T-type calcium channel inhibition restores sensitivity to MAPK inhibitors in de-differentiated and adaptive melanoma cells

Background Drug resistance remains as one of the major challenges in melanoma therapy. It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi). Methods We investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi. Results Here, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers. To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors. De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis. Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells. Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo. Conclusion In summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi. Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.


BACKGROUND
Malignant melanoma is an aggressive type of skin cancer where the survival rates and treatments vary depending on tumour stages. While early stages have a good prognosis, unresectable stage III and IV melanomas are often fatal and therapy resistance is a major challenge. Around 50% of melanoma patients carry mutations in the BRAF gene and around 30% of patients in the NRAS gene resulting in an over-activation of the mitogenactivated protein kinase (MAPK) pathway, 1 making this signalling cascade one of the most important targets for melanoma therapy. 2,3 The clinical use of BRAF inhibitors (vemurafenib, dabrafenib, encorafenib), MEK inhibitors (trametinib, cobimetinib, binimetinib) or their combinations significantly increase progression-free and overall survival of patients. 3,4 Unfortunately, most patients develop resistance to these inhibitors soon after the start of therapy 5,6 because of different factors including tumour heterogeneity and plasticity. 7 The high cellular heterogeneity seen in melanomas is partially due to a degree of phenotypic plasticity. Melanoma cells switch between proliferative/differentiated and invasive/de-differentiated phenotypes during metastasis progression, mimicking the epithelial-to-mesenchymal transition, which facilitates invasion to secondary tumour sites. [8][9][10] Indeed, induction of phenotype switching towards a de-differentiated state is likely one of the most common mechanisms underlying the development of resistance to therapies in melanoma patients. 9 Drug resistance in melanoma has been classified as intrinsic, adaptive or acquired, depending on whether the resistance is present already before treatment starts or it develops within hours or late upon treatment. 2,11 Several mechanisms have been reported to promote resistance in melanoma, including reactivation of extracellular-signal regulated kinases (ERK) signalling or activation of alternative pathways. 2,5 Identification of new targets that enhance efficacy of current cancer treatments and prevent development of resistance is essential. T-type calcium channels have been suggested as therapeutic targets in different types of cancer, [12][13][14] including melanoma. 15 These calcium channels play a role in proliferation, survival, regulation of cell cycle and differentiation. 16,17 Blockage www.nature.com/bjc of T-type calcium channels have shown promising results by inducing cytostatic effects on cancer cells that are resistant to standard anti-cancer therapies; 18,19 however, the use of T-type calcium channels inhibitors in MAPKi-adaptive melanomas, have not been yet described.
Here, we use partial reprogramming, 20 which represents an intermediate phase of cell fate reversion before fully pluripotency is attained, to investigate de-differentiation and drug adaptation in melanoma. We have found that inhibition of T-type calcium channels in adaptive cells induces differentiation, cell death in vitro and restores sensitisation to MAPKi in vivo. Therefore, we propose use of the calcium channel blocker mibefradil as a novel treatment strategy to overcome adaptation in melanoma.
Quantitative real-time PCR Total RNA was isolated using RNeasy kit (Qiagen), DNase I digestion to remove genomic DNA was performed. cDNA was synthesised using cDNA Reverse Transcription kit (Thermo Fisher Scientific, Massachusetts, USA). Quantitative RT-PCR was performed using Applied Biosystems 7500 Real-Time PCR Systems and SYBR Green PCR master mix (Thermo Fisher Scientific, Massachusetts, USA). All reactions were performed at least in triplicates and the amplification signal from the target gene was normalised to GAPDH. PCR conditions were: 50°C 2 min, 95°C 10 min, 40 × cycles of 95°C 15 s, 60°C 1 min and 72°C 7 s. The list of primers is shown in Supplementary Table S1.
Western blot Total protein lysates were collected using RIPA buffer in presence of phosphatase inhibitors. 20-30 µg of lysates were run on SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with primary antibodies (1: 1 000 dilutions) overnight at 4°C and incubated with secondary antibodies (1: 10,000 dilution) for 1 h at room temperature. Chemiluminescence reagents were used. Antibodies used were GAPDH, ERK, p-ERK, SOX2, MITF, Caspase-3 (Cell Signaling Technology, Massachusetts, USA) and CACNA1H (Santa Cruz Biotechnologies, California, USA).
Cell viability assay Cells were seeded in triplicates at a density of 1 × 10 3 -1 × 10 5 cells into 96-well plates. Treatments were tested using a range of concentrations between 0.0001 μM and 10 μM, during 24, 48 and/ or 72 h. Cell viability was determined using alamar blue solution (Invitrogen, Thermo Fisher Scientific, USA). After incubation at 37°C for 4 h, fluorescence emission was measured at 590 nm using a microplate reader (Tecan, Switzerland).

