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

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).


| INTRODUC TI ON
Cutaneous melanoma is a malignant neoplasm derived from skin melanocytes. Melanoma cells have a high ability of local invasion and metastasis, even when arise from very small volume tumors (Gray-Schopfer, Wellbrock, & Marais, 2007). Once disseminated, melanoma is highly resistant to conventional anticancer treatments, and its prognosis is poor (Pollack et al., 2011). As melanoma can present in young and medium age adults (Lee et al., 2016;Ríos et al., 2013), it causes disproportionate mortality in such population being responsible of one of the highest rate of loss of potential life for adult-onset cancers (Ekwueme et al., 2011). Although available therapies for metastatic melanoma have evolved substantially in the last years, especially with the introduction of targeted-and immunotherapies (Hao et al., 2015), melanoma is still a serious healthcare problem, and melanoma cell behavior is an important subject of research.
About 20% of melanomas harbor mutations in the NRAS gene, mostly affecting codon Q61 (Jakob et al., 2012). Moreover, many other genetic alterations have been described that confer gain/ loss function of genes that trigger abnormal signaling pathways like KIT, GNAQ/GNA11, CDKN2A, PTEN, NF1, BAP1, among others (Griewank et al., 2014). Some studies advocate that patients with primary BRAFV600E or NRAS melanoma have worse survival rate compared with wild-type (WT) BRAF melanoma patients (Davies et al., 2002;Long et al., 2011). However it seems that, once disseminated, BRAF and NRAS mutation status does not influence survival in metastatic melanoma (Carlino et al., 2014).

| Expression of TTCCs in melanoma cells with different genetic profile
We first studied the expression of TTCCs by RT-PCR in a wide range of melanoma cell lines bearing different molecular features (Table S1). Significance T-type calcium channels (TTCCs) are overexpressed during melanoma progression, but the role they play is still unclear. This study reports an impairment of migration and invasion abilities of the most common genetic melanoma subgroup (BRAF/V600E) when subjected to pharmacological TTCC blockade. It has been long described that BRAF/V600E melanomas have upregulated autophagic flux, and similar results have been observed when autophagy is blocked. These findings show that autophagy and TTCC regulation intersect at some point which seems to be controlled by Snail1. Therefore, this work is at the frontier between basic and translational science and open new therapeutic strategies.

| TTCC blockers block basal autophagy in all melanoma cell lines
Autophagy in melanoma cells is constitutively active (Arindam Das et al., 2013;Maes & Agostinis, 2014). To study autophagy, we measured LC3I/II, an autophagic marker (Sahani, Itakura, & Mizushima, 2014), by Western blot (WB) in melanoma cell lines. We observed an increase of LC3II levels in most of the BRAFV600E melanoma ( Figure 1g). When cells were treated with TTCC blockers (mibefradil (Mib) and pimozide (Pim)), increased levels of LC3II and p62 were detected in BRAFV600E (Figure 1h, Figure S1a) and NRAS (Figure 1i, Figure S1b) melanoma cell lines. These data are suggestive of autophagy blockade at a step following autophagosome biogenesis.
Treatment of the melanoma cell lines with chloroquine (CQ), that prevents the fusion of autophagosomes (AP) with lysosomes (Boya et al., 2005), led to similar increases of LC3II and p62 proteins in all cell lines (Figure 1j,k, Figure S1c,d). To further confirm these changes on autophagy observed by Western blot, LC3B immunofluorescence (IF) was used as a marker for AP (Mizushima, Yoshimori, & Levine, 2010). A significant increase of AP puncta was observed both in M3 (BRAFV600E) and in Skmel-147 (NRASQ61R) cell lines when they were subjected to Mib or CQ treatment ( Figure 1l). Moreover, addition of chloroquine to mibefradil-treated cultures did not further increase the levels of p62 and LC3II ( Figure S1e). Taken together, these results indicate that TTCC blockers block autophagy, regardless of the specific mutation present in melanoma cell lines.

