Alabdullah, Mussalam Layth
(2021)
Exploring the role of cell signalling and DNA damage pathways in primary surgically resected ovarian cancer.
PhD thesis, University of Nottingham.
Abstract
Background:
Ovarian cancer (OC) is the most lethal gynaecologic malignancy. It is a heterogeneous disease at the molecular, histological and clinical levels. It encompasses a collection of neoplasms, which have distinct molecular profiles and clinicopathological features. Despite there being a wide variety of OC subtypes, all are treated similarly with no categorisation. In addition, although there is initial sensitivity to chemotherapy especially the platinum-based regimens, majority of patients developed resistance to treatments and subsequent recurrence of the disease. Tremendous efforts have been made to identify novel pathways and prognostic biomarkers for potential therapeutic strategies. However, to date these parameters are not sufficient to reflect the degree of heterogeneity and to guide management decisions at individual patient level. Therefore, further refinement of the existing prognostic tools is urgently needed. Several studies have shown that the major histologic subtypes of OC have different risk factors, genetic susceptibilities, and clinical response to therapy. Moreover, they are all different at the molecular level. In this current study, we hypothesised that both cellular and DNA damage signalling pathways can influence ovarian tumour progression and prognosis. This study aimed to investigate a large cohort of surgically treated primary OC patients with their available, electronically recorded follow-up data and test the aberrant expression of different proteins directly related to cell signalling and DNA damage pathways. This is to further understand not only the biology of the tumour, but also to identify their clinical significance. Furthermore, to identify which of these key proteins merits to be dominantly functioning when co-expressed.
Patients and Methods:
This study included 525 primary OC cases treated surgically at Nottingham University Hospitals (NUH) between 1997 and 2010. Clinicopathological and outcome data was collected. All cases were histologically reviewed, and the representative tumour blocks were retrieved. Tissue microarrays (TMAs) were constructed from the selected and marked tumour tissue sites. Then, assessment of two main pathways with known intimate relations to cancer development and progression, cell signalling and DNA damage repair (DDR) was performed. Key pathways’ protein expression status was investigated then, correlated with the clinicopathological variables, platinum sensitivity and patients’ outcome. For this purpose, we used an exploratory design for this research, pursuing both the clinical and pre-clinical experiments. Immunohistochemistry (IHC) was utilised to stain a number of key targets: 1) cell signalling pathway proteins (p-AKT, p-mTOR, p-4EBP1, p-P70S6K), and (ATM, PTEN, P85ἁ, XIAP) and 2) DDR pathway (PARP1, XRCC1, polβ), and (LIG1, LIG3, LIG4), then their prognostic value was analysed. Preclinical analysis by gene knockdown using siRNA, nuclear and cytoplasmic extraction and western blot (WB) in a panel of platinum sensitive and platinum resistant OC cell lines (A2780 and A2780cis, respectively) were also utilised.
Results:
The majority of cell signalling pathway members (p-AKT, p-mTOR, p-4EBP1, p-P70S6K) and (ATM, P85ἁ and XIAP) showed positive associations with poor prognostic variables and shorter survival in the whole (unselected) cohort and in platinum sensitive tumours. Interestingly, p-P70S6K negative expression was highly significantly associated with improved sensitivity to platinum agents. On the other hand, p-AKT overexpression showed significance with resistance to chemotherapy and higher risk of residual disease. In addition, p-AKT and p-4EBP1 were independent predictors of unfavourable outcome. Cases with low/negative expression of both AKT and 4EBP1 exhibited a significantly improved survival.
Interestingly, we have identified PTEN as a marker of improved outcome. Its high expression was correlated with favourable patients’ prognosis. Interestingly, ATM and XIAP were additional independent factors for progression free survival (PFS). Importantly, when ATM status was positive, PTEN and p85α lost their significance on the patients’ outcome. Similar findings were observed with XIAP positivity. This could imply PTEN and p85α context specific function in relation to the corresponding expression status of ATM and XIAP.
Regarding the DNA signalling pathway proteins, PARP1, XRCC1 and polβ, all showed negative association with favourable outcome in the unselected (whole) cohort and platinum sensitive cases. In addition, PARP1 and XRCC1 positive cases exhibited a higher chance of residual disease after surgery. Interestingly, negative XRCC1 expression improved the response to platinating agents. Importantly, in multivariate analysis we have identified XRCC1 as an independent factor for poor recurrence outcome. Preclinical XRCC1 knockdown increased PARP1 levels, a novel finding that may identify a group of patients who may preferentially benefit from PARP inhibitor therapy.
When the biological significance of the three human ligases was investigated, both LIG1 and cytoplasmic LIG3 were associated with poor prognostic variables and adverse clinical outcome in the unselected and platinum sensitive cohorts. Overexpression of both proteins significantly enhanced the risk of residual tumour following surgery. Interestingly, low/negative cytoplasmic LIG3 improved the response to platinum therapy. LIG4 overexpression was a predictor of prolonged survival and improved response to chemotherapy. Importantly, in this thesis we have identified that LIG3 prognostic impact is dependent on its cellular sub-localisation status. Its cytoplasmic form exhibited tumour-encouraging qualities and worse patients’ outcome, unlike its nuclear isoform. Preclinically, we observed a significant increase in the cytoplasmic fraction of LIG3 after cisplatin treatment. Therefore, we speculate that the cytoplasmic LIG3 can influence the response to platinum therapy and may contribute to the development of chemotherapy resistance. In addition and importantly, LIG1 showed an independent poor prognostic significance in multivariate analysis, another novel finding in this research.
Conclusions:
To conclude, this thesis illustrates that OC is can exhibit different variations in the expression of tumour key protein markers, which can be utilised to predict tumour behaviour and stratify patients for therapy. This explorative study combined both clinical and pre-clinical experiments, and provided novel findings in primary ovarian tumour cases that can be utilised to improve the disease prognosis. In the future, elaborated and well-designed functional studies may reveal better insights on the contributions for each identified biomarker candidate, and their relevant pathway in driving or inhibiting platinum resistance mechanisms, and improve the outcome in primary OC patients.
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