EdU incorporation assay
Cell proliferation was determined with Click-iT EdU Cell Proliferation Kit according to the manufacturer's instructions (Invitrogen, Thermo Fisher Scientific, USA). Briefly, EdU (10 μM) was added to the medium for 2 h. Cells were harvested, washed and fixed for 15 min at room temperature in the dark. After fixation cells were washed and stained with Click-iT™ Plus reaction cocktail containing Alexa Fluor™ 647, for 30 min in the dark. Finally, cells were treated with Ribonuclease A, stained with propidium iodide (PI) (50 μg/mL) and analysed by flow cytometry. Data were analysed with FlowJo10x Software.
Apoptosis assay Detection of apoptosis was performed using FITC-annexin V apoptosis detection Kit I (RUO) (BD Biosciences, New Jersey, USA). After treatment, supernatants were collected, cells were washed twice with PBS and resuspended in Binding Buffer (1 × ) at a concentration of 1 × 10 6 cells/mL. Then, cell suspension (100 μL) was stained with FITC-annexin V and propidium iodide (PI). Cells were incubated for 15 min at room temperature in the dark. Then, 400 μL of Binding Buffer (1×) was added to each tube and then analysed by flow cytometry. Doxorubicin (0.003 μM) was used to induce apoptosis as a positive control. Data were analysed using the FlowJo10X Software.
Clonogenic assay Colony formation assay was performed following the method described by Franken and co-workers. 21 Briefly, cells were seeded in 6-well plates for 24 h, after cells were attached, treatments were added for another 24 h. Treatments were withdrawn and fresh medium was added to cells every 3-5 days. After 10-15 days, colonies were fixed and stained with crystal violet staining solution (0.5%) dissolved in methanol. Colony area was calculated using ImageJ software.   In vivo experiments and drug administration In vivo experiments were assessed using NSG xenograft mouse model. Four to six-week-old female mice were injected subcutaneously (SC) with 100 μL PBS containing 5 × 10 6 HT144 cells or 1 × 10 6 A375 cells. Tumour-bearing mice (100-300 mm 3 ) were randomised (n = 40) and treated in one of the following treatments: (1) mibefradil (0.25 mg/mL) in drinking water for 5 days; (2) vemurafenib (100 mg/kg) dissolved in HPMC solution (0.5%) by oral gavage once a day for 5 days; (3) sequential treatment: vemurafenib by oral gavage once a day for 5 days and mibefradil in drinking water on the next 5 days and (4) vehicle: 0.5% HPMC solution by oral gavage once a day for 5 days. Animals were treated in 2 cycles. Tumour volume, animal weight (18-20 Kg) and welfare were monitored daily. Tumour measurements were conducted using a calliper and tumour volume was calculated using the following formula = (length × width 2 ) × 0.5. Animals were killed by cervical dislocation once the tumour volume reached 1000 mm 3 . Drug administration or euthanasia did not require the use of anaesthesia. Animals were maintained in the animal house facility under optimal conditions and with food and water ad libitum. All animal experiments were performed following according to procedures approved by the German authorities (Animal grant approval number: 35-9185.5/G-208/18). Statistical analysis Data are expressed as the means ± SEM from three or more independent experiments. Differences were analysed by one-way ANOVA followed by post-hoc test Bonferroni or Tukey. Data analyses were performed with GraphPad Prism (GraphPad Software, Inc.). P < 0.05 was considered statistically significant. Logrank test was used to compare survival curves. Tumour growth curves were compared between treatments by fitting a linear mixed model for tumour volume with predictor time, treatment and interaction between time and treatment as fixed effects, and random intercept and slope effect for each mouse. The interaction term was tested to compare the growth rate relative to the vehicle group. P-values were adjusted for multiple comparisons using Holm correction. Software R 3.5 was used for analysis.

RESULTS
Partial reprogramming of melanoma cells induces dedifferentiation and an invasive phenotype in murine melanoma cells Melanoma progression has been described as a step-wise transformation of melanocytes to malignant melanoma triggered by a phenotypic switch toward a more aggressive status. 24 To determine whether our in vitro model of cellular reprogramming could simulate features of melanoma phenotype switching and the intermediate stages during melanoma progression, we partially reprogrammed Nras-mutated C790 and Braf-mutated 4434 murine melanoma cells for up to 20 days using a lentiviral vector carrying Oct4, Klf4 and Sox2 genes (Fig. 1a, b).
