RESUMO
Migration and Invasion Enhancer (MIEN1) is an oncogene which is involved in facilitating motility of cancer cells through actin dynamics and gene expression. Increased MIEN1 expression in many types of tumors leads to disease progression and metastatic propensity. It is unclear precisely how MIEN1 is involved in this process and more studies are required to tease out the mechanisms. Here we show that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genome editing effectively produced specific genomic deletions in the MIEN1 gene which led to the abrogation of its expression in breast cancer cells. The single guide RNAs (sgRNAs) mediated targeting of MIEN1 was specific and none of the clones screened for off-target cleavage revealed any insertions or deletions (indels). Additionally, disruption of the MIEN1 gene did not alter the cell morphology, growth, proliferation or survival. Knocking out MIEN1 in these breast cancer cells will allow future studies to determine the exact role MIEN1 plays in breast tumor metastasis, which might lead to production of novel therapeutics to treat this and other cancers.
Assuntos
Neoplasias da Mama/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Genômica , HumanosRESUMO
Migration and invasion enhancer 1 (MIEN1) is a novel gene involved in prostate cancer progression by enhancing prostate cancer cell migration and invasion. DNA methylation, an important epigenetic regulation, is one of the most widely altered mechanisms in prostate cancer. This phenomenon frames the basis to study the DNA methylation patterns in the promoter region of MIEN1. Bisulfite pyrosequencing demonstrates the MIEN1 promoter contains a short interspersed nuclear Alu element (SINE Alu) repeat sequence. Validation of methylation inhibition on MIEN1 was performed using nucleoside analogs and non-nucleoside inhibitors and resulted in an increase in both MIEN1 RNA and protein in normal cells. MIEN1 mRNA and protein increases upon inhibition of individual DNA methyltransferases using RNA interference technologies. Furthermore, dual luciferase reporter assays, in silico analysis, and chromatin immunoprecipitation assays identified a sequence upstream of the transcription start site that has a site for binding of the USF transcription factors. These results suggest the MIEN1 promoter has a SINE Alu region that is hypermethylated in normal cells leading to repression of the gene. In cancer, the hypomethylation of a part of this repeat, in addition to the binding of USF, results in MIEN1 expression.
Assuntos
Elementos Alu/genética , Sítios de Ligação/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Repressão Epigenética/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Fatores Estimuladores Upstream/metabolismoRESUMO
Migration and invasion enhancer 1 (MIEN1) is an important regulator of cell migration and invasion. MIEN1 overexpression represents an oncogenic event that promotes tumor cell dissemination and metastasis. The underlying mechanism by which MIEN1 regulates migration and invasion has yet to be deciphered. Here, we demonstrate that MIEN1 acts as a cytoskeletal-signaling adapter protein to drive breast cancer cell migration. MIEN1 localization is concentrated underneath the actin-enriched protrusive structures of the migrating breast cancer cells. Depletion of MIEN1 led to the loss of actin-protrusive structures whereas the over-expression of MIEN1 resulted in rich and thick membrane extensions. Knockdown of MIEN1 also decreased the cell-substratum adhesion, suggesting a role for MIEN1 in actin cytoskeletal dynamics. Our results show that MIEN1 supports the transition of G-actin to F-actin polymerization and stabilizes F-actin polymers. Additionally, MIEN1 promotes cellular adhesion and actin dynamics by inducing phosphorylation of FAK at Tyr-925 and reducing phosphorylation of cofilin at Ser-3, which results in breast cancer cell migration. Collectively, our data show that MIEN1 plays an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, which leads to an increase in cell motility. Hence, targeting MIEN1 might represent a promising means to prevent breast tumor metastasis.