| Autophagy blockade inhibits collective migration of BRAFV600E melanoma cell lines
Cell migration is a key process during melanoma metastasis. We assessed the effect of TTCC blockers on melanoma migration using a wound-healing assay that measures collective migration. Both TTCC blockers decreased the percentage of BRAFV600E melanoma cells migrating into the wound ( Figure 2). Consistently, treatment of BRAFV600E cell lines with CQ also reduced the migration rate Using time-lapse microscopy, we found slower migration of BRAFV600E cells (M3 and M249) treated with mibefradil or CQ when compared to control, even after 8-h treatment ( Figure S3a,b). Furthermore, the healing speed of BRAFV600E cells decreased after treatment with either mibefradil or CQ (Figure 2a,b, Figure S3a,b and Movies S1-S3 (M3)).
In contrast, mibefradil or CQ treatments did not affect neither the percentage of wound healing nor the healing speed of the NRAS mutant cells (Figure 2c,d, Figure S3c,d and Movies S4-S6 (Skmel147)).
To further study how autophagy inhibition affects melanoma cell migration, we knocked down Atg5, an autophagy-related protein required for autophagosome formation (Pyo et al., 2005), by lentivirus-driven shRNA. Silencing of Atg5 induced a decrease of LC3II in both BRAFV600E and NRAS mutant cell lines ( Figure 2e).
It also decreased the migration of BRAFV600E (M3) melanoma F I G U R E 2 Blocking macroautophagy inhibits migration of BRAFV600E melanoma cells. Representative pictures and graphs of wound-healing assay of (a, b) BRAFV600E and (c, d) NRAS mutant cells treated with mibefradil (10 μM, Mib), pimozide (10 μM, Pim), and CQ (25 μM) during 24 hr. Panels on the left of each cell line represent the percentage of wound closure as a result of at least three independent experiments. Panels on the right represent the healing speed (μm 2 / hr) of cells after treatment compared with the control analyzed by estimated linear regression. (e) WB analysis shows the downregulation of ATG5 and LC3I/II levels. β-actin was used as a loading control. Percentage of wound closure in (f) M3 and (g) WM-1366 cells control (Vector) or upon ATG5 silencing (shATG5). Statistical analysis was performed using ANOVA and Bonferroni tests or t test (*p < .05; **p < .01; ***p < .001; n.s., nonsignificant) cells ( Figure 2f), but not of the NRAS mutant cells (WM-1366) ( Figure 2g).
Overall, these results show that the BRAFV600E cell lines are less motile after treatment with mibefradil, pimozide, CQ, or silencing Atg5 expression, suggesting that TTCC blockers inhibit the migration of BRAFV600E cells by blocking the autophagic process.

| Mibefradil and CQ inhibit single-cell migration in BRAFV600E melanoma cells
To analyze the effect of TTCC blockers on cell migration independent of cell-cell interactions, we studied single-cell random migration by time-lapse. For these experiments, melanoma cells were plated at lower density compared with previous approaches, and mibefradil and CQ concentrations were halved (12.5 μM and 5 μM, respectively) in order to maximize cell survival and also halted basal autophagy ( Figure S4). Analysis of individually tracked cells revealed that the accumulated distance and the migration speed of BRAFV600E cells were significantly reduced when treated with mibefradil or CQ (Figure 3a,b, Movies S7-S9 [M3]). In line with our findings, neither the distance nor the velocity was significantly affected by either treatment in NRAS mutant cells (Figure 3c,d, Movies S10-S12 [Skmel147]).
These results show that mibefradil and CQ decrease single-cell migration in BRAFV600E melanoma cells by blocking the autophagic flux.