Morphological changes were observed during partial reprogramming (Fig. 1c). Additionally, analysis of markers of stemness and melanocytic lineage differentiation showed that Mitf and Pmel expression was significantly decreased while expression of Sox2 and Ssea-1 increased, confirming that cells were de-differentiated ( Fig. 1d and Supplementary Fig. S1a). RNA array data further supported the partial reprogramming of C790 and 4434 murine cells ( Supplementary Fig. S1b, c). Moreover, C790 and 4434 parental cells were treated with doxycycline for 20 days to evaluate whether doxycycline has an effect on the expression of Fig. 1 Partial reprogramming of murine cells leads to a less proliferative and more de-differentiated cell population. a Structure of lentiviral vector used to reprogram cells. Cells were infected with M2mCherry vector plus an expression vector carrying murine Oct4, Klf4 and Sox2 under the control of a tetracycline-responsible element (TRE) and blasticidin-resistant gene. Cells not induced with doxycycline but expressing both vectors were used as a control. b Schematic representation of partial reprogramming method. After infection, cells were treated with doxycycline and selected with blasticidin at day 3. Medium without doxycycline was used for control cells during all experiments. c Representative images of morphological changes during partial reprogramming. Scale bars represent 10 μm. d Real-Time qPCR analysis for stemness markers Sox2 and Ssea-1, as well as for melanocytic lineage differentiation markers Mitf and Pmel, for C790 and 4434 at day 20 of reprogramming. Data were normalised using control cells ("− dox") as reference and Gapdh as housekeeping gene. e Cell proliferation measured by EdU incorporation. Proliferation was analysed at different time points over partial reprogramming as indicated. After incubation with EdU cells were analysed by flow cytometry. Percentage of EdU-positive cells is shown. f Representative images of fluorescence microscopy for cell invasion assay in C790 and 4434 cells. Scale bars represent 500 μm. g Cell invasion assessed by the FluoroBlok invasion assay. Invasion was evaluated at different time points during partial reprogramming. After 20 h incubation, invading cells were labelled and relative fluorescence units were obtained (RFUs = relative fluorescence units). All data are represented as mean ± SEM of three or more independent experiments. Statistical analyses were performed with One-Way ANOVA and post-hoc test; **p < 0.01, ***p < 0.001 and ****p < 0.0001.
During melanoma phenotype switching, cells transit to a more invasive, slow-cycling state. 25 EdU incorporation and Transwell invasion assays revealed a significant reduction in proliferation of reprogrammed cells (Fig. 1e) and an increased number of invading cells (Fig. 1f, g); confirming a phenotypic switch towards a dedifferentiated, aggressive cell type.
Additional Braf-mutated murine melanoma cells (5555) were partially reprogrammed for 20 days. Expression of markers of stemness was enhanced while expression of melanocytic lineage differentiation was decreased ( Supplementary Fig. S2a).    Phenotypic switch was also confirmed with a reduction in proliferation ( Supplementary Fig. S2b) and with a more invasive phenotype ( Supplementary Fig. S2c, d).
Partially reprogrammed melanoma cells adapt to MAPK inhibitors De-differentiation of melanoma tumour cells has been associated with development of resistance to therapy. 26,27 We evaluated the effect of BRAF and MEK inhibitors on the cell viability and cell death of C790 and 4434 partially reprogrammed cells. Cell viability was measured after 72 h of treatment with trametinib, vemurafenib or the combination of trametinib and vemurafenib. Cell viability increased in partially reprogrammed cells (" + dox") throughout the days of reprogramming compared to control cells ("− dox"). Treatment with trametinib alone revealed that C790 control cells are more sensitive (mean IC50 = 2.09 ± 0.48 μM) compared to C790 reprogrammed cells at day 20 (mean IC50 > 10 μM) (Fig. 2a). Combination of vemurafenib and trametinib in 4434 reprogrammed cells also showed a higher IC50 value at day 20 (mean IC50 = 4.38 ± 0.03 μM) compared to 4434 control cells (mean IC50 = 0.0029 ± 0.0010 μM) ( Supplementary Fig. S3a). Mean of IC50 values for all treatments were presented in Supplementary  Tables S2 and S3.
