RNA maturation, particularly splicing, depends on coordinated actions of RNA-binding proteins through post-transcriptional processing and constitutes a central mechanism of gene regulation. Aberrant splicing is associated with various diseases, including cancer. Here, we show that the CoREST complex, in coordination with c-MYC, transcriptionally regulates a subset of RNA processing genes, including those encoding essential small nuclear ribonucleoproteins (snRNPs) required for proper spliceosome function. Genetic depletion or the pharmacological inhibition of the CoREST complex in melanoma cells disrupted spliceosome activity, leading to widespread changes in alternative mRNA isoform expression and reduced cell viability. These splicing alterations were associated with changes in the 2′-O-methylation (Nm) of U1 snRNA, a modification critical for spliceosomal function. The ectopic expression of the nucleolar protein NOLC1, a downstream target of the CoREST complex and known for its role in ribosomal RNA processing, partially rescued viability, splicing patterns, and U1 snRNA methylation in CoREST-deficient melanoma cells. Conversely, NOLC1 depletion sensitized melanoma cells to the MEK inhibitor trametinib, a clinical drug approved for treating advanced melanoma. Together, these findings uncover a novel CoREST-NOLC1 axis which is a transcriptional regulatory mechanism playing a significant role in RNA splicing, highlighting that NOLC1 is a downstream effector of the CoREST complex and a potential therapeutic target for melanoma treatment.

The CoREST complex is a multi-subunit epigenetic regulator implicated in histone modification and transcriptional repression, but its role in tumorigenesis is not well-defined. Here, we show that the CoREST complex directly interacts with and stabilizes the MYC oncoprotein in cancer cells through site-specific deacetylation of lysine residues, primarily mediated by HDAC1/2. These modifications protect MYC from proteasomal degradation independently of transcriptional regulation, maintaining high MYC protein levels in cancer cells. Transcriptomic analysis reveals that the CoREST-mediated MYC stabilization activates transcription of genes critical for DNA replication and mitotic chromosome segregation, and enhances melanoma cell viability. These findings suggest that the CoREST complex maintains cancer cell genome stability and promotes survival by sustaining MYC oncogenic activity, highlighting it as a potential therapeutic target in MYC-driven malignancies.

S1. Kaplan-Meier survival analysis of patients expressing high and low levels of BMP family genes. S2. Kaplan-Meier PFS analyses of cutaneous melanoma patients with BRAF-mutant and wild-type melanomas. S3. Kaplan-Meier OS analyses of cutaneous melanoma patients with BRAF-mutant and wild-type melanomas. S4. ChIP-PCR analysis demonstrating the binding of SIN3A-HDAC1/2 repressor complex in the proximal promoter regions of BMP1, BMP2 and BMP6 genes in 1205Lu melanoma cells. S5. Physiological changes of melanoma cells with stable expression of BMP6. S6. Establishment of mouse lung-specific metastatic melanoma cells for metastasis model generation. S7. Transcriptionally decreased FAM83G by BMP6 suppresses invasion capacity of melanoma cells via F-Actin deregulation. S8. Evaluation of functional roles of FAM83G in a metastasis model.

Supplementary Table S2 showing DNA sequences of primers and probes used in the study

Supplementary Figures 1-3 from Integration of Genotypic and Phenotypic Screening Reveals Molecular Mediators of Melanoma–Stromal Interaction

Supplemental Methods and Figure Legends

Supplementary Table S1 showing pathological and demographic information of patient samples

Supplementary Video 1 from Integration of Genotypic and Phenotypic Screening Reveals Molecular Mediators of Melanoma–Stromal Interaction

Supplementary Methods and Materials from Integration of Genotypic and Phenotypic Screening Reveals Molecular Mediators of Melanoma–Stromal Interaction

Abstract Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300–MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300–MITF–FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. Significance: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.

