MITF controls the TCA cycle to modulate the melanoma hypoxia response

Abstract

In response to the dynamic intra-tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia-associated transcription factor (MITF). The response to hypoxia is driven by hypoxia-inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis. HIF1α indirectly represses MITF that can activate HIF1α expression. Although HIF and MITF share a highly related DNA-binding specificity, it is unclear whether they co-regulate subset of target genes. Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown. Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia. Surprisingly, under hypoxia MITF is transiently up-regulated by HIF1α and co-regulates a subset of HIF targets including VEGFA. Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo-hypoxia. Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.

Keywords: MITF; genomewide; glucose limitation; hypoxia; melanoma.

Figure 1

Identification of a common set of melanoma hypoxia‐regulated genes. (a) Heatmaps derived from triplicate RNA‐seq analysis showing differential gene expression in three melanoma cell lines grown in normoxia or in 1% oxygen for indicated times. Only those genes differentially regulated by more than twofold and p = < 0.05 are shown. (b) GSEA analyses showing enrichment of the Elvidge hypoxia gene expression signature in indicated cell lines grown in normoxia or 1% oxygen for 12 hr. (c–e) Heatmaps showing results of GSVA for indicated gene sets for three melanoma cell lines grown in normoxia or 1% oxygen for indicated times. (f) Western blot of indicated cell lines grown in normoxia or hypoxia for 24 hr. (g) Venn diagrams showing unique and co‐regulated genes up‐ or down‐regulated by at least twofold (p < 0.05) in response to 1% oxygen in the three melanoma cell lines after 12 hr in hypoxia. (h) List of genes commonly up‐regulated in response to hypoxia in the three melanoma cell lines examined after 12 hr in hypoxia. (i) Bar charts indicating potential reasons why few genes in common are regulated by hypoxia 12 hr after exposure to 1% oxygen in all three cell lines

Figure 2

Genomewide binding of the HIFs in 501mel cells (a) Number of binding sites with peak score above 10 for each of the three HIFs derived from the ChIP‐seq analysis in the 501mel cell line. (b) Consensus motifs for each of the three HIFs determined from the ChIP‐seq analysis. (c) Venn diagram showing common and uniquely occupied sites. (d) Genome ontology of HIF binding sites. (e) UCSC genome browser screenshots of occupied sites in the 501mel cell line for three of the genes commonly regulated in the three melanoma cell lines. (f) Venn diagrams integrating ChIP‐seq and RNA‐seq datasets from 501mel cells for genes bound by the HIFs and twofold up‐ or down‐regulated at 12 hr post‐hypoxia as indicated

Figure 3

MITF is positively regulated by HIF1α. (a) Cell pellet from IGR37 cells grown in normoxia or hypoxia as indicated. (b–d) Heatmaps derived from triplicate RNA‐seq analysis showing relative expression of indicated genes from 501mel, 501mel‐shHIF1α, and IGR37 cells in normoxia or after indicated times in hypoxia. (e) Western blot of 501mel cells grown for indicated times in normoxia or hypoxia treated with control or siRNA specific for MITF. (f) Western blot of 501mel cells or 501mel‐shHIF1α cells grown for indicated times in 1% oxygen. (g) UCSC genome browser screenshot of HIF ChIP‐seq showing all three HIFs bound upstream from the MITF‐M promoter. (h,i) Heatmaps showing relative expression of indicated genes or gene sets from 501mel, 501mel‐shHIF1α cell lines grown in normoxia or after indicated times in hypoxia. (j) Western blot of extracts from 501mel cells grown in RPMI (15 mM glucose) or in medium lacking glucose (‐glucose) for indicated times, or after re‐addition of 15 mM glucose for 30 min as indicated

