The histone variant macroH2A suppresses melanoma progression through regulation of CDK8.

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin1, 7, 8. The H2A family is the most diverse and includes vertebrate-specific mH2A1 (splice variants mH2A1.1 and 1.2) and mH2A21, 8-11, which are generally associated with transcriptionally repressed chromatin12, 13. However, mH2A is widely distributed throughout chromatin2-4 and exists in post-translationally modified forms8, 14, suggesting additional unidentified functions for this variant.

Given increasing evidence for variant-mediated transcriptional control1, 8 and recent reports describing variants as prognostic markers in cancer15, 16, we hypothesized that global alteration of variants could contribute to malignant melanoma - the most lethal form of skin cancer with rising incidence17, 18. Its radial growth phase (RGP) is characterized by lateral melanocyte growth and vertical growth phase (VGP) by spread of melanoma cells into the dermis and subcutis, upon which, metastasis can occur19.

Using well characterised, paired series of murine and human melanoma cells lines, we probed the H2A variant profile. The murine B16 series represents cells of increasing metastatic potential20 and the human series of a primary melanoma (WM115) and two subsequent skin metastases derived from this same patient (WM266-4 and WM165-1)21. In highly malignant cells of the murine and human series, a global decrease of mH2A1 and mH2A2 protein and mRNA was observed (Fig. 1a, Supplementary Fig. S1, S2). Analysis of histones from both series using multiplexed quantitative mass spectrometry (Q-MS)22 confirmed these findings (Supplementary Fig. S1). Furthermore, mH2A1 and mH2A2 loss was observed in a panel of primary and metastatic melanoma cells (Supplementary Fig. S1). Interestingly, a 1.5 to 3-fold increase in H2A.Z levels (often associated with promoters of active genes)1, 8 was also observed (Fig. 1a, Supplementary Fig. S3), implicating possible H2A variant exchange during melanoma progression. Consistent with a global loss of mH2A and increased H2A.Z levels, we observed highly decondensed chromatin in B16-F10 cells by micrococcal nuclease digestion (Supplementary Fig. S2).

Figure 1

mH2A loss correlates with increasing melanoma malignancy

a, Melanoma cells probed for H2A variants; core histones used for loading. b, IHC of human tissue with mH2A2 (left), and histone H3 (right). 20x and 40x shown. mH2A2 visualized using DAB (brown) and hemotoxalin (blue). Arrows depict mH2A2 staining in non-melanocytic cells. c, qRT-PCR of mH2A1 and mH2A2 in benign nevi and melanocytes (brown circles) and metastatic melanoma (black squares); P < 0.0001 d, DNA methylation of mH2A2 promoter in nevi (n=6, 10-12 clones per nevus) and metastatic melanoma tissues (n=7, 10-14 clones per tissue); 16 CpG sites shown. Open circles (unmethylated), black circles (methylated); P-values determined by Mann-Whitney U-test.

Next, we performed immunohistochemistry (IHC) on ~115 human tissues ranging from benign nevi to metastatic melanoma (Tissue set 1, Supplementary Fig. S4). mH2A2 antibody was used for IHC, as it produced clear nuclear staining, and tissues were independently scored (0-3) by two blinded dermatopathologists with excellent inter-observer consistency (Kappa = 0.80). IHC demonstrated that while mH2A2 is abundant in melanocytes of benign nevi and RGP lesions, its expression is dramatically lost in >80% of VGP and metastatic melanomas (P < 0.001) (Fig. 1b, Supplementary Fig. S4). This suggests mH2A loss occurs during the critical RGP-VGP transition. IHC was also performed on 25 melanomas with known BRAF status (D.P. unpublished data)23. An activating mutation of BRAF, V600E, is present in approximately 65% of melanomas24. While this data set did not reveal a significant correlation between mH2A2 loss and V600E mutation, it produced similar mH2A2 results as the first cohort, as did a tissue microarray (Supplementary Fig. S4). Using fresh tissues, we observed significantly reduced levels of mH2A1 and mH2A2 mRNA in metastatic melanoma specimens as compared to nevi and primary melanocytes (Fig. 1c).

Due to the transcriptional downregulation of mH2A in human melanoma, combined with its re-expression in metastatic melanoma cells upon 5-Aza-2′-Deoxycytidine treatment (Supplementary Fig. S5), we hypothesized that DNA methylation may enable silencing of mH2A. Indeed, through extensive bisulfite sequencing analysis, we identified a region of the mH2A2 promoter that is significantly methylated in metastatic melanoma tissues and cell lines, but not in primary melanocytes, WM115 cells, or benign nevi (Fig. 1d; Supplementary Fig. S5).