Mammalian genomes are highly populated with silent virus-like repeat elements evolutionally originated from integrated retroviruses. Lsh, a member of the SNF2 chromatin-remodelling family, is involved in the control of DNA methylation during embryonic development. Here, we show that in the epidermis, the Lsh protein is expressed in the basal epidermal keratinocytes (KCs). Constitutive and epidermal-specific Lsh ablation in mice leads to severe skin inflammation associated with epidermal hyperplasia and marked alterations in the epidermal structure. Primary KCs isolated from newborn Lsh KO mice prior to the development of inflammatory phenotype showed substantial alterations in the genome-wide DNA methylation patterns compared to controls. Bisulfite sequencing revealed that in Lsh-deficient KCs, the majority of hypomethylated DNA sites are found at the repeat sequences containing Long Terminal Repeats (LTR), Long Interspersed Elements (LINEs) and minor satellites. The global transcriptome profiling and GSEA analysis of Lsh-null KCs revealed a dramatic upregulation of the genes involved in anti-viral defence response, type I interferon- and interferon g-mediated signalling pathways, as well as increased expression of Keratins 16/17 and downregulation of Keratin 10, Loricrin, Involucrin, Filaggrin compared to controls. These data reveal Lsh as a critical determinant controlling DNA methylation and silencing of the repetitive elements in epidermal KCs and suggest a new model for analyses of the role of the endogenous retroviral-like elements as pathogenic agents in autoimmune skin diseases.

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin’s favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Earlier identification of aggressive melanoma remains a goal in the field of melanoma research. With new targeted and immune therapies that have revolutionized the care of patients with melanoma, the ability to predict progression and monitor or predict response to therapy has become the new focus of research into biomarkers in melanoma. In this review, promising biomarkers are highlighted. These biomarkers have been used to diagnose melanoma as well as predict progression to advanced disease and response to therapy. The biomarkers take various forms, including protein expression at the level of tissue, genetic mutations of cancer cells, and detection of circulating DNA. First, a brief description is provided about the conventional tissue markers used to stage melanoma, including tumor depth. Next, protein biomarkers, which provide both diagnostic and prognostic information, are described. This is followed by a discussion of important genetic mutations, microRNA, and epigenetic modifications that can provide therapeutic and prognostic material. Finally, emerging serologic biomarkers are reviewed, including circulating melanoma cells and exosomes. Overall the goal is to identify biomarkers that aid in the earlier identification and improved treatment of aggressive melanoma.

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called ‘dynamic stemness’. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma–endothelium co-culture model, where the MSLC niche is recapitulated in vitro , we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

Abstract Epigenetic agents have drawn great attention as anti-cancer therapies, with several HDAC inhibitors approved for a subset of hematologic malignancies. One of the biggest challenges in targeting epigenetic mechanisms of tumorigenesis is the wide spectrum of effects which restrict the therapeutic window for these compounds. We have developed a series of potent small molecule inhibitors with specificity towards the CoREST epigenetic corepressor complex through a dual-action mechanism targeting LSD1 and HDAC1. These compounds show a unique profile of pharmacologic action with an improved therapeutic window in a variety of cell types. Screening of tumor cell lines for growth inhibitory effects revealed variable efficacy in a broad spectrum of cancers with the most consistent and potent effects seen in human melanomas. The growth of a number of melanoma cell lines was found to be potently inhibited by one of these compounds, Corin 2; however, primary human melanocytes were relatively resistant to this agent. Transcriptomic analysis revealed that Corin2 was a more potent inducer of tumor suppressor genes compared to the parent HDAC and LSD1 compounds. Genetic knockdown of CoREST or LSD1 in cancer cell lines abolished the differences in potency of Corin2 vs. the parent HDAC inhibitor, Entinostat, suggesting that Corin2's favorable pharmacologic effects rely on an intact CoREST complex. Corin2 was also effective in slowing tumor growth in a melanoma mouse xenograft model. These dual action inhibitors demonstrate a novel, potent, and specific therapeutic approach to targeting epigenetic pathways in human melanomas which may lead to improved therapeutic benefits in patients with advanced disease. Note: This abstract was not presented at the meeting. Citation Format: Muzhou WU, Jay Kalin, Byungwoo Ryu, Philip Cole, Rhoda Alani. A novel dual action inhibitor of histone deacetylase and demethylase in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5068. doi:10.1158/1538-7445.AM2017-5068