We further evaluated whether cell death is also affected in partially reprogrammed cells after treatment with MAPKi by measuring apoptosis with FITC-annexin V/PI staining. After treatment with trametinib (15 μM), we found that the percentage of apoptotic cells was significantly reduced at day 12 and 20 of reprogramming in C790 de-differentiated cells ( + dox) compared to control cells (−dox). Percentage of apoptosis was also reduced in reprogrammed cells after treatment with doxorubicin (0.003 μM) (Fig. 2b, c). Similar results were obtained at day 20 for 4434 reprogrammed cells, treated with trametinib (8 μM), vemurafenib (15 μM) or the combination of both at a ratio 1:1 (8 μM) ( Supplementary Fig. S3b, c). In addition, activation of caspase-3 was suppressed in reprogrammed cells after treatment with MAPKi compared to control cells ( Fig. 2d and Supplementary  Fig. S3d). We also confirmed a decrease in cell death (Supplementary Fig. S2e, f) and activation of caspase-3 ( Supplementary  Fig. S2g) in 5555 partially reprogrammed cells at day 20 after treatment with the combination of trametinib and vemurafenib. All these findings support that partially reprogrammed cells become less sensitive to MAPKi.
MAPKi-adaptive cells overexpress T-type calcium channels Since partially reprogrammed cells were less sensitive to MAPKi we wondered whether ERK activation was also affected following treatment with trametinib or with the combination of trametinib and vemurafenib. Western blot analysis revealed that phosphorylation of ERK is impaired in both C790 and 4434 partially reprogrammed cells and control cells (Fig. 3a, b), indicating that MAPK pathway inhibition is independent from the dedifferentiation status of the cells.
We further investigated the mechanisms of adaptation in dedifferentiated cells using gene expression array analysis for C790 reprogrammed cells at day 20 after the treatment with trametinib (Fig. 3c). The dendrogram showed some of the genes differentially expressed in reprogrammed cells compared to control cells, +dox vs −dox. Moreover, IPA software was used to analyse upstream regulators that might be involved in regulation of drug sensitivity after partial reprograming of melanoma cells (Fig. 3d). Interestingly, T-type calcium channels were predicted to be one of the significant upstream regulators in adaptive cells, suggesting them as a target to alleviate therapy adaptation in melanoma. Moreover, analysis from TCGA-SKCM database showed that melanoma patients with high expression of CACNA1H have a worse survival outcome compared with those with low CACNA1H expression (p = 0.029) ( Supplementary Fig. S4a) Furthermore, we confirmed that Cacna1h was highly expressed in both C790 and 4434 partially reprogrammed cells ( Fig. 3e and Supplementary Fig. S4b), highlighting the importance of calcium in adaptation to MAPKi.
Based on these data, we were interested to further explore the effect of T-type calcium channels in de-differentiated and adaptive melanoma cells. We speculated that adaptation to MAPKi could be regulated by an alternative pathway, in which T-type calcium channels may be involved.
Furthermore, single treatments with mibefradil and lomerizine slightly increased the percentage of apoptotic cells after 24 h in both C790 (Fig. 4b, c) and 4434 partially reprogrammed cells ( Supplementary Fig. S5b, c). Together, these data indicated that inhibition of T-type calcium channels has the potential to promote apoptosis and affect cell viability in de-differentiated and adaptive cells.
Considering these results and the high expression of T-type calcium channel in de-differentiated cells (Fig. 3e and Supplementary Fig. S4b), we hypothesised that these channels could be potential candidates to re-sensitise adaptive cells to MAPKi. Therefore, we evaluated drug sensitisation by treating reprogramed cells with mibefradil or lomerizine for 24 h followed by treatment with MAPKi for another 24 h (sequential treatment). The percentage of apoptotic cells was significantly increased in partially reprogrammed cells after the sequential treatment ( Fig. 4d and Supplementary Fig. S5d), and the capacity of colony formation was significantly reduced in C790 partially Fig. 2 Partially reprogrammed cells adapt to MAPK inhibitors (MAPKi). a Cell viability performed using alamar blue assay. Nras-mutant cells (C790) were treated with trametinib (10-0.0001 μM) for 72 h, after 3 h incubation with alamar blue, fluorescence emission (read at 590 nm) was obtained. Cell viability was evaluated at days 6, 12 and 20 of reprogramming of C790 cells. Cytotoxicity curves represent as mean ± SEM (n = 6). b Representative scatter plots of PI (y-axis) vs. annexin V (x-axis) for day 6 and 20 of partial reprogramming. Early apoptotic cells are shown in the lower right quadrant and late apoptotic cells are shown in the upper right quadrant. c Apoptosis after staining with FITC-Annexin V/PI. Cells were treated with trametinib (15 μM) for 72 h and with doxorubicin (0.003 μM) to induce apoptosis (positive control). Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 3). d Western blot analysis. Whole cell lysates were immunoblotted with GAPDH and caspase-3 antibodies at day 20 of reprogramming after treatment with trametinib. GAPDH loading control is identical in western blots of Figs. 2d and 3a at day 20, since it was analysed on the same blot. Statistical analyses were performed with One-Way ANOVA and posthoc test; **p < 0.01, ***p < 0.001 and ****p < 0.0001.