Collectively, these findings prompted us to examine the functional consequences of mH2A loss. We established multiple stable shRNA lines in murine B16-F0 and F1 and human WM115 melanoma cells, targeting mH2A1, mH2A2, and control GFP (Supplementary Fig. S6, 7). Two shRNA-transduced cell lines from mouse and human were utilized for in-depth analysis: B16-F1 mH2A1_91 and mH2A2_25 and WM115 mH2A1_90 and mH2A2_05 (Supplementary Fig. S6, 7 for additional shRNA lines and isoform-specific knockdown).

Proliferation was examined in shRNA-expressing cells by colony formation and MTS cell viability assays. The loss of mH2A increases proliferation of murine and human melanoma cells (Fig. 2a, Supplementary Fig. S6, 7), as well as anchorage-independent growth of WM115 cells (Fig. 2b, Supplementary Fig. S6). In order to examine growth potential in vivo, B16-F1 shRNA cell lines were injected subcutaneously into mice; mH2A-deficient cells exhibited significantly enhanced tumour growth compared to controls (Fig. 2c). mH2A knockdown was confirmed by immunoblotting lysates from tumours (Supplementary Fig. S8).

Figure 2

mH2A depletion and ectopic expression alter malignant properties of melanoma cells in vitro and in vivo

a, Colony assay of B16-F1 shRNA-expressing cells. Quantified (below); *P < 0.0000005. Mean ± s.e.m; (n=4). b, Soft agar assay of WM115 shRNA-expressing cells. Quantified (below); *P < 0.0005. Mean ± s.d; (n=4). c, Tumour volume (mm3) post-subcutaneous injections of B16-F1 shRNA cells; *P < 0.05 at day 14. Mean ± s.e.m; (n=10 mice per group). d, Trans-well migration assay in B16-F1 and WM115. Quantification below, *P < 0.0001 Mean ± s.d; (n=5). e, Representative lungs of B16-F1 shRNA cells injected intravenously, 14 days post-injection. Mean ± s.e.m; nodules/lung/mouse shown (n=6-8 mice per group). f, Representative lungs of B16-F10 mCherry cells injected intravenously, 10 days post-injection. Quantified (right); *P < 0.005. Mean ± s.e.m; (n=6-9 mice per group).

Because cell motility contributes to metastasis and melanocytes originate from migratory neural crest cells, shRNA lines were analyzed for migratory behaviour. Loss of mH2A in murine and human cells enhanced migration through an 8μ trans-well and the ability to close an artificial wound, as compared to control cells (Fig. 2d, Supplementary Fig. S6, 7). Next, shRNA cell lines were injected into the lateral tail veins of mice to assay metastatic potential. Fourteen days post-injection, mice were sacrificed and lungs dissected for macro- and microscopic histology (Fig. 2e). Lungs of mice injected with mH2A1_91 and mH2A2_25 cells showed a five- and thirty-fold increase, respectively, in the number of macroscopic metastases as lungs from control mice (Fig. 2e). Hematoxylin and eosin (H&E) and Ki-67 staining revealed metastatic disease with proliferation, respectively, in mH2A shRNA-expressing tumours; mH2A knockdown in lungs was confirmed by IHC (Supplementary Fig. S8).

Next, mH2A expression was stably restored in malignant B16-F10 and human WM266-4 and A375 cells. The core histone H2A, mH2A1 (1.2) and mH2A2 were fused to mCherry and functional stable lines generated (Supplementary Fig. S9, 11, 13). Ectopic expression of mH2A1.2 and mH2A2, but not H2A or mCherry alone, resulted in reduced proliferation (without evidence of apoptosis) and migration (Supplementary Fig. S10, 12, 14). Human A375 cells expressing the mCherry series were injected subcutaneously into the flanks of immunocompromised mice; expression of mH2A1.2 and mH2A2 suppressed growth (Supplementary Fig. S14). Furthermore, B16-F10 cells expressing mCherry fusions were injected into tail veins of mice; mH2A1.2 and mH2A2 significantly suppressed metastasis to the lungs (Fig. 2f, Supplementary Fig. S10).

