Tumor cells often utilize developmental processes in order to progress towards

Tumor cells often utilize developmental processes in order to progress towards advanced disease. Together, these studies mechanistically demonstrate a previously unrecognized interplay between ERK1/2, TWIST1, and MMP-1 which is likely significant in the progression of melanoma towards metastasis. (4). In mammals, TWIST1 expression during a precise time frame in embryogenesis allows for the migration and differentiation of 16837-52-8 manufacture several mesodermal and neural crest cell lineages (5, 6). Many of the phenotypes attributed to TWIST1 occur as a result of its binding to E-box consensus sites in gene promoters, ultimately leading to transcriptional activation or repression (4, 7). TWIST1 is overexpressed in many primary tumors including colon, breast, prostate, and gastric carcinomas (8C11). In agreement with its role in embryonic cell migration, TWIST1 overexpression has been linked to increased tumor cell migration, invasion, and metastasis (7, 11C13). These actions of TWIST1 have been correlated with changes in classical EMT targets such as E-cadherin and N-cadherin (7, 11, 12); however, the extent to which TWIST1 regulates non-EMT targets is not fully understood. Recently, TWIST1 was found to be highly up-regulated in the vast majority of melanoma tumors and cell lines, and was correlative to worse patient survival (8, 14). Melanoma is an aggressive skin cancer which arises from neural crest-derived melanocytes (15, 16837-52-8 manufacture 16). Invasion plays a critical role in melanoma progression. If cells are mainly confined 16837-52-8 manufacture to expansion within the epidermis (radial growth phase, RGP), melanoma is easily cured through surgical intervention (15, 16). If undiagnosed and properties of invasion begin to emerge, cells escape the basement membrane and expand through the deeper dermal layers. This conversion to vertical growth phase (VGP) is the direct precursor to metastasis (15). The depth of melanoma 16837-52-8 manufacture invasion and tumor thickness are used as predictors of poor clinical prognosis (17, 18); however, the mechanisms underlying melanoma invasion from the epidermis into the dermis remain poorly characterized. Up-regulation of the RAS-RAF-MEK-ERK1/2 signaling pathway may be critically important in this process. Hyperactivation of this pathway is common in multiple cancer types but especially in melanoma, where mutations in N-RAS (15C20%) or B-RAF (40C60%) are prevalent (15, 16, 19). Additionally, mutant B-RAF, especially B-RAFV600E, is required for enhanced growth and invasion of melanoma cells (20). Many of the factors influencing increased melanoma invasion downstream of RAS-RAF-MEK-ERK1/2 are unknown. Since TWIST1 plays important roles in the developing and highly migratory neural crest, and since tumor cells often aberrantly regulate developmental pathways, we sought to determine the role of TWIST1 in melanoma invasive growth. In this study, we have found that TWIST1 promotes invasion in 3D dermal-mimetic assays and reveal an ERK1/2-TWIST1-MMP-1 pathway which likely will have a major impact on invasion and metastasis. Materials and Methods siRNA Transfection WM793 and WM115 cells were transfected for 4 hours with chemically synthesized siRNAs (Dharmacon, Lafayette, CO) at a final concentration of 25nM using Oligofectamine (Invitrogen). Transfections were harvested at 72 hours. siRNA sequences are listed in Supplementary Table S1. Quantitative RT-PCR RNA was extracted from cells using PerfectPure RNA Cultured Cell Kit (5Prime, Gaithersburg, MD) as per the manufacturers instructions. Conversion to cDNA was achieved through the iScript cDNA Synthesis Kit (Biorad). Quantitative RT-PCR was carried out using iQ SYBR Green Supermix (Biorad), 0.4M oligonucleotide primers, and 0.1g cDNA. CENPA Primer sets can be found in Supplementary Table S1. Relative fold change in mRNA levels were calculated after normalization to -Actin using the comparative Ct method (21). Statistical Analysis Statistical analysis was performed using a two-tailed Students t test calculated with Excel (Microsoft). A p value < 0.05 was considered statistically significant. Additional methods Detailed methods for cell culture, patient samples, lentiviral and adenoviral construction/transduction, invasion/migration assays, spheroid outgrowth asssays, live/dead staining, 16837-52-8 manufacture EdU incorporation assays, dual-luciferase assays, ChIP, biotinylated oligonucleotide pulldown assays, inhibitors, and western blot analysis are available in Supplementary Materials and Methods. Results TWIST1 is up-regulated in melanoma cell lines, particularly in VGP, downstream of oncogenic B-RAF and is positively regulated by active ERK1/2 signaling We explored the TWIST1 expression profile across an extensive panel of melanoma cell lines representing various tumor stages and genotypes. Our results show that TWIST1 protein is up-regulated in all melanoma cell lines tested compared to neonatal human epidermal melanocytes (NHEM) (Fig. 1A). Furthermore, TWIST1 protein is especially high in.