T-type calcium channel inhibition restores sensitivity to MAPK inhibitors. . . K Granados et al. reprogrammed cells after sequential treatment, (Fig. 4e, f), suggesting that previous sensitisation with mibefradil increased cell death and decreased the capacity of unlimited cell reproduction after treatment with MAPKi. Similar results were obtained in 4434 partially reprogrammed cells ( Supplementary Fig. S5e, f). Since calcium participates as a second messenger in a wide variety of cellular mechanisms, identifying how calcium regulates cellular capacity for self-renewal is challenging. We speculated that T-type calcium channels affect the pluripotency status of reprogrammed cells. Treatment with mibefradil and lomerizine in C790 and 4434 partially reprogrammed cells revealed that the expression of stemness markers Sox2, Ssea-1 and CD271 were significantly reduced ( Supplementary Fig. S6a, b), leading into a more differentiated phenotype. Together these data suggest that Calcium channel antagonists sensitise human BRAF-adaptive melanoma cells To establish the role T-type calcium channels play in adaptive human melanoma cells, we used BRAFi-adaptive melanoma cells A375, SK-MEL-28 and HT144 treated for 24 h with vemurafenib (3 µM). We confirmed that human vemurafenib-adaptive melanoma cells increased expression of T-type calcium channels genes, CACNA1G and CACNA1H compared to parental cells (Fig. 5a).
These results were consistent with observations in partially reprogrammed murine cells and confirmed the possibility of using calcium channels blockers to sensitise adaptive cells to MAPK inhibitors.
In agreement with earlier results, treatment with mibefradil on human adaptive cells suppressed expression of differentiation markers associated with adaptive resistance 28,29 in both parental and adaptive cells ( Supplementary Fig. S9a, b, c). Sequential treatment ("Vem24h » Mibe24h » Vem24h") showed a strong reduction of SOX2 expression, mRNA and protein levels, in SK-MEL-28 and HT144 adaptive cell lines ( Supplementary Fig. S9d), supporting a possible connection between calcium signalling and stemness maintenance.
Silencing of CACNA1H promotes cell death and differentiation in human BRAF-adaptive melanoma cells To test whether CACNA1H is responsible for the increase in apoptosis and sensitisation in adaptive cells, we silenced CACNA1H gene in human melanoma cells (A375, SK-MEL-28 and HT144) and analysed the effect on cell death and differentiation after treatment with vemurafenib. CACNA1H knockdown was verified by qPCR and western blot in all cell lines (Fig. 6a, b and Supplementary Fig. S10a, b).
We evaluated apoptosis in CACNA1H knockdown cell lines after 24 h of treatment with vemurafenib or the combination treatment with vemurafenib and trametinib. CACNA1H knockdown increased apoptosis in CACNA1H knockdown cell lines compared to the scramble control (Fig. 6c), thereby confirming that induction of apoptosis in BRAFi-adaptive cells was driven by inhibition of CACNA1H calcium channel. Due to limited efficiency of knockdown ( Supplementary Fig. S10b) there was no significant difference in cell death for HT144 CACNA1H knockdown cells ( Supplementary Fig. S10c). Moreover, colony formation capacity was affected in CACNA1H knockdown cells lines A375 and SK-MEL-28 after treatment with vemurafenib (Fig. 6d, e), supporting that CACNA1H calcium channel knockdown increased sensitivity of adaptive cells to BRAFi.
To confirm that knockdown of CACNA1H was responsible for changes in differentiation status, we measured SOX2 expression in human melanoma cells. mRNA and protein levels were substantially reduced in both A375 and SK-MEL-28 cells indicating decreased stemness phenotype ( Supplementary Fig. S10d, e). Taking together, CACNA1H silencing induced a similar effect on cell death and differentiation as the pharmacological inhibition of calcium channels with mibefradil. Sequential treatment using mibefradil and vemurafenib reduces tumour growth and improves survival in HT144 and A375 adaptive xenografts model To determine the anti-tumour effect of the sequential treatment in vivo, HT144 and A375 xenografts model were used. Mice were inoculated subcutaneously either with HT144 or A375 cells treated for 24 h with vemurafenib. After tumour volume reached 100-300 mm 3 , animals (n = 40) were randomised to four different treatment groups. Sequential treatment consists in oral administration of vemurafenib for 5 days, follow by mibefradil for another 5 days. This workflow was repeat until two cycles were completed and finally, vemurafenib was apply for the last 5 days of the experiment (Fig. 6f and Supplementary Fig. S10f). In parallel, single treatments with vemurafenib, mibefradil or vehicle were tested.
