Overcoming melanoma resistance to vemurafenib by targeting CCL2-induced miR-34a, miR-100 and miR-125b

Abstract

In melanoma, the adaptative cell response to BRAF inhibitors includes altered patterns of cytokine production contributing to tumor progression and drug resistance. Among the factors produced by PLX4032-resistant melanoma cell lines, CCL2 was higher compared to the sensitive parental cell lines and increased upon drug treatment. CCL2 acted as an autocrine growth factor for melanoma cells, stimulating the proliferation and resistance to apoptosis. In patients, CCL2 is detected in melanoma cells in tumors and in plasma at levels that correlate with tumor burden and lactate dehydrogenase. Vemurafenib treatment increased the CCL2 levels in plasma, whereas the long-term clinical response was associated with low CCL2 levels.Increased CCL2 production was associated with miRNA deregulation in the resistant cells. miR-34a, miR-100 and miR-125b showed high expression in both resistant cells and in tumor biopsies that were obtained from treated patients, and they were involved in the control of cell proliferation and apoptosis. Inhibition of CCL2 and of the selected miRNAs restored both the cell apoptosis and the drug efficacy in resistant melanoma cells. Therefore, CCL2 and miRNAs are potential prognostic factors and attractive targets for counteracting treatment resistance in metastatic melanoma.

Keywords: BRAF inhibitor; CCL2; drug resistance; melanoma; miRNAs.

Figure 1. CCL2 is upregulated in PLX4032-resistant cell lines and is induced upon drug treatment

A. CCL2 gene expression in seven resistant cell lines compared to their sensitive counterparts as detected by quantitative real time RT-PCR and shown as 2^−ΔCt (left); CCL2 release in medium shown as pg/mL/10⁵ cells (right). _*p< 0.05 by Mann-Whitney U-test and unpaired t-test. B. Induction of CCL2 by short-term (72 h) PLX4032 treatment in resistant and sensitive cell lines, plotted as the fold increase compared to untreated controls. S, sensitive cell lines; R, resistant cell lines. _*p<0.05, unpaired t-test.

Figure 2. CCL2 acts as an autocrine factor for melanoma cells

A. Treatment with rCCL2 (100 ng/mL) increased the number of viable LM16-S cells (left) and reduced the number of caspase-3 positive cells upon treatment with PLX4032, as assessed by FACS analysis (right). B. pCCL2/LM16-S transfectant cells show an increase in cell growth (left), and a reduction of caspase-3 positive cells after treatment with PLX4032 (right) compared to mock transfectants (pmock). pCCL2/LM16-S transfectant cells showed a significant increase in CCL2 gene expression (137-fold) and protein release (200-fold) when compared to pmock/LM16-S cells. _***p<0.001 and _**p<0.01 by two-way ANOVA. C. Upregulation of phosphorylated AKT (S473) and p70S6K (T389) in LM16-S cells treated with rCCL2 as detected by western blot analysis; vinculin was used as a loading control. D. Proliferation of LM16-S cells in the bottom chamber of transwell plates with LM16-R cells loaded into the upper wells; the fold change relative to untreated controls is shown. _***p<0.0001 by one-way ANOVA followed by Bonferroni correction. E. Left, transfection with siRNA for CCL2 increases the responsiveness of LM16-R, LM17-R and LM47-R cells to PLX4032 cell growth inhibition, as detected by MTT assays. Two different siCCL2s (A and B) are shown. The results are shown relative to scrambled control. _***p<0.0001 and _*p<0.05 by one-way ANOVA followed by Bonferroni correction. Right, PLX4032 treatment increased the number of LM16-R, LM17-R and LM47-R cells that were positive for caspase-3 when treated with siRNA for CCL2.

Figure 3. CCL2 is expressed by melanoma cells in tumor tissues from patients

A. CCL2 gene expression (orange bars) and protein (blue bars) detected in fresh surgical melanoma biopsies, including one excised before treatment (A10) and seven during treatment (A2, SE1, SE2, B3, and G8), two of which resulting non-tumoral at histopathology (NM1, NM2). A10 and A2 are matched biopsies obtained before and during treatment, respectively, whereas SE1 and SE2 are biopsies excised from the same patient at 2 and 12 months of treatment. CCL2 gene expression was detected by qRT-PCR and calculated as ΔCt ± SD relative to β-actin. CCL2 protein was quantified by bead-based FACS analysis: values of μg of CCL2 detected in 50 μg of protein lysates are shown. _***p<0.0001 and _**p<0.01 by unpaired t-test. B. CCL2 immunostaining of three representative tumor lesions showing low (upper), moderate (middle), or high (lower) staining intensity; these lesions were all stained positive for BRAFV600E. At the bottom, ISH showing the CCL2-specific signal in the tumor cells (black arrows); UBC staining was used as a control.

Figure 4. CCL2 plasma levels are high in melanoma patients, depend on tumor burden, and increase during treatment with BRAFi

A. CCL2 plasma levels in 32 patients (Pts) and in age-and gender-matched healthy donors (HD). _*p<0.05 by unpaired t-test. B. Association of CCL2 levels with tumor burden (LTB, low tumor burden; HTB, high tumor burden). _***p<0.0001 by unpaired t-test. C. Correlation between plasmatic CCL2 and LDH values. The r_s and p values resulting from Spearman analysis are shown. D. Higher CCL2 levels were found in patients with a short clinical response to treatment (SR, n=23) compared to those in long-term responders (LR, n=10). _*p<0.05, unpaired t-test; ns, not significant. E. CCL2 levels in LR patients measured at baseline and at different time points during treatment. CCL2 plasma levels in two different set of samples from LR patients are shown, LRa (n=10) and LRb (n=12). _**p<0.01 by paired t-test.

