MIEN1 PROMOTES CANCER CELL MIGRATION AND INVASION THROUGH ENHANCED ACTIN DYNAMICS

Surgical resection and adjuvant therapies have so far, only incrementally improved patient survival; as metastatic disease remains incurable. Knowledge has been in part limited because metastasis is a ‘hidden’ process that occurs inside the body and is inherently difficult to observe. Migration and invasion are critical parameters in the dissemination of cancer cells and the formation of distant metastases. Hence identifying migratory and invasive genes and their action mechanisms may provide new insights into the pathogenesis and management of tumor metastasis. In the present investigation, we report functional studies of one of the prime regulators of cancer cell migration and invasion, which is also prenylated and contains an ITAM like the atypical Rho GTPase, RhoH. Migration and invasion enhancer1 (MIEN1) also known as C35, C17orf37, RDX12, and MGC14832, is frequently amplified and overexpressed in breast tumors. Purpose (a): Migration and Invasion Enhancer 1 (MIEN1), previously known as C35, C17orf37, RDX12 and MGC14832, is a novel gene located in the chromosomal region 17q12-21. While absent or low in normal tissues, MIEN1 is abundantly expressed in multiple cancers; including breast, prostate, oral and gastrointestinal carcinomas. A membrane-bound signaling adaptor, MIEN1 localizes to the leading edge of migrating cells and promotes migration and invasion by increasing filopodium formation. MIEN1 contains several functional motifs including a prenylation motif and an immunoreceptor tyrosine-based activation motif (ITAM). While prenylation of MIEN1 is shown to be important for its functions, little is known about the importance of its ITAM. The overall goal of the present study is to dissect the mechanisms by which MIEN1 regulates breast cell motility and whether the ITAM is important. Methods (b): Using site-directed mutagenesis, we introduced point mutations in amino acid sequences in MIEN1-ITAM domains. We established NIH3T3 stable cell lines over-expressing the wild type or mutant proteins. We performed immunofluorescence, migration and invasion assays using the established stable cell lines and breast cancer cells to investigate the mechanisms by which different domains of MIEN1 potentiate cell motility. Results (c): Analyses of in vitro migration and invasion assays showed that stable cells over-expressing MIEN1 phosphorylation mutants failed to induce significant migration and invasion compared to cells over-expressing MIEN1 wild type protein. Immunofluorescence staining with rhodamine conjugated-phalladoin confirmed that MIEN1 induced- migration is associated with actin filaments; and post-translational modifications at the ITAM domains is critical for eliciting MIEN1 functions. Conclusions (d): Our results confirm that MIEN1 regulates cancer cell migration and invasion through filopodia formation. Furthermore we showed that MIEN1 is involved in cell-cell adhesion, a process required for cell motility. MIEN1 is a prime regulator of cancer cell motility; hence understanding the molecular mechanisms by which it is aiding the invasion-metastasis cascade will enable the design of novel and effective treatments for metastatic tumors.