An autophagy-driven pathway of ATP secretion supports the aggressive phenotype of BRAFV600E 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^V600E. 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^V600E 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^V600E 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^V600E 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.

Keywords: BRAFV600E inhibitor-resistance; P2RX7 receptor; Vemurafenib; extracellular ATP; invasion; macroautophagy; melanoma.

Figure 1.

PLX-acquired resistance promotes migration, invasion and resistance in an eATP-dependent manner. The indicated isogenic cell models were assessed after a 72-h incubation by light microscopy-based scratch assays (A-B), transwell cell migration (C), invasion (D) and eATP (E). The capacity of 451-Lu/Res conditioned media (CM) to stimulate migration of the parental 451-Lu cells was assessed by transwell migration assays in the presence or absence of 2 U/ml apyrase (APY) (F). The ability of eATP to stimulate cell migration (G, I) or invasion (H, J) were monitored by transwell assays. Cells were exposed to either APY (G, H) or ATP (50 µM, I, J) and then monitored after 72 h. The SM1-based isogenic cell models were assessed for their eATP alone or following 72-h exposure to 10 µM PLX; RLU, relative luciferase units. (K). The effect of extracellular ATP modulation of migration (L) and invasion (M) of the SM1 isogenic cell lines was assessed 72 h post ATP or APY additions. Scale bars: 100 µm. All experiments are representative of 3 independent experiments and expressed as mean ± SD. *^/$ = P < 0.05, ** = P < 0.01, ***^/$$$ = P < 0.001, **** P < 0.0001.

Figure 2.

Extracellular ATP phenotype extends in vitro to naturally resistant tumor cells. Patient-derived melanoma cells demonstrating natural PLX-sensitive (M229, M249) and -resistant (M233, M263) phenotypes were exposed to increasing doses of PLX4032 (PLX) for 72 h and resistance assessed by MUH assay-based cell viability assays (A) or extracellular ATP analyzed (B, 10 µM PLX for 72 h; RLU, relative luciferase units). The capacity of exogenous ATP to facilitate migration (C, E) or invasion (D, F) were assessed following either APY (C, D; 2 U/ml) or ATP (E, F; 50 µM) exposure for 72 h. Data are the mean ± SD of 3 independent experiments. * = P < 0.05, **^/$$ = P < 0.01.

Figure 3.

PLX-acquired resistance promotes an aggressive, drug-resistant signature in vitro. 451-Lu isogenic models were assessed, after a 72-h incubation with either extracellular APY (A, 2 U/ml) or ATP (B, 50 µM), for adaptations in side population (SP) subsets. The 451-Lu isogenic cell models were screened for a panel of drug resistance, stemness and tumor aggression markers by q-RTpcr (C-I, including; ABCB1, ABCG2, KDM5B/JARID1B, SOX10, TERT, NGFR, MYC normalized to GAPDH). All experiments are representative of 3 independent experiments and expressed as mean ± SD. *^/$ = P < 0.05, **^/$$ = P < 0.01, ***^/$$$ = P < 0.001, **** P < 0.0001.

Figure 4.

Elevated secretion of ATP by PLX-resistant melanoma cells is an autophagy-dependent process. 451-Lu PLX isogenic cell models were stably knocked down in ATG5 expression, in comparison to control (shCon) by shRNA and confirmed by western blot analysis of ATG5, SQSTM1 and MAP1LC3B/LC3B-II, normalized to ACTB (A). Following stable ATG5 knockdown, eATP was stained and assessed using a FlexStation 3 microplate reader; RLU, relative luciferase units (B). The effects of ATG5 knockdown on the cell migration or invasion potential were characterized by transwell assays (C, D). Hoechst 33342-based flow cytometry was performed on 451-Lu/RES cells, stably transduced with shCon vs. shATG5, and the percentage of Hoechst^low cells determined (E). All experiments are representative of 3 independent experiments ± SD. *^/$ = P < 0.05, **^/$$ = P < 0.01, ***^/$$$ = P < 0.001.

Figure 5.

Autophagy governs ATP secretion of the PLX-resistant melanoma cells. Following knockdown of either ULK1 (A, C) or ATG7 (B, D) in 451-Lu or 451-Lu/RES melanoma cells, eATP (A, B), or migration by transwell assays (C, D) were assessed; RLU, relative luciferase units. The capacity of exogenously added ATP (50 µM) to restore migration was assessed by transwell migration assays (C, D). 451-Lu and 451-Lu/Res isogenic melanoma models were treated with 1 µM quinacrine (green) for 2 h at 37°C alone or in combination with Baf A1 (10 nM, 1 h pre-treatment), before colocalization with autophagosomes was assessed with MAP1LC3B/LC3B-II (red) (E), light microscopy overlays (differential interference contrast/DIC) are provided. Colocalization was calculated using imageJ (F). All experiments are representative of 3 independent experiments and expressed as mean ± SD. Scale bar: 10 µM. *^/$ = P < 0.05, **^/$$ = P < 0.01, *** = P < 0.001.

Figure 6.

Extracellular ATP promotes PLX-resistant melanoma cell migration and invasion in a purinergic receptor-dependent manner. eATP was analyzed in the 451-Lu isogenic models following treatment with either 5 µM suramin or 10 µM A740003; RLU, relative luciferase units. (A). 451-Lu and 451-Lu/RES cell migration (B) and invasion (C) was assessed by scratch and transwell invasion assays following treatment with either 5 µM suramin or 10 µM A740003. 451-Lu/RES cells (shCon vs. shATG5) were analyzed by Hoechst 33342-based flow cytometry and changes in the percentage of Hoechst^low cells determined (D). Data are the mean ± SD of 3 independent experiments. ** = P < 0.01, *** = P < 0.001, **** P < 0.0001.

Figure 7.

Extracellular ATP promotes cellular uptake via P2RX7. 451Lu and 451-Lu/Res were treated with 50 µM ATP for 24 h alone or in combination with 10 µM A740003 (A74) and Rh123 (2 µM for 1 h) positivity assessed by flow cytometry (A). 451-Lu isogenic cells were exposed to 2 µM Rh123 alone or in combination with APY (2 U/ml, overnight pre-incubation) for 1 h and uptake assessed (B). 451Lu and 451-Lu/Res cells were treated with 50 µM ATP for 24 h alone or in combination with 10 µM A740003 (A74) and FITC-dextran (C, 40 µg/ml for 2 h) uptake assessed by flow cytometry. 451-Lu/Res cells harboring shCon compared with shATG5 were analyzed for Rh123 uptake potential at basal conditions (D) or following exposure to 50 µM ATP alone or combined with 10 µM A74 (E). Data are the mean ± SD of 3 independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. MFI, mean fluorescence intensity.