Investigation of the PTP4A3 oncogene and how it mechanistically regulates autophagy in ovarian cancer

Abstract

Ovarian cancer has the highest mortality rates of all gynaecologic malignancies. High mortality rates are associated with poor levels of early detection due to asymptomatic tumour growth and limited screening programmes. As a result, most women are diagnosed at very advanced disease stages, when treatment and recovery can prove challenging. Two-thirds of ovarian cancer mortalities are specifically due to high-grade serous ovarian carcinoma (HGSOC), which is also the most common subtype. The Protein Tyrosine Phosphatase 4A3 (PTP4A3) oncogene is a phosphatase that activates PI3K and RAS signalling pathways to promote cellular growth and proliferation. PTP4A3 is overexpressed in ovarian cancer and this correlates with poorer disease survival. A previous study has linked PTP4A3 overexpression in ovarian cancer cells with increased macroautophagy and showed that high co-expression of PTP4A3 and the autophagy genes PIK3C3 and BECN1 correlated with even worse patient survival. In the current study the focus was on understanding the molecular mechanisms which govern PTP4A3-mediated activation of cell growth, proliferation, and autophagy pathways in HGSOC. PTP4A3 expression levels and autophagy activity were analysed in three HGSOC cell lines; OVCAR 3, OVCAR 4 and Kuramochi. PTP4A3 protein was highly expressed in Kuramochi, while OVCAR 4 expressed 10 % of Kuramochi expression levels, with no detectable levels observed in OVCAR 3. While OVCAR 3 and OVCAR 4 cells showed rapid autophagy induction in response to amino acid deprivation, Kuramochi cells were unable to activate autophagy. Consistent with KRAS expression levels in each cell line, RAS-dependent basal autophagy was present in both OVCAR 3 and Kuramochi, but not OVCAR 4 cells. Modulation of PTP4A3 levels, by overexpression in OVCAR 3 or depletion in Kuramochi cells, did not alter autophagy profiles. In contrast, silencing of PTP4A3 in OVCAR 4 cells resulted in increased basal autophagy levels (RAS independent) and decreased ability to activate autophagy . Use of the pan-PTP4A1-3 inhibitor (JMS-053) in Kuramochi and OVCAR 4 cells had opposing effects on PI3K and RAS pathway signalling in each cell line, revealing possible compensatory mechanisms occurring when targeting all PTP4A1-3 phosphatases that could depend on KRAS status. Moreover, opposite responses in those pathways were also observed when comparing PTP4A3 silenced vs pan-PTP4A inhibition in OVCAR 4 cells, again suggesting compensatory mechanisms of PTP4A1 and/or PTP4A2 in response to PTP4A3 loss of protein expression. Analysis of the anti-cancer effects of pan-PTP4A1-3 inhibition/PTP4A3 knockdown in each of the cell lines was carried out, both alone and in combination with MEK inhibition or the chemotherapeutic drugs, fluorouracil (5FU), cisplatin (CDDP) and paclitaxel (PTX). Results showed that PTP4A3 knockdown increased drug-mediated cytotoxicity, with the exception of CDDP in OVCAR 4 cells. Moreover, the absence of PTP4A3 in OVCAR 3 cells correlated with their high sensitivity to CDDP and PTX. Taken together, these results suggest that targeting PTP4A1-3 phosphatases in ovarian cancer is highly dependent on KRAS status. In KRAS-driven tumours, compensatory mechanisms to increase/maintain signalling through RAS, PI3K and autophagy pathways appear to exist. Specifically targeting PTP4A3 expression may result in compensation by the alternative phosphatases, PTP4A1 and/or PTP4A2. However, increased sensitivity of PTP4A3 knockdown cell lines to chemotherapeutic drugs suggests that PTP4A3 depletion and/or inhibition may be a promising approach to sensitise HGSOC cells to chemotherapeutic drugs and limit development of drug resistance in the clinical setting.