Given the striking phenotypes of mH2A manipulation in melanoma cells, we hypothesized that loss of mH2A may alter the transcriptional state of proliferation- and metastasis-related genes. We performed gene expression profiling using Affymetrix microarrays with B16-F1 cells (mH2A1_91, mH2A2_25 and sh_GFP). As expected for mH2A’s role in fine-tuning of gene expression2, many genes showed < 2-fold change, (Supplementary Fig. S15, 16 and 17 for Venn diagrams, heatmaps, and Gene Ontology). Fifteen genes showed ≥ 2-fold change, common to both shRNA lines in two independent experiments, including Integrin alpha 4 (Itga4), transcriptional regulators CDK8 (Mediator complex component) and Cited1 (CBP/p300 transactivator) (Fig. 3a). In concordance with our data, Itga4 expression is inversely correlated with invasive potential of B16 cells25 and expression profiling of human melanoma cells identified Cited1 loss in metastatic cells26. CDK8, however, is a new player in melanoma malignancy.

Figure 3

Microarray and ChIP analysis identify CDK8 as a direct mH2A-regulated gene in melanoma

a, Heat map representing gene expression changes (≥2-fold) in B16-F1 mH2A-deficient cells. qRT-PCR of CDK8 (bottom); Mean ± s.d; (n=3). b, Immunoblots of CDK8 in murine shRNA lines; actin for loading. c, Immunoblot and qRT-PCR of CDK8 in human shRNA lines; Mean ± s.d; (n=3). d, CDK8 and DsRed immunoblots of mH2A2_24 line expressing H2A- and mH2A2-cherry; asterisk depicts fusion proteins. e, CDK8 qRT-PCR analysis in A375 subcutaneous tumours; Mean ± s.d; (n=3). f, mH2A1 ChIP analysis of the −1kb position from TSS for CDK8, PACS2, ATP5G1, and GAPDH; intergenic control. IgG used as control antibody; Mean ± s.d; (n=3).

In concordance, CDK8 mRNA and protein levels were elevated in both murine and human cells depleted of mH2A (Fig. 3a-c), as well as subcutaneous tumours and lung metastases derived from mH2A-deficient lines (Supplementary Fig. S18). Next we stably expressed RNAi-resistant mH2A2 and H2A in the B16-F1 mH2A2_24 shRNA line, which targets the 3′UTR of mH2A2. While CDK8 levels remained high in mH2A2_24 cells expressing H2A-mCherry, addition of mH2A-mCherry rescued CDK8 expression to B16-F1 levels (Fig. 3d). Moreover, CDK8 was downregulated in subcutaneous tumours derived from A375 cells expressing mH2A1- and mH2A2-mCherry (Fig. 3e, Supplementary Fig. S14).

Intrigued by transcriptional up-regulation of CDK8, a colorectal cancer oncogene5,6, we enquired if CDK8 is a direct target of mH2A. Chromatin Immunoprecipitation (ChIP) analysis demonstrated that the CDK8 promoter is enriched in mH2A1-containing nucleosomes in B16-F1, but absent in B16-F10 cells and a control shRNA line (Fig. 3f, Supplementary Fig. S18). ChIP analysis of additional mH2A target genes (but not an intergenic locus or GAPDH) also revealed enrichment, and demonstrated that CDK8 is a highly enriched mH2A target gene (Fig. 3f).

By examining a panel of human cell lines, we observed high CDK8 protein levels in metastatic melanoma cells, comparable to that of colon cancer cells (Fig. 4a). We utilized shRNAs to deplete CDK8 from B16-F10 and human A375 and WM165-1 cells, which contain high levels of CDK8 (Fig. 4b, Supplementary Fig. S19, 20). The loss of CDK8 significantly reduced proliferation (Fig. 4b) mediated by G2/M arrest in human cells (Supplementary Fig. S20). Conversely, ectopic expression of CDK8 (and a kinase-defective mutant, D173A)5 in B16-F1 cells resulted in significantly increased proliferation (Supplementary Fig. S21). While proliferation in murine melanoma cells appears independent of CDK8’s kinase activity, it may be consistent with recent studies demonstrating a kinase-independent role of CDK827.

Figure 4

CDK8 is a major effector of mH2A loss

a, Melanocytes, metastatic melanoma, and colon cancer cells probed for CDK8; actin for loading. b, Immunoblot of A375 cells expressing CDK8 shRNA (left), MTS assay (right), Mean ± s.d; (n=5). c, Tumour volume (mm3) post-subcutaneous injections; *P < 0.05 at day 12. Mean ± s.e.m; (n=9 mice per group). d, MTS assay of WM115 mH2A1_90 line co-expressing CDK8 or Med12 shRNAs, Mean ± s.d; (n=5). e, qRT-PCR of mH2A2 and CDK8 in 36 melanoma tissues (from 30 patients; Supplementary Table S4); Pearson’s r= −0.406 with p=0.014. Mean ± s.d; (n=3).