Single treatments with vemurafenib (p = 0.013) and mibefradil (p = 0.025) significantly reduced tumour growth compared to control group (vehicle) while sequential treatment (p < 0.001) showed the most significant reduction on tumour volume compared to control mice (vehicle) in HT144 xenografts (Fig. 6g). Fig. 3 Expression analysis of MAPKi-adaptive cells shows upregulation of T-Type calcium channels. a, b Western blot analysis. Whole cell lysates were immunoblotted with GAPDH, ERK and P-ERK antibodies in C790 and 4434 melanoma cells. GAPDH loading control is identical in western blots of Figs. 2d and 3a at day 20, since it was analysed on the same blot c Differential gene expression analysis. Heat map of microarray data showing hierarchical clustering of 2700 differentially expressed genes in partially reprogrammed cells C790 after 72 h treatment with DMSO (control) and trametinib; control ("−dox") and reprogrammed cells (" + dox") were evaluated both at day 20. Blue or yellow colours indicate differentially up-or downregulated genes, respectively (FC > 2-fold). d Analysis of gene expression data by a Venn diagram showing the analysis of treatments between control cells and reprogrammed cells (2700 genes). The blue circle (474 genes) indicates the number of genes exclusively expressed in control vs. trametinib ("−dox"); yellow circle (1406 genes) indicates the number of genes exclusively expressed in control vs. trametinib (" + dox"). Some of the deregulated genes are listed; green and red colours indicate differentially up-or downregulated genes, respectively. Moreover, results from IPA analysis of upstream regulators from C790 partially reprogrammed cells at day 20 after treatment with trametinib are listed. e Real-Time qPCR analysis for Cacna1h at different days of reprogramming for both C790 and 4434 cells. Data were normalised using control cells as reference and GAPDH as housekeeping gene. All data are represented as mean ± SEM of three or more independent experiments. Statistical analyses were performed with One-Way ANOVA and post-hoc test; **p < 0.01 and ***p < 0.001.
T-type calcium channel inhibition restores sensitivity to MAPK inhibitors. . . K Granados et al. Moreover, survival data showed a better outcome for mice treated with sequential treatment compared to control group (p < 0.001) (Fig. 6h). In addition, single treatments of vemurafenib (p = 0.001) and mibefradil (p < 0.001) showed a significant difference in survival compared to vehicle group. These results support the use of mibefradil to enhance the anti-tumour effects of vemurafenib in vivo. In A375 xenografts, single treatment with vemurafenib alone led to a slight reduction in tumour growth compared to control group (vehicle). In contrast, mice treated with mibefradil (p =    Fig. S10g). Considering the survival data, mice treated with sequential treatment showed a better outcome, compared to control group (p < 0.001) ( Supplementary Fig. S10h), similar to the results obtained in HT144 xenografts. Single treatments of vemurafenib (p = 0.053) and mibefradil (p = 0.063) did not show significant difference in survival compared to vehicle group. These in vivo effects are consistent with our previous in vitro data in human adaptive cells and mouse reprogrammed cells, suggesting that the inhibition of T-type calcium channels is a promising strategy to sensitise de-differentiated and adaptive melanoma cells to MAPKi.

DISCUSSION
Here, we have demonstrated that partial reprogramming of melanoma cells induced a de-differentiated phenotype and increased adaptation against MAPKi. We observed that T-type calcium channels expression increased in MAPKi-adaptive melanoma cells and that inhibition of T-type calcium channels enhanced cell death and differentiation in reprogrammed melanoma cells and in human BRAFi-adaptive melanoma cells. Based on the reduction of tumour growth and the improvement in overall survival after sequential treatment in vivo, we suggest that T-type calcium channels are potential targets to eliminate adaptive melanoma cells by restoring sensitivity to MAPKi.
In vitro de-differentiation or reprogramming of melanoma cells has been described previously. 20,[30][31][32] Nras and Braf-mutated melanoma cells were partially reprogrammed to study genetic and molecular changes during the intermediate stages of tumour progression. We observed a switch in gene expression of partially reprogrammed cells with increased expression of stemness related genes, reduced levels of melanocytic markers as well as decreased proliferation and increased invasiveness, which are consistent with the classical features of phenotype switching in melanoma. 24,33 Considering that cellular de-differentiation has been correlated with development of resistance to therapies, 6,33,34 and that progressive de-differentiation status of melanoma has been observed in patient samples under standard treatments with MAPKi, 26 we hypothesised that partially reprogrammed melanoma cells would be less sensitive to BRAF or MEK inhibitors. Consistent with other reports where de-differentiation of human melanoma cells conveyed increased adaptation to MAPKi, 31 our results indicated that partially reprogrammed murine melanoma cells also increased survival after treatment with trametinib, vemurafenib or with their combination.
