Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma

Abstract

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Keywords: MITF; TNFα; invasiveness; melanoma; phenotype-switching.

Figure 1.

Glutamine limitation activates then suppresses MITF. (A) Western blot using anti-MITF cell lines grown in DMEM or MEM supplemented with the indicated amino acids. ERK was used as a loading control. (B) Western blot of IGR37 cells grown in DMEM (t = 0) or MEM. (C) Western blot of melanoma cells grown in DMEM or MEM supplemented with serine and glycine with the indicated concentrations of glutamine for 48 h. (D) Quantitative RT–PCR (qRT–PCR) using MITF-M-specific primers using mRNA from IGR37 cells grown in DMEM or MEM. (E) Western blot of IGR37 cells grown in DMEM or MEM supplemented with serine and glycine.

Figure 2.

ATF4 couples MITF to the ISR. (A) Heat map from gene array data of well-characterized MITF targets from IGR37 cells grown in DMEM or MEM for the indicated times. (B) Western blot of IGR37 cells grown in DMEM or MEM supplemented with serine and glycine (−Gln). All samples are from the same experiment. (C) Heat map from gene array data of well-characterized ATF4 target genes. (D) Immunofluorescence using the indicated antibodies of SKmel28 cells expressing doxycycline-inducible ATF4 grown with or without 100 ng of doxycycline for 24 h. (E) Luciferase activity of an MITF promoter luciferase reporter cotransfected with an ATF4 expression vector. Results are expressed as average ± SD. n = 3. The inset shows a Western blot of ectopically expressed ATF4 with increasing amounts of transfected expression vector. (F) Flow cytometry profiles of iATF4 501mel cells before and after addition of doxycycline. (G) qRT–PCR of mRNA extracted from IGR37 cells grown with or without glutamine and exposed or not to anti-ATF4 siRNA (left panel) and a Western blot of the same cells (right panel). (H) Analysis of The Cancer Genome Atlas (TCGA) RNA sequencing (RNA-seq) data. The black line indicates TCGA samples ranked by average expression of a 103-gene glutamine starvation signature (GSS) score derived from the 6-h gene array time point (see Supplemental Table S4). Vertical gray lines indicate expression of the indicated gene in each melanoma sample. The colored line indicates the moving average of the indicated gene across 20 melanomas. (I) Model depicting early response to glutamine limitation.

Figure 3.

Glutamine limitation drives ATF4-mediated senescence bypass and invasiveness. (A) SA-β-gal activity in SKmel28 cells grown in DMEM and depleted for MITF or in MEM supplemented with serine (S) and glycine (G) or glutamine (Q) as indicated. Bars, 10 µm. (B) Flow cytometry of the indicated melanoma cell lines grown in the presence or absence of glutamine as indicated. (C) Western blot using the indicated antibodies of IGR37 cells depleted for glutamine and refed as indicated. (D) SA-β-gal activity in B16 cells expressing doxycycline-inducible ATF4 and mCherry. Cells were depleted for MITF using siRNA, and, 24 h later, ATF4 was induced using 100 ng of doxycycline. SA-β-gal is false-colored green to facilitate visualization of colocalization. Arrowheads indicate SA-β-gal-positive cells not expressing ATF4. Bars, 10 µm. (E) Analysis of TCGA human melanoma samples or melanoma single-cell RNA-seq data (Tirosh et al. 2016) for the presence of the Hoek (Hoek et al. 2006) or Verfallie (Verfaillie et al. 2015) invasiveness signature in samples ranked by the GSS score. Vertical gray lines indicate expression of the invasive signatures in each melanoma sample. Colored line indicates the moving average of the invasive signature across 20 melanoma samples. Venn diagrams indicate the number of genes in each signature (see also Supplemental Table S4). (F) Matrigel invasiveness assay in melanoma cells grown in DMEM or MEM supplemented with serine, glycine, or glutamine or the same cell lines expressing doxycycline-inducible ATF4. Results are expressed as mean ± SD of at least three biological replicates. (***) P = <0.001. (G) Cell numbers in an iMITF 501mel cell line grown in the presence or absence of doxycyline or minus glutamine as indicated. Results are expressed as mean ± SD of three biological replicates. (H, top panel) Matrigel invasiveness assays using the indicated cell lines grown with or without glutamine deprivation for 72 h as indicated. Results are expressed as mean ± SD of three biological replicates. (***) P = <0.001. (Bottom panel) Western blot at the indicated times of cell lines grown in the presence or absence of glutamine as indicated.

Figure 4.