To dissect the relationship between mH2A and CDK8, we first depleted CDK8 in mH2A shRNA-expressing cells (B16-F1 and WM115 lines). Knockdown of CDK8 was able to suppress the enhanced proliferation induced by mH2A loss in vitro and in vivo (Fig. 4c, d; Supplementary Fig. S21, S22). Knockdown of Med12, a subunit of the CDK8 submodule of Mediator 27, showed a similar effect (Fig. 4d; Supplementary Fig. S22), suggesting that CDK8 functions within the Mediator subcomplex in melanoma.

Next, we performed qRT-PCR of mH2A and CDK8 in 36 melanoma specimens. This analysis demonstrated a statistically significant inverse correlation of mH2A2 and CDK8 at the mRNA level (Pearson’s r= −0.406; p = 0.014; Fig. 4e). We performed IHC for CDK8 in human tissues previously scored for mH2A2 (Kappa = 0.58), and observed strong CDK8 staining (scored 2-3) in a large fraction of mH2A2 negative (scored 0) melanomas (29/38= 76%; Supplementary Fig. S23). A similar trend was observed in a panel of human melanoma cell lines (Supplementary Fig. S23). Finally, by probing fresh benign nevus tissues, we observed high mH2A and low CDK8 protein levels (Supplementary Fig. S23). Collectively, these results strongly suggest that CDK8 is a major effector of mH2A-mediated melanoma progression.

Here, we demonstrate that mH2A is globally lost during melanoma progression. Similar findings have recently been described in lung cancer; mH2A1.1 is enriched in pre-cancerous senescent cells, but lost upon bypass of senescence16. However, the mechanism by which this occurs and its biological consequences remain unclear. Our study suggests mH2A loss in melanoma, mediated in part by DNA methylation, occurs after a potential senescence bypass (i.e. in a nevus), but rather during the critical RGP to VGP transition. Nevertheless, mH2A isoforms may serve as important biomarkers for melanoma, and/or other cancers.

The data presented here point towards a novel mechanism whereby CDK8 is regulated by the unique histone variant mH2A. We look forward to future studies focused on CDK8 function and its inhibition in melanoma. Our findings support emerging links between chromatin structure and cancer, and for the first time, demonstrate a direct role of mH2A in this process.

Methods Summary

Cell culture, plasmids, infections and RNAi

Detailed information is described in Methods.

Chromatin fractionation, acid extraction of histones and immunoblotting

Chromatin fractionation and acid extraction of histones performed as described14. Antibodies used for immunoblotting can be found in Methods.

Quantitative mass spectrometry

Q-MS performed as previously described22.

Immunohistochemistry, pathology and statistical analysis

Specimens were obtained from MSSM’s Division of Dermatopathology (Project# HSD08-00565), NYU (IRB# 10362), and melanoma tissue microarray (Imgenex #IMH-369). Details on staining, pathology and statistical analyses described in Methods.

Clinical Specimens

Human specimens were collected at the time of surgery. Approval to collect melanoma specimens was granted by Mount Sinai Biorepository Cooperative and the NYU IMCG (Project #’s above). Approval to collect benign nevi was granted by MSSM’s Division of Dermatopathology (Project # 08-0964).

Bisulfite sequencing

Performed according to manufacturer’s instructions (Zymo Research). Details described in Methods.

Cell proliferation, migration and mouse injections

MTS performed according to manufacturer’s instructions (Promega). Colony formation and soft agar assays performed as described28. Trans-well migration assay described in Methods. In vivo metastasis assays performed as described28. For subcutaneous injections, 2.5×105 B16-F1 cells were injected into 6-week old C57BL/6J mice and 2×106 A375 cells injected into NOG mice (Jackson Laboratories); tumour volume measured over a 14 and 20-day period, respectively.

Microarray hybridization, data analysis and hierarchical clustering

Microarray was performed using two biological replicates according to Affymetrix GeneChip protocol. Initial data extraction performed at the Microarray Shared Research Facility at MSSM. Heatmaps generated using Cluster and Tree View programs.

Quantitative PCR and Chromatin Immunoprecipitation

qPCR performed in triplicate on Stratagene Opticon 2 using FastStart SYBR Green Mix (Roche). Expression levels normalized to TATA Binding Protein (TBP) or GAPDH. ChIP assays performed using Magna ChIP™ Kit (Millipore) as per manufacturer’s instructions.