Several mechanisms have been implicated 2,5,35,36 in acquired, adaptive and intrinsic resistance in melanoma, although most have included reactivation of ERK signalling with some exceptions. 36 Here we showed that the inhibition of MAPK pathway in partially reprogrammed cells affected the phosphorylation status of ERK protein in both C790 and 4434 melanoma cells independently of the de-differentiated state of the cells. Moreover, gene expression analysis of C790 reprogrammed cells revealed an upregulation of T-type calcium channels suggesting an association between the de-differentiated state of the cells and sensitivity to calcium channel antagonists. T-type calcium channels belong to a wide family of voltage gated calcium channels (VGCCs); each subfamily involves several isoforms (Cav1, Cav2 or Cav3) that display different electrophysiological properties. 37 Channels with low voltage-activated and transient currents are known as T-type channels, which are expressed in numerous cell types including non-excitable cells. 38 Alteration in the expression of any of the VGCCs is associated with neurological diseases, such as epilepsies, 39 or cardiac conditions like arrhythmias. 40 Since T-type calcium channels were reported to be upregulated in cancer cells, 41 growing evidence has shown that VGCCs are widely expressed in many types of cancer with a particularly significant increase of T-type calcium channels in tumour cells. This overexpression has been confirmed in glioblastoma, 19,42 ovarian cancer, 18 breast cancer, 13 leukaemia 17 and melanoma. 15 Accordingly, our results showed upregulation of T-type calcium channels in MAPKi-adaptive melanoma cells and the effect of mibefradil by inducing apoptosis and reducing the capacity of colony formation.
Previous investigations have shown that administration of Ttype calcium channel antagonists (mibefradil or NNC-55-0396) inhibits proliferation and induces apoptosis in several cancer cells, 12 i.e. U87MG glioma, 43 colon cancer, 16 human lung adenocarcinoma (A549), pancreatic cancer (MiaPaCa2), 14 melanoma 44 and leukaemia cells. 17 Moreover, biopsies from melanoma patients show a gradual increase in T-type calcium channel expression, which relates to poor prognosis. 45 In accordance with this, the TCGA analysis also confirmed that melanoma patients with high expression of the CACNA1H gene have poorer survival compared to patients with low expression.
Inhibition of T-type calcium channels has been used to sensitise resistant cancer cells to specific therapies or conventional chemotherapy. However, there are no previous reports of using T-type calcium channel antagonists in de-differentiated and adaptive melanoma cells in order to restore sensitivity to MAPKi. Investigations in ovarian cancer show that mibefradil enhances anti-tumour activity of carboplatin in vitro and in vivo. 18,46 In glioblastoma, mibefradil treatment enhanced the anti-tumour effects of ionizing radiation. 47 The combination of mibefradil with temozolomide has shown a stronger therapeutic effect and survival. 19 Consistent with these findings our results show that mibefradil potentiates the lethal effect of MAPKi, when administered sequentially, in MAPKi-adaptive melanoma cells in vitro and in vivo. This was further supported through silencing of CACNA1H transcript in human BRAFi-adaptive melanoma cells.
The role of T-type calcium channels in de-differentiation and adaptation to therapy remains unclear. Zang et al. (2017) showed that T-type calcium channels are enriched in glioblastoma stemlike cells (GSC), which are resistant to temozolomide. They   Fig. 4 Inhibition of calcium channels increases sensitivity to MAPK inhibitors in partially reprogrammed cells. a Cell viability was performed using alamar blue assay. Partially reprogrammed cells C790 were treated with mibefradil (10-1.25 μM) for 24 h. After 3 h incubation with alamar blue, fluorescence (at 590 nm) was obtained. Cell viability was evaluated at days 6, 12 and 20 of reprogramming. b Representative scatter plots of PI (y-axis) vs. annexin V (x-axis) for day 20 of partial reprogramming. Early apoptotic cells are shown in the lower right quadrant and late apoptotic cells are shown in the upper right quadrant. Mibe: mibefradil (7 μM). Lome: lomerizine (7 μM). Mibe (Lome) »Tra: sequential treatment with mibefradil (lomerizine) 24 h, followed by trametinib (15 μM) for another 24 h. c Apoptosis analysis using annexinV/PI staining. Cells were treated with mibefradil (7 μM) and lomerizine (7 μM) for 24 h. Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 3). d. Apoptosis analysis using sequential treatment with calcium channel blockers and MAPKi. Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 3). e Clonogenic assay of partially reprogrammed cells C790 treated with DMSO (0.01%), mibefradil (7 μM), lomerizine (7 μM), Mibe (Lome) »Tra: sequential treatment with mibefradil (lomerizine), and trametinib (15 μM) for 24 h. Representative images of wells stained with crystal violet are shown. f Percentage of colony area for all treatments is shown as mean ± SEM (n = 5) for partially reprogrammed cells C790. All data are represented as mean ± SEM of three or more independent experiments. Statistical analyses were performed with One-Way ANOVA and post-hoc test; *p < 0.05, **p < 0.01 and ***p < 0.001.