| Autophagy blockade inhibits invasion of BRAFV600E melanoma cell lines
We next investigated the effect of TTCC blockers on the invasive capacity of melanoma cells. Treatment with mibefradil or CQ for 24 hr inhibited the invasive capacity of BRAFV600E melanoma cells (Figure 4a,b). Similar results were observed upon 24-h treatment with pimozide ( Figure S5a F I G U R E 3 Mibefradil and CQ reduce single-cell migration in BRAFV600E melanoma cells. Cell tracking analysis was carried out for 24 hr at a rate of 1 frame per 20 min. The total accumulated length migrated (μm) and the migration speed (μm/hr) of treated and untreated cells were analyzed in (a,b) BRAFV600E and (c,d) NRAS mutant cells. Around 25 significant tracks of control and treated cells were plotted in the trajectory graphs in the right side of the figure. Trajectories of each group of cells were standardized to all begin at the same starting point. The statistical analysis was performed using ANOVA and Bonferroni tests (*p < .05; **p < .01; ***p < .001)

| Gene silencing of TTCCs reduces the invasion ability of BRAFV600E melanoma cell lines
Previously, we showed that TTCC silencing leads to autophagy impairment that mimics the effect of TTCC blockers (Arindam Das et al., 2013). To investigate the possible involvement of TTCCs in the invasion capacity of BRAFV600E melanoma cells, we knocked down TTCCs in M3 cells using lentiviral constructs carrying shRNA specific to Cav3.1 and Cav3.2 ( Figure S5c-e). When measured in transwell assays, both TTCC silencing inhibited the invasive capability of M3 cells (Figure 4e,f). An increase of p62 and LC3II protein levels was observed when either Cav3.1 or Cav3.2 isoforms were silenced ( Figure S5f), thus mimicking the effects of mibefradil, pimozide, or CQ. These results indicate that TTCCs leads to induce invasion in BRAFV600E melanoma cell lines.

| Analysis of expression of TTCCs in biopsies from patients with melanoma
To extend our observations to the clinical settings, we used a cohort of 33 primary and 28 metastatic melanomas biopsies. Such cohort was divided into two main groups according to their BRAF genetic status (mutant BRAFV600E/K vs BRAFWT) analyzed by PCR sequencing (Table S2). The expression levels (histoscore) of TTCCs (Cav3.1 and Cav3.2) and LC3 were assessed by IHC. All melanoma samples bearing BRAFV600E/K gene mutations showed a higher immunoexpression of Cav3.1 compared with the WT BRAFWT cohort (Figure 5a,b), thus confirming our previous results (Maiques et al., 2017). In contrast, Cav3.2 immunoexpression did not show significant differences between the two groups (Figure 5c,d).
We additionally checked whether the mutation status of melanoma cells had an impact on macroautophagy, by quantifying the expression of LC3. The immunoexpression of LC3 was elevated in the BRAFV600E/K biopsies compared with the BRAFWT group (Figure 5e,f), suggesting an enhanced basal autophagy of BRAFV600E/K biopsies.

