Beyond the genome: challenges and potential for epigenetics-driven therapeutic approaches in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease of the cardiopulmonary system caused by the narrowing of the pulmonary arteries, leading to increased vascular resistance and pressure. This leads to right ventricle remodeling, dysfunction, and eventually, death. While conventional therapies have largely focused on targeting vasodilation, other pathological features of PAH including aberrant inflammation, mitochondrial dynamics, cell proliferation, and migration have not been well explored. Thus, despite some recent improvements in PAH treatment, the life expectancy and quality of life for patients with PAH remains poor. Showing many similarities to cancers, PAH is characterized by increased pulmonary arterial smooth muscle cell proliferation, decreased apoptotic signaling pathways, and changes in metabolism. The recent successes of therapies targeting epigenetic modifiers for the treatment of cancer has prompted epigenetic research in PAH, revealing many new potential therapeutic targets. In this minireview we discuss the emergence of epigenetic dysregulation in PAH and highlight epigenetic-targeting compounds that may be effective for the treatment of PAH.

Loss of prdm1a accelerates melanoma onset and progression.

Melanoma is an aggressive, deadly skin cancer derived from melanocytes, a neural crest cell derivative. Melanoma cells mirror the developmental program of neural crest cells in that they exhibit the same gene expression patterns and utilize similar cellular mechanisms, including increased cell proliferation, epithelial-mesenchymal transition, and migration. Here we studied the role of neural crest regulator PRDM1 in melanoma onset and progression. In development, Prdm1a functions to promote neural crest progenitor fate, and in melanoma, we found that PRDM1 has reduced copy number and is recurrently deleted in both zebrafish and humans. When examining expression of neural crest and melanocyte development genes, we show that sox10 progenitor expression is high in prdm1a-/- mutants, while more differentiated melanocyte markers are reduced, suggesting that normally Prdm1a is required for differentiation. Data mining of human melanoma datasets indicates that high PRDM1 expression in human melanoma is correlated with better patient survival and decreased PRDM1 expression is common in metastatic tumors. When one copy of prdm1a is lost in the zebrafish melanoma model Tg(mitfa:BRAFV600E );p53-/- ;prdm1a+/- , melanoma onset occurs more quickly, and the tumors that form have a larger area with increased expression of sox10. These data demonstrate a novel role for PRDM1 as a tumor suppressor in melanoma.

Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration.

Melanocytes derive from neural crest cells, which are a highly migratory population of cells that play an important role in pigmentation of the skin and epidermal appendages. In most vertebrates, melanocyte precursor cells migrate solely along the dorsolateral pathway to populate the skin. However, zebrafish melanocyte precursors also migrate along the ventromedial pathway, in route to the yolk, where they interact with other neural crest derivative populations. Here, we demonstrate the requirement for zebrafish paralogs pcdh10a and pcdh10b in zebrafish melanocyte precursor migration. pcdh10a and pcdh10b are expressed in a subset of melanocyte precursor and somatic cells respectively, and knockdown and TALEN mediated gene disruption of pcdh10a results in aberrant migration of melanocyte precursors resulting in fully melanized melanocytes that differentiate precociously in the ventromedial pathway. Live cell imaging analysis demonstrates that loss of pchd10a results in a reduction of directed cell migration of melanocyte precursors, caused by both increased adhesion and a loss of cell-cell contact with other migratory neural crest cells. Also, we determined that the paralog pcdh10b is upregulated and can compensate for the genetic loss of pcdh10a. Disruption of pcdh10b alone by CRISPR mutagenesis results in somite defects, while the loss of both paralogs results in enhanced migratory melanocyte precursor phenotype and embryonic lethality. These results reveal a novel role for pcdh10a and pcdh10b in zebrafish melanocyte precursor migration and suggest that pcdh10 paralogs potentially interact for proper transient migration along the ventromedial pathway.

SIX1 and EWS/FLI1 co-regulate an anti-metastatic gene network in Ewing Sarcoma.

Ewing sarcoma (ES), which is characterized by the presence of oncogenic fusion proteins such as EWS/FLI1, is an aggressive pediatric malignancy with a high rate of early dissemination and poor outcome after distant spread. Here we demonstrate that the SIX1 homeoprotein, which enhances metastasis in most tumor types, suppresses ES metastasis by co-regulating EWS/FLI1 target genes. Like EWS/FLI1, SIX1 promotes cell growth/transformation, yet dramatically inhibits migration and invasion, as well as metastasis in vivo. We show that EWS/FLI1 promotes SIX1 protein expression, and that the two proteins share genome-wide binding profiles and transcriptional regulatory targets, including many metastasis-associated genes such as integrins, which they co-regulate. We further show that SIX1 downregulation of integrins is critical to its ability to inhibit invasion, a key characteristic of metastatic cells. These data demonstrate an unexpected anti-metastatic function for SIX1, through coordinate gene regulation with the key oncoprotein in ES, EWS/FLI1.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk.

It is well established that a subset of cells within primary breast cancers can undergo an epithelial-to-mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration, and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFß signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFß signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis in vivo without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.