There is a significant need for the development of diagnostic tools that can precisely distinguish Spitz nevi and spitzoid melanomas. Here, we report the development of a PCR-based quantitative diagnostic assay for spitzoid melanocytic lesions utilizing the expression ratio of neuropilin-2 and melan-A genes in primary tumor specimens. We find that the expression ratio of neuropilin-2/melan-A is significantly increased in spitzoid melanomas compared with Spitz nevi. The diagnostic potential of this quantitative assay was validated in two independent sets of patient samples as demonstrated in a receiver operating characteristic curve analysis showing an area under the curve value of 91.8%. Furthermore, the assay was found to quantitatively distinguish the clinical nature of atypical spitzoid melanocytic lesions that were diagnostically undetermined using histopathologic criteria alone. Our data indicate that this quantitative assay may be used as a tool in determining the diagnostic classification of histologically challenging spitzoid tumors.

In the epidermis, progenitor cells residing in the basal layer proliferate and differentiate, forming multi-layered epithelium. Execution of cell type–specific differentiation program in multi-potent progenitor cells requires a high degree of coordination between the distinct levels of chromatin organization including covalent DNA/histone modifications and higher-order chromatin remodeling. Lsh, a member of the SNF2 chromatin-remodelling family is involved in the control of DNA methylation patterns during embryonic development. Here, we show that in the skin, the Lsh protein is expressed in the basal epidermal layer and in the hair matrix keratinocytes. Skin obtained from newborn Lsh-null mice that die shortly after birth was grafted onto nude immunodeficient mice and showed significant hyperplasia of the interfollicular epidermis with major alterations in the epidermal structure occurring 3 weeks after grafting. In contrast to WT grafts, the Lsh-null grafts showed dramatic hyperproliferation and thickening of the basal epidermal layer. Global transcriptome profiling revealed a marked decrease in the expression of the keratinocyte-specific genes, including Keratin 10, Loricrin, Involucrin, Filaggrin in the epidermis of Lsh-null grafts compared to controls. However, upregulation of Keratin 16 and 17 was also seen in the epidermis of Lsh-null skin versus controls. Lsh ChIP-seq data revealed its binding to numerous target genes including those that control epidermal proliferation and differentiation. Furthermore, Lsh-null skin grafts showed accelerated regeneration after wounding compared to WT controls. These data suggest Lsh as an essential chromatin regulator controlling epidermal proliferation, differentiation and response to injury in postnatal skin.

How metastatic cancer lesions survive and grow in secondary locations is not fully understood. There is a growing appreciation for the importance of tumor components, i.e. microenvironmental cells, in this process. Here, we used a simple microfabricated dual cell culture platform with a 500 μm gap to assess interactions between two different metastatic melanoma cell lines (1205Lu isolated from a lung lesion established through a mouse xenograft; and WM852 derived from a stage III metastatic lesion of skin) and microenvironmental cells derived from either skin (fibroblasts), lung (epithelial cells) or liver (hepatocytes). We observed differential bi-directional migration between microenvironmental cells and melanoma, depending on the melanoma cell line. Lung epithelial cells and skin fibroblasts, but not hepatocytes, stimulated higher 1205Lu migration than without microenvironmental cells; in the opposite direction, 1205Lu cells induced hepatocytes to migrate, but had no effect on skin fibroblasts and slightly inhibited lung epithelial cells. In contrast, none of the microenvironments had a significant effect on WM852; in this case, skin fibroblasts and hepatocytes—but not lung epithelial cells— exhibited directed migration toward WM852. These observations reveal significant effects a given microenvironmental cell line has on the two different melanoma lines, as well as how melanoma effects different microenvironmental cell lines. Our simple platform thus has potential to provide complex insights into different strategies used by cancerous cells to survive in and colonize metastatic sites.

