Cross talk between CD271 expressing tumour subpopulations and autophagy in the drug resistance, invasion and survival of cutaneous metastatic melanoma

Abstract

Cutaneous malignant melanoma remains an increasing world health problem. Although targeted therapy to hyper-activated MAPK signalling has significantly increased survival for patients with BRAF/NRAS mutant melanomas, the development of acquired resistance is inevitable and associated with the emergence of chemo-resistant subpopulations expressing the neurotrophin receptor CD271. The relationship between drug-induced CD271 expressing subpopulations and autophagy however, remains undefined. The central aim of the present study was to define the relationship between CD271 (both constitutive and drug-induced) expression and autophagy, and the impact of CD271 inhibition or autophagy modulation on the potential re-sensitisation of BRAF mutant metastatic melanoma cells to MEK inhibition. Semi-quantitative immuno-histochemical analysis (IHC) of CD271 expression in primary cutaneous melanomas of different AJCC stage, revealed a significant stepwise increase in expression in advanced, stage III melanomas, paralleled with biphasic expression of p62 and consistent with the paradoxical role of autophagy in cancer. Additionally, chemical inhibition of autophagy with chloroquine or a specific Vps34 inhibitor, selectively inhibited cell viability CD271 positive but not negative subpopulations, isolated from BRAF mutant melanoma cell line, collectively suggesting constitutive CD271 expression is associated with an increase in basal pro-survival autophagy. Prolonged exposure of BRAFV600E mutant melanoma cells to the MEK 1/2 specific inhibitor trametinib resulted in increased CD271 expression, LC3-II accumulation and reduced p62 expression, again highlighting the relationship between CD271 expression and autophagy activation. Genetic or chemical inhibition of CD271 in trametinib-induced drug-resistant BRAF mutant melanoma subpopulations resulted in significant inhibition of cell viability and resensitisation to trametinib-induced apoptosis. Furthermore, Inhibition of autophagy with chloroquine/ Vps34 inhibitor also resulted in reduced colony forming capacity, invasion and the re-sensitization of CD271 expressing BRAF mutant subpopulations to the cytotoxic effects of trametinib in vitro, with Vps34 inhibition additionally promoting trametinib-induced death and the prevention of tumour invasion and dissemination of trametinib-induced drug resistant subpopulations in a zebra fish xenograft model. iv Attempts to harness cytotoxic autophagy to overcome drug-induced resistance to trametinib additionally revealed treatment of drug-induced resistant CD271 expressing BRAF mutant melanoma subpopulations with 9-tetrahydrocannabinol (THC) resulted in significant inhibition of cell viability. Collectively these data underpin the intimate relationship between trametinib-induced CD271 drug-resistant subpopulations and pro-survival autophagy and suggest the targeting of CD271 or indirect clinical modulation of autophagy (either through selective inhibition or the harnessing of its cytotoxic effects induced by THC) may provide valuable therapeutic strategies through which to overcome the resistance of BRAF/NRAS mutant metastatic melanoma to MEK inhibition.