numbers of these cells. A compensatory increase of the CKI protein p16, as well as the cellular senescence level was also observed in p53 deleted MOSE cells, suggesting that p53 deletion alone was not sufficient to bypass p16- mediated tumor defense mechanisms in MOSE cells. Taken together, single deletion of p27 and p53 significantly amplified the phenotype of benign tubular adenomas in Wv/Wv mouse. However, neither p27 nor p53 deletion was sufficient to induce the development of malignant ovarian carcinomas in Wv/Wv mice, probably due to the up-regulation of CKI family proteins such as p21, p16 or p19.

Ovarian cancer remains the deadliest gynecologic malignancy. Current treatment has low efficacy in the long term, leading to low 5-year survival rates of 20-40%. Treatment-free periods between cycles of chemotherapy are accepted in standard treatment. These periods lead to accelerated tumor cell proliferation, angiogenesis and drug resistance development. Studies presented herein show advantages of continuous carboplatin dosing schedule over conventional intermittent regimen, both administered intraperitoneally. Continuous carboplatin therapy blocked acceleration of cell proliferation observed during treatment-free period of intermittent therapy. Moreover, continuous carboplatin led to 57% inhibition of SKOV3 tumors grown intraperitoneally in SCID mice, a significant advantage over the 33% tumor suppression observed with intermittent carboplatin. Immunohistochemical analysis revealed continuous carboplatin led to greater tumor cell death while suppressing tumor cell proliferation and angiogenesis when compared to intermittent administration. These results show that the suppression of tumor growth with carboplatin can be enhanced by the elimination of treatment free periods.

Ovarian cancer remains the deadliest gynecologic malignancy. Current treatment has low efficacy in the long term, leading to low 5-year survival rates of 20-40%. Treatment-free periods between cycles of chemotherapy are accepted in standard treatment. These periods lead to accelerated tumor cell proliferation, angiogenesis and drug resistance development. Studies presented herein show advantages of continuous carboplatin dosing schedule over conventional intermittent regimen, both administered intraperitoneally. Continuous carboplatin therapy blocked acceleration of cell proliferation observed during treatment-free period of intermittent therapy. Moreover, continuous carboplatin led to 57% inhibition of SKOV3 tumors grown intraperitoneally in SCID mice, a significant advantage over the 33% tumor suppression observed with intermittent carboplatin. Immunohistochemical analysis revealed continuous carboplatin led to greater tumor cell death while suppressing tumor cell proliferation and angiogenesis when compared to intermittent administration. These results show that the suppression of tumor growth with carboplatin can be enhanced by the elimination of treatment free periods.

No description available.

This thesis is an investigation of the use of thrombospondin-1 mimetic peptides for the treatment of epithelial ovarian cancer. The current standard of care for women diagnosed with ovarian cancer is surgical de-bulking followed by chemotherapeutics. Initially, this treatment regimen results in a reduction in the primary tumor, unfortunately chemoresistance and disease recurrence are problematic. Recent data has suggested a potential role for anti-angiogenic therapy for the treatment of various cancers. Therefore, the purpose of this study was to investigate the use of mimetics consisting of the anti-angiogenic domain of thrombospondin-1 (TSP-1) for the treatment of epithelial ovarian cancer (EOC) using a mouse model of the disease. The peptides were applied at various stages of tumor progression and a significant reduction in tumor size following treatment was observed. We found that not only were the peptides capable of slowing down tumor progression but they also played a role in reducing the size of established tumors. Treatment with TSP-1 mimetics also resulted in a significant reduction in secondary lesions and ascites fluid in the peritoneal cavity of animals. A significant increase in disease-free survival was also identified following long-term treatment with the peptide. Various histological studies revealed that the anti-angiogenic peptide was in fact inducing apoptosis of the endothelial cells and also re-organizing the vasculature. To determine whether this resulted in increased blood vessel profusion we applied standard chemotherapeutics in combination with TSP-1 mimetics. Experiments with radiolabelled and fluorescent chemotherapeutics demonstrated that pre-treating with TSP-1 mimetics allowed the vasculature to become normalized and resulted in an increased uptake of chemotherapeutics. Lastly, we investigated the mechanism of action of anti-angiogenic peptides. Most of the anti-tumor effects appeared to be due to the apoptotic effects of TSP-1 mimetics on the vasculature. A direct apoptotic effect on epithelial cells also was observed; however, it is uncertain how much of a role this plays. In conclusion, this study was important for identifying TSP-1 mimetic peptides as a potential therapeutic treatment for women suffering from EOC.

Epidemiological studies have described a link between chronic inflammatory conditions, such as diabetes or obesity, and EOC suggesting that systemic inflammation may increase the risk of the disease. The purpose of this study was to identify the impact of prolonged exposure to low-grade inflammation on EOC tumorigenicity. We hypothesized that exposure to this inflammation would accelerate ovarian tumor growth. In vitro, normal and transformed ovarian epithelial cells had limited responsiveness to inflammatory cytokines. In vivo, LPS-induced low-grade chronic systemic inflammation accelerated EOC progression primarily through enhanced angiogenesis. Evaluation of the relationships between chronic systemic inflammation and EOC may provide a role for anti-inflammatory treatment in combinational EOC therapies. Additionally, as the rate of metabolic disorders increases in the Western world the results from this work may facilitate the advancement of complimentary therapeutic interventions for other cancers that are influenced by inflammation.

Ovarian cancer is the deadliest gynaecologic cancer in New Zealand. Its high mortality rate is due to the fact that it is usually diagnosed at an advanced stage. Advanced ovarian cancer is less responsive to current cytotoxic treatment. Thus, there is an urgent need for novel anti-cancer drugs that can improve patient longevity and quality of life. One of the clinical features of advanced ovarian cancer is the growth of secondary tumours due to the highly metastatic nature of the disease. Cancer cells disseminate from the ovary, some form cell clusters that travel through the abdominal cavity by physiological movement of body fluid and then deposit on the abdominal wall and internal organs to generate secondary tumours. The exact mechanisms of how these cells metastasize are unclear, but prognosis typically worsens if levels of vascular endothelial growth factor (VEGF) are elevated. This study investigated the anti-tumour activities of naturally occurring food compounds resveratrol, acetyl resveratrol and (-)-Epicatechin-3-gallate (EGCG), in cell spheroids/clusters of ovarian cancer. It also examined the protein expression of various proteins involved in the NF-κB signalling pathway. This pathway has been suggested to mediate the secretion of VEGF and is a possible target for the naturally occurring compounds. Results show that resveratrol and acetyl resveratrol reduce cell growth and cellular metabolism in a dose-, time- and cell line- dependent fashion. In addition, the reduction of VEGF is also dose-, time- and cell line- dependent. Paradoxically, another angiogenic protein interleukin-8 (IL-8) secretion is increased. Resveratrol and acetyl resveratrol attenuate the expression of NF-κB but this effect is cell line specific. EGCG has limited effect on cell growth, cellular metabolism and the secretion of VEGF and IL-8. These findings suggest that resveratrol and its derivative may have the ability to supress the angiogenic activity of ovarian cancer cells and warrant further in vivo study.