Aberrant HGF-MET (hepatocyte growth factor-met proto-oncogene) signaling activation via interactions with surrounding stromal cells in tumor microenvironment has significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation, which is influenced by the tumor microenvironment, and its consequential effects on melanoma malignancy remain uncharacterized. In this study, we identified SPINT2 (serine peptidase inhibitor Kunitz type 2), a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which has a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared with those in early-stage primary melanomas, which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA-hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits the HGF-induced MET-AKT (v-Akt murine thymoma viral oncogene) signaling pathway and decreases malignant phenotype potential such as cell motility and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor-suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF, which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.

Abstract Cancer mortality varies widely across different cancer types, but over 90% of cancer deaths, regardless of tumor origin, are caused by metastasis. Recent work to elucidate the mechanisms of metastasis has focused on the early stages of metastasis, specifically migration within a primary tumor and intravasation into vessels of the circulatory system. What is still relatively unknown is how these circulating cancer cells extravasate from the vessels to form micrometastases in distant organs. Chemokine gradients have long been associated with cell migration in embryogenesis and have also been implicated in promoting migration of cancer cells in vitro. This study was designed to investigate how competing gradients of chemotactic cytokines produced by cells in the metastatic niche communicate with cancer cells trapped in the vasculature to promote extravasation and colonization of new tumors. A microfluidic channel having five inlets and outlets was fabricated from polydimethylsiloxane (PDMS) bound to glass, and metastatic breast cancer cells (MDA-MB-231-4175 cell line, ATCC) were seeded into the device. Following cell attachment, a syringe pump supplied cells with a constant gradient of either epidermal growth factor (EGF) or stromal cell-derived factor 1 (SDF-1)-two known breast cancer chemoattractants-in serum-free media. Cells in the device were imaged regularly for 8 hours, and the timelapse data was used to quantify velocity, directionality, and chemotactic index of the cells. Following single-chemokine gradient studies, EGF and SDF-1 gradients of equal strength but opposite direction were introduced to the chip simultaneously. Cells in the device were again imaged for 8 hours, and velocity, directionality, and chemotactic index of cells in the competing gradients were quantified. To study chemokine gradients involved specifically in hematogenous metastasis, an extravasation microenvironment was built inside a microfluidic channel similar to the device previously described. Evenly spaced PDMS posts extending the height of the channel separated the channel into 5 lanes. A collagen gel was polymerized in two lanes to simulate niche extracellular matrix and to provide structural support for endothelial cells grown to confluence in the shape of a vessel. The shape of the EGF diffusion gradient formed across the endothelial monolayer was characterized using fluorescein isothiocyanate (FITC)–dextran as a diffusion marker. These experiments test the ability of both EGF and SDF-1 to promote directional migration of MDA-MB-231-4175 both individually and when applied in opposition, allowing us to test the hypotheses that chemotaxis of metastatic breast cancer cells can be controlled by chemokine gradients and that cells can sense and respond to multiple gradients simultaneously. In regard to hematogenous metastasis, characterization of an EGF gradient across an endothelial monolayer in both the absence and presence of cancer cells will assess the function of endothelial cell tight junctions in establishing chemokine gradients in the metastasis microenvironment. Combined, these studies will evaluate the hypotheses that: (1) Metastatic cancer cells trapped in the vasculature must initiate communication by modifying tight junctions between endothelial cells and (2) Once chemokines are free to diffuse into the vessel, extravasation and colonization are influenced by communication via a variety of competing chemokines that the cancer cells can sense and respond to simultaneously. Which chemokines ultimately “win” the competition and succeed in attracting cancer cells to colonize a target organ is the driving question of this research and will be the focus of future work. Citation Format: Laura Blaha, Chentian Zhang, Byungwoo Ryu, Rhoda Alani, Mario Cabodi, Joyce Wong. A microfluidic platform to evaluate soluble signaling in the metastasis microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B11. doi:10.1158/1538-7445.CHTME14-B11