Harnessing autophagy to overcome mitogen‐activated protein kinase kinase inhibitor‐induced resistance in metastatic melanoma

Background Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen‐activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance. Objectives To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy. Methods Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib‐induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib‐resistant melanoma cell survival and invasion, respectively. Results CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF‐mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271‐expressing subpopulations. Trametinib‐induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271‐expressing melanoma subpopulations with RNA interference and small‐molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents – including Δ9‐tetrahydrocannabinol and Vps34 – reduced survival of MEKi‐resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi‐resistant cells in an in vivo zebrafish xenograft. Conclusions These results highlight a novel mechanism of MEKi‐induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug‐resistant melanoma cells to the cytotoxic effects of MEKi

The Impact of an Extreme Storm Event on the Barrier Beach of the Lefkada Lagoon, NE Ionian Sea (Greece)

The present investigation examines the characteristics of a high energy storm event, that took place on November 9-11, 2007 in the NE Ionian Sea (eastern Mediterranean), and its impact upon the barrier beach that separates the Lefkada lagoon from the open Ionian Sea. The storm event was caused by NW winds with speeds exceeding 20 m/s (40 knots), which have an annual frequency of occurrence less than 0.015%. This high energy event produced waves with >5 m significant offshore height and 9.5 s period; these waves developed on 10th November during the rapid rise of barometric pressure (~1.4 hPa/hr), which followed the barometric pressure drop from 1020.5 hPa at 06:00 (UTC) of 9th November to 1001.7 hPa at 06:00 h (UTC) of 10th November. Secondary breaking at the shoreline produced wave heights >1.5 m, associated with a surge of >0.4 m and a run-up capability of >2.4 m. The waves managed to overtop the barrier beach (elevations ~2.5 m), lowering the seaward side of the barrier beach by 10-30 cm and causing a coastline retreat of 0.9 to 2.2 m; these morphological changes correspond volumetrically to a sediment loss of approximately 8 m3/m of coastline length from the sub-aerial part of the beach. During the last three decades a significant change in the frequency of occurrence and direction (from S-SW-W to N-NW-NE) of severe storms with wind speeds exceeding 40 knots has been recorded, affecting the sediment transport pattern and contributing to the erosion of the north beaches of Lefkada