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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:735642 |
| Date | January 2016 |
| Creators | Francis, Kyle Evan |
| Contributors | Langdon, Simon ; Harrison, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/25956 |
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