Abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered on single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells' response to combinatorial treatment. We here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a likely cause of antitumor activity. We propose that the inhibition of the autophagic flux on combinatorial treatment is attributed to the major cytoskeletal changes induced on ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.
Highlights
We used phosphoproteomics to reveal the underlying mechanisms of drug synergy on epidermal growth factor receptor (EGFR) and ROCK co-inhibition in Triple-negative breast cancer (TNBC) cells
Based on previous work [18], which revealed that the combination of EGFR and ROCK inhibitors effectively reduced TNBC cell growth by inducing cell cycle arrest, we here complement these previous findings by providing an insight into the molecular mechanisms triggered by the combinatorial EGFRiϩ ROCKi treatment
We show that EGFR inhibition by gefitinib induces autophagy activation in TNBC cells, which was evident from the increased expression levels of several autophagy protein markers (e.g. MAP1LC3, GABARAP) and the formation of autophagic vacuoles
Summary
We used phosphoproteomics to reveal the underlying mechanisms of drug synergy on EGFR and ROCK co-inhibition in TNBC cells. No EGFR therapies are currently approved for TNBC because of low response rates, necessitating better markers for patient stratification [4] as well as the exploration of combination therapies [5, 6]. In this light, two independent studies recently showed great synergistic antitumor activity when inhibitors of the RAF-MEK-ERK cascade were combined with autophagy inhibition in pancreatic and other RAS-driven cancers [7, 8]. Macroautophagy (hereafter referred to as autophagy) is a highly dynamic multi-step biological process of self-cannibalization that involves the degradation of damaged organelles, misfolded proteins and long-lived macromolecules in lysosomes.
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