The clinical significance of cyclin E1 deregulation in high grade serous ovarian cancer and basal like breast cancer
AuthorAziz, Diar G Aziz
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2022-04-28.
© 2019 Diar G Aziz Aziz
High grade serous ovarian cancer (HGSOC) and basal like breast cancer (BLBC) are genomically unstable and aggressive cancers that frequently co-occur and share common molecular features. Of these molecular characteristics are P53 inactivation occurring in almost all cases of HGSOC and BLBC, BRCA1/2 inactivation reported in more than 50% of both cancers and CCNE1 amplification reported in up to 30% and 8% of HGSOC and BLBC respectively. Both HGSOC and BLBC are currently grouped in many clinical trials to test new drugs or drug combination, for instance, PARP inhibitors in the context of BRCA1/BRCA2 mutation. We asked whether deregulated cyclin E1 (CCNE1 amplification and/or its encoding protein, cyclin E1, overexpression) is an additional biomarker that can potentially be used to group patients with both diseases for therapeutic purposes. We studied two well characterised cohorts of 262 HGSOC and 222 familial breast cancer (BLBC enriched) samples of formalin fixed paraffin embedded sections. HGSOC and the BLBC enriched cohort were from patients enrolled in the Australian ovarian (AOCS) and the Kathleen Cuningham Foundation Consortium for research into Familial Breast cancer (KConFab) respectively. Using automated tissue based assay and an in situ hybridization probe that spans 19q12 locus harbouring CCNE1, we assessed the level of CCNE1 amplification. We also assessed the expression of cyclin E1 and a cyclin E1 degradation associated protein FBXW7 and a cyclin E1 deubiquitinase, USP28, by immunohistochemistry, as possible drivers of high cyclin E1 expression in amplified and non-amplified cyclin E1hi subsets. We also assessed the expression of URI1 in our HGSOC cohort. URI1 is a protein encoded by the URI1 gene which co-localise with CCNE1 on 19q12 locus. In HGSOC, we identified seemingly two separate subsets of cyclin E1hi tumors that have different pathological and biological characteristics as well as different clinical outcomes. These are the amplified/cyclin E1hi group that had amplification and high expression of cyclin E1, low expression of FBXW7, higher genomic instability, intact BRCA1/2 and worse outcome. The other is the non-amplified/cyclin E1hi tumors that typically had high expression of cyclin E1 in the absence of amplification, high USP28 expression, lower genomic instability, more prevalent BRCA1/2 loss and more favorable outcome compared to the amplified group. Next we assessed cyclin E1 deregulation in the overlapping groups BRCA1 mutant breast cancer and BLBC. Both subtypes had significantly higher expression of cyclin E1 and amplification compared to other breast cancer types. However, the intensity of cyclin E1 expression and level of 19q12 amplification were lower in BLBC compared to those observed in HGSOC. Moreover, in BRCA1 mutant breast cancer and BLBC patients, only high expression of cyclin E1 was associated with lower overall survival while amplification did not seem to impact outcome. These observations were further supported by our meta-analysis that included our cohorts as well as other published datasets. In the meta-analysis, both CCNE1 amplification and cyclin E1 expression were found to be adverse prognostic factors in HGSOC while only high expression was associated with worse survival in BLBC patients. In fact, both amplified and non-amplified cyclin E1hi BLBC subsets shared almost all cyclin E1 deregulation associated features as well as many features with the non-amplified cyclin E1hi HGSOC subset. Of these are the prevalence of high expression of the cyclin E1 deubiquitinase, USP28, BRCA inactivation, the lower genomic instability and cyclin E1hi linked adverse outcome. In order to provide better therapeutic options for cyclin E1hi BRCA1 mutant breast cancer/BLBC patients, we sought to further assess mechanisms behind the co-occurrence of cyclin E1 overexpression and BRCA1 inactivation. Using the KConFab cohort we have found that BRCA1 loss correlated with decreased phosphorylation of cyclin E1, on Threonine 62, assessed by immunohistochemistry. We also showed by in vitro analysis that BRCA1 loss in cell lines led to cell cycle specific stabilisation of cyclin E1 by reducing cyclin E1 T62 phosphorylation. Conversely, BRCA1 overexpression increased T62 phosphorylation. Overexpression of cyclin E1 with an inactivated T62 site, to mimic loss of phosphorylation, increased cyclin E1 stability and resistance to Paclitaxel. These findings suggest that BRCA1 regulates cyclin E1 stability in breast cancer cells via regulating T62 phosphorylation. We next assessed a combination therapy that target cyclin E1 and BRCA1 inactivation using CDK2 and PARP inhibitors. CDK inhibitors are suggested to induce DNA damage and therefore we hypothesised that CDK2 inhibition would enhance sensitivity of BRCA1 deficient cells to PARP inhibition. Our finding is that CDK2 inhibition induced DNA damage and synergised with the PARP inhibitor Rucaparib in BRCA1 mutated cell lines. Combination treatment of xenograft are in progress but the preliminary data is supportive of our hypothesis. Our results propose a new therapeutic strategy for BRCA1-mutant breast cancer/BLBC by combining CDK2 and PARP inhibitors to enhance synthetic lethality. As this group shares similarities with non-amplified cyclin E1hi HGSOC subset, we suggest that this combination is likely to be effective in the comparable HGSOC subset.
KeywordsHigh grade serous ovarian cancer (HGSOC), Basal like breast cancer (BLBC), cyclin E1 degradation, CCNE1, cyclin E1, FBXW7, DNA damage response (DDR), PARP inhibitors, CDK inhibitors, Synthetic lethality, BRCA1, BRCA2, USP28, T62, targeted therapy, synthetic lethality, genomic instaibility
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