Zhang, Siwei
(2018)
Calpain in ovarian cancer progression and chemotherapeutic response.
PhD thesis, University of Nottingham.
Abstract
The calpain system is associated with cancer chemotherapeutic response in both in vivo and in vitro studies. Previous immunohistochemistry (IHC) data conducted in our group indicated that high calpain-2 expression was associated with both the resistance to platinum-based adjuvant chemotherapy and worse patient outcome; moreover calpain-2 appeared as an independent prognostic factor in multivariate analysis.
To test the hypothesis that conventional calpain subunits, especially calpain-2, are associated with the chemo-resistance of ovarian cancer cells to platinum-based chemotherapy (cisplatin and/or carboplatin), five ovarian cancer cell lines, with varying platinum-based chemotherapy sensitivities, were chosen as in vitro models: the platinum-sensitive A2780 cells and its resistant counterpart A2780-cis cells; the platinum-resistant SKOV3 cells; and the platinum-sensitive PEO1 cells and its platinum-resistant counterpart PEO4 cells. Western blotting was used to assess the expression of the conventional calpain subunits (i.e. calpain-1, -2 and -4) and calpastatin in this panel of cell lines. Calpain activity was regulated by inhibitor calpeptin and calpain-2 short hairpin RNA (shRNA) was used in attempt to specifically downregulate calpain-2 expression. Calpain activity was assessed by an activity assay using fluorogenic peptidase substrate t-BOC. The role of calpain in proliferation and resistance to platinum-based chemotherapy were examined in vitro using growth curves and colony formation. Moreover the study of calpain-4 expression was added into the current project, in addition to verifying the association between the expression of calpain-1, -2 and calpastatin and clinicopathologic variables (e.g. chemo-resistance and patient outcome) by standard immunohistochemistry (IHC) with a larger patient cohort (n=575). To test the hypothesis that conventional calpain subunits and calpastatin are associated with ovarian tumour metastasis, the effect of calpain inhibition (by calpain inhibitors) and activation (by calcium ionophore) on ovarian cancer cell migration was examined using haptotaxis (scratch wound) migration assay. Based on information from 2016 FASEB calpain conference, microtubule-associated protein 4 (MAP4) and spleen tyrosine kinase (Syk) appeared as potential calpain-related proteins associated with angiogenesis and epithelial-mesenchymal transition. Using IHC, their protein expression (i.e. MAP4 and Syk) was assessed and their associations with clinicopathological variables in ovarian cancer patient samples were studied; besides, their correlations with conventional calpain subunits and calpastatin, together with EMT (epithelial-to-mesenchymal transition)-associated proteins and angiogenesis-associated proteins (n=87, data provided by Dr S. Deen) were analysed.
Significant variations of calpain system protein expression levels were observed between the different cell lines. Among the 5 cell lines, A2780 and A2780-cis cells (likely to be endometrioid carcinoma cell lines) expressed very low levels of the conventional calpain subunits and calpastatin; whilst PEO1 and PEO4 cells (high-grade serous carcinoma cell lines) expressed comparatively higher level of these proteins. Thus, different expression of the calpain system seemed to be associated with ovarian cancer histological subtypes, which was supported by the IHC study. No significant difference of the calpain system expression was detected and calpeptin caused a similar inhibition of cell proliferation between chemo-sensitive ovarian cancer cells and their resistant counterparts. Because SKOV3 and PEO4 cells expressed the highest levels of calpain-2, shRNA was used for specific knockdown of calpain 2 in these two cell lines, unfortunately numerous attempts proved unsuccessful. Although with the optimised concentration and treatment duration, calpeptin could inhibit approximately 30% of calpain activity, down-regulation of calpain activity via calpeptin could not sensitise ovarian cancer SKOV3, PEO1 and PEO4 cells to cisplatin and carboplatin. Hence, to revisit the question as to whether conventional calpains and calpastatin are associated with chemoresponse and patient survival, a larger cohort was used to validate the previous study. In the current study, the expression of the conventional calpain subunits and calpastatin were positively associated with each other. Calpain-2, -4 and calpastatin expression were associated with overall survival (OS) but none of them was an independent marker of OS in multivariate analysis. Neither the conventional calpain subunits nor calpastatin expression was found associated with resistance to platinum-based adjuvant chemotherapy. Since low calpain-1 expression was associated with low tumour stage, cellular processes that involved in cancer spread were studied. Again calpeptin was used for the inhibition of calpain activity, but no significant inhibition was observed on ovarian cancer cell migration. In contrast, upregulating calpain activity by A23187 using optimised concentration was found able to significantly inhibit the migration of SKOV3 cells. The recently found calpain-related proteins MAP4 and Syk were then included into the current study. Like calpain-1, neither MAP4 nor Syk expression was associated with patient outcome. Next, cases were grouped by clinicopathological variables for the examination of the association between the protein expression and survival; in such a case only high nuclear Syk expression was significantly associated with better patient outcome in certain subgroups. Low calpain-1 expression was associated with low tumour stage, so were MAP4 and Syk expression. MAP4, Syk and calpain-1 expression were significantly associated with tumour histological subtypes and their expression were significantly correlated with each other. Integrin α2β3 was moderately correlated with calpain-1, MAP4 and cytoplasmic DARC expression.
In conclusion, although in both the previous and the current study, calpain-2 expression was adversely associated with OS of ovarian cancer patients, the current results did not support the initial hypothesis that calpain can sensitise ovarian cancer cells to cisplatin/carboplatin. The roles that calpain system played in cancer cell haptotactic migration appeared to vary with cell context. Calpain-1, MAP4 and Syk expression were significantly correlated with each other and were closely related to ovarian cancer spread.
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