Exploring mTOR signalling as a targeted approach against ovarian cancer

Abstract

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer suffers from a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH-2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. We demonstrate that MDAH-2774 cells have higher proliferative capacity than SKOV3 cells, suggesting different inherent signalling capacities. Using a wound healing assay we show that inhibition of the mTOR pathway using Rapamycin, rapalogues, Resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 hours in these cell lines. We extended these findings up to 72 hours with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 or 3 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer. We also show that increased DEPTOR is a positive prognostic predictor in ovarian cancer and is decreased in later stage disease. Finally, we demonstrate that DEPTOR upregulation is mirrored in RNA extracted from whole blood of ovarian cancer patients. This in conjunction with the development of ultra-fast qPCR instrumentation can provide a valuable point of care testing platform for non-invasive diagnostics. A screening method that could provide early detection of ovarian cancer would represent a sea change in the diagnosis and treatment of this commonly lethal disease.