An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAF^V600E inhibitor-resistant metastatic melanoma cells

Abstract

The ingrained capacity of melanoma cells to rapidly evolve toward an aggressive phenotype is manifested by their increased ability to develop drug-resistance, evident in the case of vemurafenib, a therapeutic-agent targeting BRAF. Previous studies indicated a tight correlation between heightened melanoma-associated macroautophagy/autophagy and acquired Vemurafenib resistance. However, how this vesicular trafficking pathway supports Vemurafenib resistance remains unclear. Here, using isogenic human and murine melanoma cell lines of Vemurafenib-resistant and patient-derived melanoma cells with primary resistance to the BRAF inhibitor, we found that the enhanced migration and invasion of the resistant melanoma cells correlated with an enhanced autophagic capacity and autophagosome-mediated secretion of ATP. Extracellular ATP (eATP) was instrumental for the invasive phenotype and the expansion of a subset of Vemurafenib-resistant melanoma cells. Compromising the heightened autophagy in these BRAF inhibitor-resistant melanoma cells through the knockdown of different autophagy genes (ATG5, ATG7, ULK1), reduced their invasive and eATP-secreting capacity. Furthermore, eATP promoted the aggressive nature of the BRAF inhibitor-resistant melanoma cells by signaling through the purinergic receptor P2RX7. This autophagy-propelled eATP-dependent autocrine-paracrine pathway supported the maintenance and expansion of a drug-resistant melanoma phenotype. In conclusion, we have identified an autophagy-driven response that relies on the secretion of ATP to drive P2RX7-based migration and expansion of the Vemurafenib-resistant phenotype. This emphasizes the potential of targeting autophagy in the treatment and management of metastatic melanoma.

Characterization of Melanoma Cell Lines Resistant to Vemurafenib and Evaluation of Their Responsiveness to EGFR- and MET-Inhibitor Treatment

“…The A375 and WM9 resistant cell lines that we generated significantly differed from their parental counterparts in terms of motile abilities, both in the case of spontaneous migration/invasion and wound closure. Similar observations were described for both melanoma cell lines and cells derived from patients following long-term treatment with vemurafenib [42,43]. In a study by Zubrilov et al, resistant cells exhibited an elevated rate of transmigration through lung endothelial cells, which may facilitate the formation of metastases [44].…”

Non-Apoptotic Cell Death Signaling Pathways in Melanoma

“…Re-phosphorylated ERK induced activating transcription factor 4 (ATF4) to trigger cytoprotective autophagy [115]. Moreover, it has been shown that vemurafenib-resistant melanoma cells employed autophagosomes to secret ATP and enhance cell migration and invasion [116]. Compromising autophagy by downregulation of several autophagy-related genes significantly reduced secretion of ATP and, consequently, cell invasion [116].…”

“…Moreover, it has been shown that vemurafenib-resistant melanoma cells employed autophagosomes to secret ATP and enhance cell migration and invasion [116]. Compromising autophagy by downregulation of several autophagy-related genes significantly reduced secretion of ATP and, consequently, cell invasion [116]. Inhibition of AMPK-α1 in vemurafenib-resistant melanoma cells impaired autophagy that was associated with decreased formation of the autophagosomes [117].…”

T-Type Calcium Channels: A Potential Novel Target in Melanoma

“…In this context, we set out to study the modulation of TTCCs and/or the inhibition of autophagy as a possible therapy for Vemurafenib-resistant melanoma. In fact, our group recently demonstrated that chronic exposure to Vemurafenib-induced drug resistance in BRAF V600E -mutant melanoma cells was due to a Vemurafenib-promoting autophagic process, a mechanism that contributes to BRAFi resistance in melanoma cells [22,[81][82][83]. In addition, Barceló and colleagues showed that both Vemurafenib-resistant BRAF V600E -mutant melanoma cells and biopsies from human melanoma relapsing under BRAFi expressed higher levels of the Cav3.1 isoform and the LC3 protein compared with their parental Vemurafenib-sensitive cell line or pre-treatment melanoma tumors, respectively (Table 1; Figures 1A and 2).…”