| Snail1 expression is higher in BRAFV600E melanomas and decreases upon autophagy blockade
Snail1 plays several roles in cell migration and EMT process (Cano et al., 2000). To understand if autophagy was regulating cell migra- The statistical analysis was performed using ANOVA and Bonferroni test (*p < .05; **p < .01; ***p < .001; n.s., nonsignificant) Snail1 was reduced in BRAFV600E cell lines that were treated with TTCC blockers (Figure 6c), or subjected to gene silencing of TTCCs (Figure 6f). In addition, treatment of BRAFV600E cells with CQ ( Figure 6d) or gene silencing of ATG5 (Figure 6e) consistently decreased the levels of Snail1, showing that the expression of Snail1 was related to the status of the autophagic flux. It has been shown that Snail1 plays an important role in melanoma progression (Hao et al., 2012;Olmeda et al., 2007). Indeed, Snail1 knockdown impaired cell migration and invasion (Figure 6g (Figure 6l). These results suggest that Snail1 could be essential to impair migration/invasion when autophagy is blocked, particularly by targeting TTCCs. To complement these approaches, we used the publicly available database TCGA and found that Snail1 expression was increased in melanoma biopsies of patients harboring BRAFV600E mutation, compared to that BRAFWT (Figure 6n).
Cell migration and invasion are key steps during metastatic dissemination; therefore, we performed Snail1 IHC analysis in metastatic vs primary melanoma biopsies. Snail1 nuclear staining (active form) was significantly higher in metastatic compared with primary lesions only in patients harboring BRAFV600E/K mutations (Figure 6o).
Moreover, we observed a higher Snail1 score in BRAFV600E/K metastasis, compared with BRAFWT metastatic tumors. These results indicate an important role of Snail1 during metastatic spread of BRAFV600E melanomas.
For instance, Xie et al. showed that autophagy inhibition by deletion of Atg7 or treatment with CQ suppresses melanoma tumor growth and increases survival of mice driving oncogenic BRAFV600E expression and PTEN deficiency in melanocytes (Xie, Koh, Price, White, & Mehnert, 2015). Furthermore, CQ reduces tumor growth and impairs melanoma cell invasion and metastasis . In addition, Sharifi and coworkers revealed that autophagy inhibition reduces cell migration and invasion (in breast cancer and melanoma) and attenuates the induction of metastasis by disrupting the focal adhesion turnover . We studied the role of autophagy in the migration/invasion of BRAFV600E and NRAS melanomas. Our results demonstrate that TTCC blockers, mibefradil and pimozide, impair migration and invasion of BRAFV600E cells, an effect exerted also by CQ or Atg5 knockdown. In contrast, cell motility in vitro is largely unaffected by autophagy inhibition in NRAS melanoma cell lines.
We evaluated Snail1 expression, a master transcription factor that induces EMT and invasion in melanoma cells (Hao et al., 2012;Olmeda et al., 2007). Our results indicate that Snail1 expression is higher in BRAFV600E melanoma cells compared with BRAFWT cells. Interestingly, Snail1 levels decrease when autophagy is blocked by chloroquine or by TTCC blockers, thus inhibiting migration and invasion. Furthermore, when we overexpressed Snail1 in NRAS mutant melanoma cell line, we show an impairment of migration/invasion abilities when autophagy is blocked. Therefore, autophagy appears to regulate cell motility through different mechanisms depending on the cell type and context (Kenific, Thorburn, & Debnath, 2010).
BRAF and NRAS mutations are mutually exclusive in melanoma (Davies et al., 2002). Common to them is the direct or concomitant hyperactivation of signaling pathways like MEK-ERK and PI3K (Griewank et al., 2014;Vu & Aplin, 2016). However, our studies indicate that BRAFV600E and NRASQ61 activate distinct cellular mechanisms related to melanoma progression. It has been shown that activation of ERK, the main target of BRAFV600E, is an upstream signaling mechanism responsible for high constitutive Snail1 expression in melanoma cells (Massoumi et al., 2009). In addition, Snail1 knockdown disrupts tumor growth and impairs melanoma progression and migration, similar to results reported here (Hao et al., 2012;Massoumi et al., 2009).
In BRAFV600E melanomas, the phosphorylation of cortactin and the exocyst subunit Exo70 upon ERK activation, which regulates the secretion of matrix metalloprotease-2, appears as a relevant mechanism for cell migration (Lu et al., 2016;Sandri et al., 2016).
Our findings reveal an unknown link between autophagy and Snail1 expression that regulates the migration and invasion of BRAFV600E melanoma cells. It has been described that p62 modulates the stability of Snail1, a mediator of TGFβ/Smad signaling, through its UBA domain (Bertrand et al., 2015). New evidences indicate that autophagy is not only involved in the intracellular degradation of damaged proteins, but also plays an important role in protein secretion (Kraya et al., 2015;Narita et al., 2011).
In addition, autophagy-related secretion affects the tumor microenvironment and reflects the autophagy dynamics of tumor cells (Kraya et al., 2015). A recent paper further revealed that the TGFβ/Snail signaling pathway induces EMT-like process in a paracrine manner in melanoma (Lv et al., 2017). Moreover, knockout mice of GABARAP, an Atg8/LC3 family member implicated in the induction of autophagy, showed reduced amounts of TGFβ in serum and inhibition of tumor initiation and progression, through the enhancement of both antitumor immunity and cell death signaling (Salah et al., 2016). Therefore, all these data may suggest that the secretion of TGFβ through autophagy could lead to increased expression of Snail1, during the induction of migration/ invasion of BRAFV600E melanoma cells. Nevertheless, the mechanism by which Snail1 levels decrease upon autophagy blockade in BRAFV600E melanoma cells requires further investigation. A possibility would be that Snail1 proteasomal degradation (Muqbil, Wu, Aboukameel, Mohammad, & Azmi, 2014) is deregulated after autophagy blockade.
In conclusion, our findings indicate that BRAV600E melanoma cells display higher levels of the Cav3.1 TTCC and an increased basal autophagy, compared with other types of melanoma cells.
In addition, the migration and invasion capabilities of BRAFV600E cells are sensitive to the genetic ablation or pharmacological inhibition of autophagy and depend on Snail1 levels. Thus, chemotherapeutic strategies targeting TTCCs and/or autophagy appear especially suitable to tackle metastasis in the most common type of melanoma.