Figure 5. Concerted HIF1A and CCL2 regulation

A. HIF1A gene expression analysis showing upregulation in LM16-R cells compared to LM16-S cells, reduction upon CCL2 silencing (siCCL2), and an increase in LM16-S cells treated with rCCL2 at 100 ng/mL for 24 h. Actin was used as the internal reference and LM16-S or siControl as the calibrator. Relative quantification (RQ) values obtained by qRT-PCR are shown. _***p<0.0001 and _*p<0.05 by unpaired t-test. B. Increase in HIF1A gene expression in melanoma cell lines upon PLX4032 treatment. 2^−ΔCt values are shown. _*p<0.05 by Mann-Whitney U-test. C. Analysis of the correlation between the CCL2 and HIF1A gene expression levels in melanoma tissues from patients (n=20). The r_s and p values resulting from Spearman analysis are shown. D. Expression levels of HIF1 targets in LM16-R compared to LM16-S cell lines. FACS analysis detection of EGFR, integrin α1 and integrin α5. Percentages of protein expression are shown in the graph. E. Expression of COX2 in LM16-S and LM16-R cells as detected by western blot analysis; production of MMP-2/-9 as detected by gelatin zymography in supernatants from LM16-S and LM16-R cells.

Figure 6. miR-34a, miR-100 and miR-125b are upregulated in resistant cells

A. Expression levels of selected miRNAs in seven resistant cell lines relative to that in their sensitive counterparts. 2^−ΔCt values calculated relative to U6 used as the internal reference are shown. _*p<0.05, _**p<0.01 by Mann-Whitney U-test. B. In LM16-R cells, silencing of CCL2 reduces the expression of miR-34a, miR-100 and miR-125b. miRNA expression is shown as RQ values calculated by comparison to the scrambled control. _*p<0.05 and _***p<0.0001 by unpaired t-test. C. In LM16-S cells, treatment with rCCL2 increased the expression of miR-34a, miR-100 and miR-125b. rCCL2 was used at 100 ng/mL for 72 h. miRNA expression is shown as RQ values calculated by comparison to untreated cell control. _***p<0.0001 by unpaired t-test. D. Reduction of CCL2 release by LM16-R cells after inhibition of miR-34a, miR-100 and miR-125b in combination but not when each was individually inhibited. Data are presented as the percent change when compared to the scrambled control._***p<0.0001 by one-way ANOVA followed by Bonferroni correction. i-miR, specific miRNA inhibitors.

Figure 7. miR-34a, -100 and -125b are upregulated in tumors from patients treated with BRAFi

. A. Expression levels of miR-34a, miR-100 and miR-125b in metastatic lesions excised from patients during treatment with BRAFi (n=5) (treated) and before treatment (n=20) (untreated). _**p<0.01, _***p<0.0001 by Mann-Whitney U-test. B. Analysis of the correlation between miR-125b expression and the expression of miR-34a and miR-100 in BRAFV600E melanoma tissues (n=27). The r_s and p values resulting from Spearman analysis are shown.

Figure 8. Inhibition of miR-34a, miR-100 and miR-125b increases responsiveness to PLX4032

A. Concomitant inhibition of miR-34a, miR-100 and miR-125b increased the antiproliferative effect of BRAFi treatment as indicated by MTT assay (left) and increased caspase-3 positive cells (right). _***p<0.0001 by unpaired t-test. The expression of miR-34a, miR-100 and miR-125b was reduced by 3.3-, 100-, and 25-fold, respectively. B. The increased expression of miR-34a, miR-100 and miR-125b caused by the transfer of miRNA mimics reduced the apoptosis in LM16-S cells. The expression of miR-34a, miR-100 and miR-125b was increased by 14200-, 4227- and 812-fold, respectively. C. Left, LM16-S cell growth in transwell plates shown as the fold change compared to the untreated control: co-culture with LM16-R cells in the upper chamber reduced the antiproliferative effect of PLX4032. Right, the inhibition of miR-34a, miR-100 and miR-125b by specific miRNA inhibitors (i-miR) increases the antiproliferative effect of the drug. Fold change values compared to scrambled control are shown. _***p<0.0001 by one-way ANOVA followed by Bonferroni correction, _*p<0.05 by unpaired t-test. Microscope appearance of the co-cultured cells (magnification 10X). D. Cell apoptosis induced by soluble TRAIL (50 ng/mL for 24 h) after the treatment with inhibitors (i-miR) or mimics (m-miR) of miR-34a, miR-100 and miR-125b. The fraction of activated caspase-3-positive cells is shown.

Figure 9. Schematic representation of the CCL2/HIF1/miRNA loop associated with resistance

In resistant cells, CCL2 production is associated with HIF1 activation and miR-34a, miR-100 and miR-125b upregulation. The inhibition of CCL2 or of miRNAs restores PLX4032 responsiveness. siCCL2, CCL2 siRNA; i-miR, miRNA inhibitors.