Figure 4

MITF regulates a subset of hypoxia‐responsive genes (a) Venn diagram showing ChIP‐seq peaks for each of the three HIF factors and MITF. Numbers indicated number of unique and overlapping peaks for each combination of experiment. (b) Read density maps of the ChIP‐seq profile for each HIF and MITF ranked by peak score for HIF1α centered on the co‐occupied sites ± 2.5 kb. (c) De novo motifs derived from the ChIP‐seq analysis of overlapping sites bound by each of the hypoxia factors as well as MITF. (d) Heatmap derived from triplicate RNA‐seq of 501mel cells or a derivative line expressing shHIF1α showing a set of genes bound and co‐regulated by HIF and MITF. (e) UCSC genome browser screenshot of HIF ChIP‐seq and a biological replicate of an MITF ChIP‐seq showing all three HIFs and MITF bound upstream from the VEGFA gene. (f) Heatmap derived from triplicate RNA‐seq showing differential expression of genes associated with peaks co‐occupied by HIFs and MITF in 501mel expressing doxycycline‐inducible MITF over time following induction. (g, h) Heatmap showing peak scores for the HIF family members and MITF on the indicated genes after 12 hr in 1% Oxygen

Figure 5

MITF represses its own expression. (a) Western blot showing doxycycline‐mediated induction of ectopic HA‐tagged MITF expression and corresponding expression of endogenous MITF. (b) qRT–PCR of the endogenous human MITF mRNA in 501mel cells induced to express ectopic murine HA‐tagged Mitf using 100 ng doxycycline. Expression normalised to ACTIN mRNA. (c) Immunofluorescence of 501mel cells transfected with an FLAG‐tagged mouse Mitf expression vector. Anti‐FLAG antibody was used to detect ectopically expressed Mitf WT and anti‐human‐specific mouse monoclonal antibody D5 used to detect endogenous MITF. (d) UCSC genome browser screenshot showing a biological replicate ChIP‐seq of HA‐MITF bound to the MITF locus

Figure 6

MITF regulates SDHB to suppress pseudo‐hypoxia. (a) GSVA analysis showing relative expression of indicated gene sets in the top and bottom 20 CCLE melanoma cell lines ranked by MITF expression. (b) The TCA cycle. Green‐boxed metabolites are significantly (p < 0.01) up‐regulated both in IGR39 compared to IGR37 cells and in MITF‐depleted 501mel cells compared to control. Succinate inhibits the prolyl hydroxylase (PH) that triggers HIF1α degradation. Malonate is generated from oxaloacetate by pyruvate carboxylase by the action of ROS. (c) Enzymatic determination of relative succinate levels in IGR37 versus IGR39 cells. n = 6 biological replicates of 2 technical replicates. ****p = < 0.0001. (d) Western blot showing expression of HIF1α in indicated cell lines treated over time with malonate or succinate as indicated. (e, f) Levels of indicated metabolites in IGR39 or IGR37 cells, or 501mel cells treated with control or MITF‐specific siRNA as indicated. For each metabolite data from each of three biological replicates is presented. Error bars indicate standard deviation, n = 3, *p = <0.05, **p = < 0.01, ***p = < 0.001, **** p = < 0.0001, n.s. = not significant, t test

Figure 7

MITF regulates SDHB to suppress pseudo‐hypoxia. (a) GSVA analysis showing relative expression of indicated gene sets in the top or bottom 20 CCLE melanoma cell lines ranked by SDHB expression. (b) Analysis of TCGA human melanoma samples ranked by the MITF expression (black line) for expression of indicated SDH subunits. Gray lines indicate expression of SDH subunits in each melanoma sample. Colored lines indicate moving average of SDH subunit expression across each 20 melanoma samples. (c) Heatmap showing CCLE melanoma cell lines ranked by MITF expression and relative expression of indicated SDH subunits. Also shown is the Spearman's correlation between MIITF or HIF1α expression and each SDH subunit. (d) Duplicate Western blot of IGR37 and IGR39 cells using indicated antibodies. (e) Western blot of indicated cell lines transfected with control or MITF‐specific siRNA. (f) UCSC browser screenshot of a duplicate ChIP‐seq experiment showing MITF binding within the SDHB gene. (g) Diagram showing the interactions between the transcription factors HIF1α, DEC1 and MITF and the regulation of HIF1α by succinate. See text for details