T‐type calcium channels drive migration/invasion in BRAFV600E melanoma cells through Snail1

Abstract

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).

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

“…TTCC blockers inhibit autophagy and induce cell death in all melanoma cells, regardless of the specific mutation present [19,20]. Recently, our group performed in vivo xenograft mouse models with subcutaneous injections of melanoma cell lines, with a further administration of Mibefradil daily via oral gavage for 2 w. We showed that TTCC blocker treatment significantly reduces tumor growth and induces apoptosis via an autophagy blockade in xenograft melanoma models (Table 1) [22].…”

“…Our group was the first to show that the Cav3.1 isoform is upregulated in both melanoma cell lines and biopsies from BRAF V600E -mutant melanomas, which was accompanied by increased levels of LC3II protein-important for the autophagic process-compared with NRAS-mutant melanomas [20]. In line with this, we described a significant correlation between the presence of BRAF V600E -mutant protein with Cav3.1 expression and a positive link to LC3II protein in a cohort of primary and metastatic melanomas (Table 1; Figure 2) [21].…”

“…Cav3. ↑ mRNA expression in melanoma cell lines and biopsies compared with melanocytes [18] ↑ mRNA expression in melanoma cell lines and biopsies compared with melanocytes [18] Hypoxia ↑ mRNA expression (M16, JG, M28) [19] Hypoxia ↑ mRNA expression [19] ↑ mRNA expression in BRAFV600E melanoma [20] ↑ immunoexpression from normal skin to naevi to melanoma biopsies (tumor progression) [21] ↑ immunoexpression from normal skin to naevi to melanoma biopsies (tumor progression) [21] ↑ immunoexpression in primary melanoma tumors with Breslow thickness >1 mm [21] ↑ immunoexpression in primary melanoma tumors with Breslow thickness >1 mm [21] ↑ immunoexpression positively correlates with BRAFV600E protein expression and LC3 [21] ↑ immunoexpression positively correlates with Glut1 and Ki-67 [21] ↑ mRNA expression and immunoexpression in BRAFV600E melanoma [20] ↑ mRNA expression and immunoexpression in BRAF inhibitor-resistant melanoma [22] ↑ mRNA expression during acquisition of Vemurafenib resistance in melanoma [22] ↓ mRNA expression after TTCC blocker treatment in sensitive and Vemurafenib-resistant melanoma [22] Cellular function TTCC blocker or molecular knockdown of Cav3.1 expression arrest cells in G1 phase and decreases cell viability in melanoma cells [18,19,22] TTCC blocker or molecular knockdown of Cav3.2 expression arrest cells in G1 phase and decreases cell viability in melanoma cells [18,19,22] TTCC blocker arrests cells in G1 phase and decreases cell viability in melanoma cells [18,19,22] TTCC blocker or molecular knockdown of Cav3.1 expression impairs migration/invasion rates in BRAFV600E-mutant melanoma cells [20] TTCC blocker or molecular knockdown of Cav3.2 expression impairs migration/invasion rates in BRAFV600E-mutant melanoma cells [20] TTCC blocker impairs migration/invasion rates in BRAFV600E-mutant melanoma cells [20] TTCC blocker reduces tumor growth in vivo [5] TTCC blocker reduces cell viability and inhibit migration/invasion rates in Vemurafenib-resistant melanoma cells…”

“…It is well known that the most prevalent genetic hallmark of cutaneous melanoma is the presence of BRAF (BRAFV600E/K, 40-50%) or NRAS (NRASQ61, 20%) mutations, which are mutually exclusive [49]. Recently, the expression of TTCCs was investigated in different melanoma cell lines and human biopsies according to their genetic profile [20]. Melanoma cell lines that harbored BRAF V600E mutation showed higher mRNA levels of the Cav3.1 and Cav3.3 isoforms compared with melanoma cells with NRAS mutation, whereas no significant differences were observed regarding Cav3.2 mRNA levels.…”

“…Melanoma cell lines that harbored BRAF V600E mutation showed higher mRNA levels of the Cav3.1 and Cav3.3 isoforms compared with melanoma cells with NRAS mutation, whereas no significant differences were observed regarding Cav3.2 mRNA levels. Extending it to clinical settings, a cohort of primary and metastatic melanoma human biopsies bearing BRAF V600E/K gene mutation showed a higher immunoexpression of Cav3.1 compared with a BRAF wild-type melanoma cohort (Table 1; Figure 1b) [20].…”