| Cell lines
Twelve human malignant melanoma cell lines were used and sequenced (BRAF or NRAS mutation) (Table S1). For cell culture conditions, see Supplemental Experimental Procedures.

| Western blot
Melanoma cells were lysed in 2% sodium dodecyl sulfate (SDS), 125 mM Tris-HCl, pH 6.8. Western blot was conducted as described (Arindam Das et al., 2013). The antibodies used are detailed in Supplemental Experimental Procedures.

| Lentiviral infection
The lentiviral vector containing the sequences of the shRNA of

| Wound healing
A confluent monolayer of cells was scratched with yellow tip.
Thereafter, cells were treated, and we captured an image of the scratch at time 0 hr and after 24 hr to calculate the percentage of the wound filled by cells. For time-lapse wound-healing assay movies, see Data S1 .

| Single-cell migration assay
Cells were plated at low density (6000 cell/cm 2 ) to minimize cell-cell interactions. Cells were treated and captured an image every 20 min for 20 hr. Cells were tracked using ImageJ plugin, and the accumulated distance (μm) and the velocity (μm/hr) of the single cells were evaluated using Chemotaxis and Migration Tool (Ibidi).

| Transwell experiments
We first treated the cells during 24 hr. Then, cells were trypsinized and plated in the upper chamber of the Transwell (8 μm pore, Falcon) coated with Matrigel in serum-free medium. We used 10% of FBS as a chemoattractant. After 24 hr, cells were fixed with paraformaldehyde 4% and stained with Hoechst (5 μg/ml). Finally, cells were pictured under an epifluorescence microscope (Leica), before and after the cotton swap, and we counted (ImageJ) to have the percentage of the migrated cells.

| Tissue microarray and immunohistochemical study
One tissue microarray (TMA) was constructed from 61 formalinfixed, paraffin-embedded (FFPE) melanoma tumors (primary and metastatic Table S2). Assessment of TTCC immunostaining and LC3 was made as detailed in Maiques et al. (2017). Antibodies used and IHC protocol of Snail1 and scoring, are detailed in Data S1.

| Ethics statement
Studies using human samples were approved by the Ethics Committee on Clinical Investigation of the Hospital Universitari Arnau de Vilanova (HUAV, Lleida, Spain), and all patients gave their informed consent.

| Statistical analysis
Statistical analysis was carried out using GraphPad Prism software.
All data were expressed as mean ± SD from at least three independent experiments. Statistical significance was checked by application of Kolmogorov-Smirnov normality test followed by t test or ANOVA and Bonferroni test (parametric Test), or Mann-Whitney test or Kruskal-Wallis test (nonparametric test). p-values are indicated by asterisks *p < .05; **p < .01; ***p < .001.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.