Inhibition of eIF2B drives invasion. (A) Western blot of 501mel cells grown in DMEM and treated with 20 µM salubrinal. (B) Matrigel invasion assays for cells grown in DMEM; MEM; MEM supplemented with serine (S), glycine (G), or glutamine (Q); or DMEM plus 20 µM salubrinal as indicated. Statistical analysis used a Student's unpaired t-test assuming equal variances. Data are presented as mean ± SD of at least three biological replicates. (***) P = <0.001. (C) Matrigel invasion assays for cell lines grown in DMEM, MEM, or MEM supplemented with serine (S) and glycine (G) (−Gln) in the presence or absence of 10 µM ISRIB. Results are expressed as mean ± SD of at least three biological replicates. (***) P = <0.001. (D) Western blot using the indicated antibodies of 501mel cells stably expressing Flag-tagged wild-type or S51A mutant eIF2α and starved of glutamine for the indicated times. (E) Matrigel invasion assays for parental 501mel cells or derivatives stably expressing Flag-tagged wild-type or S51A mutant eIF2α grown in the presence or absence of glutamine. Results are expressed as mean ± SD of at least three biological replicates. (**) P = <0.01; (n.s.) not significant. (F) Zebrafish embryo expressing Foxd3-citrine in the neural crest 5 d after fertilization with or without 10 µM ISRIB. (G) Yeast invasion assay showing wild-type and mutant colonies grown on YPD agar before and after washing.

Figure 5.

TNFα activates ATF4 to trigger a GSS. (A) Analysis of TCGA or single-cell (Tirosh et al. 2016) human melanoma samples for the presence of a 219-gene TNFα response signature (see also Supplemental Table S4; Landsberg et al. 2012) in samples ranked by the GSS score (black line). Vertical gray lines indicate expression of the TNFα signature in each melanoma sample. The colored line indicates the moving average of the TNFα signature across 20 melanoma samples. The Venn diagram indicates the number of genes in each signature. (B) Western blot of melanoma cells treated with 20 ng/mL TNFα. (C) Matrigel invasiveness assay in melanoma cells grown in DMEM with 20 ng/mL TNFα. Statistical analysis was carried out using Student's unpaired t-test assuming equal variances. Data are presented as mean ± SD of at least three biological replicates. (***) P = <0.001. (D) Strategy for analysis of pmel adoptive T-cell therapy-mediated dedifferentiation and relapse of mouse melanoma (left) and Western blot (right) of murine melanoma Hcmel3 cells grown in the presence or absence of TNFα or glutamine. (E) Gene set enrichment analysis (GSEA) of gene array data derived from relapsed mouse melanomas. (NES) Normalized enrichment score; (FDR) false discovery rate. (F) Box plots showing the relative expression of the indicated genes from gene arrays of untreated or relapsed mouse melanomas after T-cell immunotherapy. The respective gene probe identities are indicated below the gene symbols. Box plot horizontal lines and whiskers indicate quartiles. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, unpaired two-sided t-test.

Figure 6.

Translation reprogramming is sufficient to trigger melanoma invasiveness and drives therapeutic resistance signatures. (A) Analysis of TCGA human melanoma samples ranked by the GSS score (black line) for AXL expression or single-cell melanoma for an AXL signature. Gray lines indicate the expression of AXL in each melanoma sample. The colored line indicates the moving average of AXL or AXL signature expression across 20 melanoma samples. (B) Western blot of 501mel cells grown in DMEM or MEM supplemented with serine (S) and glycine (G) minus Gln (Q) for the indicated times. (C) qRT–PCR of mRNA from IGR37 cells grown in DMEM or MEM supplemented with serine (S) and glycine (G) minus Gln (Q) for 48 h. (D) Western blot of 501mel cells grown in DMEM inducibly expressing ATF4 for 24 h in response to 100 ng of doxycycline. (E,F) Heat maps showing gene set variation analysis (GSVA) scores of the IPRES (innate anti-PD-1 resistance) gene signatures enriched in the top and bottom 75 TCGA melanoma samples ranked by the GSS (E) or salubrinal (F) signatures (see also Supplemental Table S4).

Figure 7.

Translation reprogramming enriches for tumor initiation capacity. (A) Lung colonization by B16 melanoma cells grown in DMEM or for 17 d in MEM prior to tail vein injection. Mice were sacrificed 20 d after injection. (B) Lung colonization by B16 melanoma cells grown in DMEM or for 17 d in MEM supplemented with serine and glycine (−Gln) or in DMEM plus 20 µM salubrinal prior to tail vein injection. Mice were sacrificed at 15 d after injection. Quantification was made by counting visible tumors using a dissecting microscope. Error bars indicate mean ± SD. n = 5. (**) P = <0.01.