T-type calcium channel inhibition restores sensitivity to MAPK inhibitors. . . K Granados et al. demonstrated that blockade of calcium channels can re-sensitise resistant glioblastoma cells to chemotherapy. 19 It is known that undifferentiated mouse embryonic stem cells (ES) have voltage dependent Ca + currents, which link to cell cycle progression and maintenance of self-renewal in ES cells. 48 In a similar way, GSC show an enrichment in the expression of the Cav3.2 channel, which correlates with resistance and poor prognosis. 19 In addition, differentiation of GSC is induced upon treatment with mibefradil, which was confirmed by downregulation of stemness markers. 19 In order to elucidate the connection between enhanced expression of T-type calcium channels and the de-differentiation status of MAPKi-adaptive cells we evaluated specific stemness markers in murine and human melanoma cells. After single treatment with mibefradil or lomerizine, we observed a significant 18   T-type calcium channel inhibition restores sensitivity to MAPK inhibitors. . . K Granados et al. Fig. 5 Mibefradil increases vulnerability to MAPK inhibitors in human BRAF-adaptive melanoma cells. a Gene expression analysis of CACNA1G and CACNA1H in human melanoma cell lines A375, SK-MEL-28 and HT144 upon 24 h treatment with vemurafenib (3 μM) (adaptation). Data were normalised using control cells as reference and 18 S as housekeeping gene. b Human melanoma cells were treated with mibefradil and lomerizine (10, 9, 7, 5, 2.5 and 1.25 μM) for 24 h (only mibefradil treatment is shown). After 3 h of incubation with alamar blue, fluorescence (590 nm) was obtained. c Apoptosis analysis of human melanoma, cells were treated with vemurafenib (3 μM) for 24 h, followed by mibefradil (7 μM) for 24 h; after this period cells were re-treated with vemurafenib (3 μM) for another 24 h ("Vem 24 h » Mibe 24 h » Vem 24 h"). Cells were stained and analysed by flow cytometry. Percentage of apoptotic cells (early and late apoptosis) is shown as mean ± SEM (n = 5). d Clonogenic assay of human cells A375, SK-MEL-28 and HT144 treated for 24 h with DMSO (0.01%), mibefradil (7 μM), vemurafenib (3 μM) and sequential treatment (Vem 24 h » Mibe 24 h » Vem 24 h). Representative images of wells stained with crystal violet are shown. e Percentage of colony area for all treatments is shown as mean ± SEM (n = 3) in all human melanoma cell lines. All data are represented as mean ± SEM of three or more independent experiments. Statistical analyses were performed with One-Way ANOVA and posthoc test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. decrease of stemness related markers in murine de-differentiated cells. These results are in line with the reduction of Oct3/4 and Nanog expression in mouse ES after pharmacological blockage of T-type calcium channels or with Cav3.2 siRNA. 48 Consistently, adaptive resistance markers SOX2, ID1 and ID3 were also decreased in human adaptive cells after treatment with mibefradil alone or with the sequential treatment. Reduction of SOX2 expression was also confirmed with CACNA1H knockdown in human melanoma cells. 28 Together, these results conclude that the inhibition of T-type calcium channels induces differentiation in MAPKi-adaptive cells, suggesting the participation of calcium signalling in the stemness maintenance and drug adaptation in melanoma cells.
In conclusion, we have demonstrated that inhibition of T-type calcium channels increases cell death, differentiation, and restores sensitivity of de-differentiated and adaptive melanoma cells to MAPKi. Sequential treatment with mibefradil and vemurafenib is more effective against MAPKi-adaptive melanoma cells in vitro and in vivo, reducing tumour growth and increasing overall survival in mice. Our results confirm the potential use of T-type calcium channels antagonists as an effective treatment to restore sensitivity to MAPKi in melanoma. Importantly, the safe and therapeutic use of mibefradil in humans have been evaluated in a sequential treatment with temozolomide in patients with recurrent high-grade gliomas, 49 supporting the suitability of this new treatment approach for melanoma patients.