• The Role of L-type Voltage Gated Calcium Channels in Ovarian Cancer

      Shiva, Subramanam; Luderman, William (University of DerbyUniversity of Derby, 2021-03)
      Ovarian cancer is the most lethal gynaecological malignancy. Of the four ovarian cancer subtypes - serous, mucinous, endometroid and clear cell - serous ovarian cancer is the most common, comprising around 70% of cases. The median stage of diagnosis of ovarian cancer is stage III and patients present with widespread metastasis, usually facilitated by peritoneal fluid accumulation in the abdomen (ascites). Live single cancer cells and aggregates from the ascites represent a population with the potential to become metastatic and are thought to be the main contributors to disease recurrence, displaying increased chemoresistance after traditional first line therapy, which consists of extensive tumour debulking followed by combination platinum and taxane chemotherapy. For this reason, research into potential therapeutic targets in this important cell population is critical for development of more efficacious treatments for ovarian cancer. The opportunity to re-purpose existing pharmaceuticals for use as chemotherapeutics is an idea that has gained traction recently in the medical research field. This method circumvents the requirement for lengthy target identification and validation and extensive toxicity testing. One class of drugs that are a possible candidate for use against ovarian cancer are the dihydropyridines, which are antagonists of voltage-gated calcium channels (CaV). In ovarian cancer, CaV have already been shown to play a role in malignant behaviours such as migration and proliferation, although most expression and functional data in the literature are from ovarian cancer cell lines. There has been very little research on the role of CaV in primary ovarian cancer cells. This work addresses the question of whether CaV are expressed in primary ovarian cancer cells and whether channels in ovarian cancer cells are functional, particularly in cells derived from malignant ascitic fluid. Here, the results from immunohistochemical staining of ovarian tumour sections and RT-qPCR using normal ovaries, tumours and cells derived from malignant ascitic fluid suggest that in the majority of cases, CaV1.2 and CaV1.3 become expressed in ovarian cancer cells only after cells have been shed from the primary tumour. CaV1.2 and CaV1.3 mRNA is expressed in ascites derived cells, although the highest expression of these mRNAs was seen in a sample from a patient with mucinous ovarian cancer. In serous ovarian cancer cells from ascites, CaV1.2 protein was shown with flow cytometry to be highly expressed and immunofluorescent staining confirmed that this expression is localised to the nucleus. Functionally, dihydropyridine antagonism with nifedipine was found to prevent cell migration only in a 2-dimensional wound-healing model, whereas invasion of cell lines and ascites derived primary cells into basement membrane extract was unchanged. Cell lines display differing apoptotic responses to nifedipine, which triggers apoptosis in SKOV-3 but no response in OVCAR-8 at the same concentration. To assess channel functionality, fluorescent measurement of cytosolic Ca2+ flux using Fluo4-AM was performed on cell lines in the presence of the CaV agonist Bay K8644 and patch clamp electrophysiology was performed on cell lines and malignant ascites derived cells. Both of these techniques confirmed that no detectable L-type CaV current is present in the ovarian cancer cell lines. CaV current was observed in cells derived from malignant ascites, from the mucinous sample. Transient receptor potential (TRP) currents were also detected in OVCAR-8 cells, as well as single cells and spheroids from the ascites derived mucinous sample and were most likely carried by the Ca2+-selective TRPV5 or TRV6. These results suggest that functional L-type CaV are present in cancer cells from malignant ascites of patients with mucinous ovarian cancer. Although CaV1.2 was nuclear expressed in serous samples, similar research from other cancers has shown that these channels may still function in migration, even when expression is restricted to the nucleus, although nuclear channels would not be amenable to therapeutic targeting. These results fit into a wider context of contemporary research which is placing a greater role for CaV and ion channels in cancer, although more research needs to be performed in ovarian cancer ascites derived cells to determine the possible function of these nuclear ion channels.