No description available.

No description available.

No description available.

Diethylstilbestrol (DES) is one of the synthetic estrogen available today for therapeutic use. It is also referred to as a carcinogenic agent, with a great number of side effects reported between maternal ingestion of DES during pregnancy, as a postovulatory agent to prevent implantation and possible occurrence of carcinoma in the progeny of women known to be aborterse. In the present study, experiments were done to determine the effects of DES on the estrogen titer of the maternal, fetal and neo-natal plasma ovarian homogenates of female Long-Evans rats. Adult and 30-day old rats were treated via stomach intubation with 35 mg/2cc/kg body wt of DES. The estrous cycle of the rats was monitored and vaginal smear cell counts were determined in 0.5 cc saline smear volume. Radioimmunoassay was used to determine the estradiol levels in the plasma and ovarian homogenates. Results showed no changes in the estrous cycle synchrony of the maternal rats, while the estrous cycle of the treated 30-dayold rats has synchronous. The vaginal smear cell count was significantly greater in the two groups of treated rats studied than in the controls. Comparing the mean estradrol value for the plasma and ovarian homogenates in the maternal rats, an obvious increase in the plasma E2 level was obtained as compared to small, decrease in the mean value of ovarian E2 level. There was no difference in the mean value of pooled ovarian homogenate E2 level of the control saline, control and DES-treated 19-1/2-day old rats. A significant decrease in the plasma E2 mean value of 30-day old treated rats was obtained when compared to both control groups. There was no significant increase in ovarian homogenate E2 level of both treated and the control groups. In light of these results, DES has a secondary effect, asynchronous cell proliferation in the vaginal epithelia leading to prolonged specific stages of the estrous cycle of the young adult female rats. Also, DES causes an increase in plasma E2 levels of treated maternal rats and a decrease in plasrna E2 levels of 30-dayold rats. Finally, DES has no effect on the estrogen level of the ovarian homogenates of the maternal, fetal and 30-clay-old young Long-Evans female rats.

The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.

The PI3K/Akt/mTOR and MAPK pathways are frequently altered in ovarian cancer cells, making them potential candidates for targeted therapy. A more complete understanding of the complex interactions between the different proteins within the two pathways would assist in developing more effective treatment strategies to help overcome therapy resistance. The purpose of this project was to understand the phosphoproteomic changes in response to PI3K/mTOR inhibition in ovarian cancer cells and to identify potential mechanisms that may lead to targeted therapy resistance. To investigate the effect of inhibiting PI3K/mTOR at the cellular level in ovarian cancer, PI3K (LY294002), mTOR (rapamycin) and dual PI3K/mTOR (BEZ235) inhibitors were used to treat a panel of ovarian cancer cell lines. All tested cells, irrespective of PI3K/Akt/mTOR and MAPK pathways mutational status, responded to the three inhibitors. BEZ235 treatment produced greater cell inhibition than the monotargeted agents, while PTENmutated cell lines were more responsive to mTOR blockade than inhibition of PI3K alone. The phosphoproteomic changes in the cell lines were evaluated over a time course after treatment with the inhibitors, stimulated by heregulin, and studied using reverse phase protein array analysis. The results revealed that the decreased expression of pAkt (Thr308) appears to be a biomarker of sensitivity for LY294002 and BEZ235 in both PEO4 and A2780 cells, while upregulation of pAkt (Ser473) is an indicator for effective rapamycin treatment within the same cell lines. Increased pAkt (Ser473) expression after rapamycin treatment in PEO4 cells is believed to be due to the S6K1-mTORC2-Akt feedback loop. It was observed that pERK was upregulated upon BEZ235 treatment, which suggested the presence of cross talk between the PI3K/Akt/mTOR and MAPK pathways. A combination of BEZ235 and PD-0325901 (MEK inhibitor) treatments inhibited both pAkt (Ser473) and pERK, which also produced significant inhibition in cell proliferation compared to monotherapy treatment. The data also revealed a novel finding in ovarian cancer that prolonged (24h) treatment with rapamycin sensitises mTORC2 in PEO4 cells under heregulin stimulation. Moreover, network correlation and clustering analysis using the phosphoproteomic data identified significant correlations between the expression of pmTOR (Ser2481), and both p-cRaf (Ser259 and Ser338). Sin1 knockdown was performed in PEO4 cells and showed significant downregulation in the expression of pAkt (Ser473) and upregulation in pERK expression, indicating the role of Sin1 to regulate both the PI3K/Akt/mTOR and MAPK pathways potentially via mTORC2 and Ras. Phosphoproteomic profiling was performed on 469 ovarian cancer tissue samples using TMA and IHC analysis. Several significant associations were discovered between the phosphoproteomic data and the different clinicopathological parameters. High expression of pmTOR (Ser2448) was correlated with poorer overall survival in patients with ovarian endometrioid carcinoma compared to patients with low expression (p < 0.024). This implies that pmTOR (Ser2448) expression may potentially be a prognostic marker for patients with ovarian endometrioid carcinoma. In conclusion, I present dynamic phosphoproteomic profiling of the PI3K/Akt/mTOR and MAPK pathways in ovarian cancer, suggesting novel feedback loops and cross talk that could play a role in resistance mechanisms to these therapies. Combination treatment showed an additive effect on cell growth offering an approach to overcome drug resistance.

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.

No description available.

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.