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Cancer cell migration and invasion involves actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as WASP (Wiskott Aldrich Syndrome Protein) and NWASP (Neural-WASP). N-WASP and WASP interacting protein (WIP) play a crucial role in integrating signaling cascades leading to actin cytoskeleton remodeling. Compared to nontumorigenic HaCaT cells, N-WASP expression was found to be low in the tumorigenic, nonmetastatic epithelial cell line, HSC5 and high in the metastatic HaCaT derivative cell line, A5RT3. Differential expression of N-WASP may play a role in determining the function of NWASP during tumorigenesis. Characterization of N-WASP promoter was carried out by using GFP reporter assay, which led to the identification of a 393 bp regulatory region in N-WASP promoter critical for N-WASP promoter activity. Yeast one hybrid screen identified Msx1, an embryonic transcription factor as a binding element in N-WASP promoter. N-WASP expression has been shown to be increased under hypoxia conditions. In the current study, it was found that N-WASP promoter activity was enhanced in the presence of Hypoxia Inducible factor (HiF1α) suggesting a possible regulatory mechanism exerted by HiF1α. Sequence analysis of the N-WASP promoter revealed presence of two hypoxia response elements (HRE) characterized by the consensus motif 5’ACGTG 3’. Site-directed mutagenesis and ChIP assay demonstrated one of the HRE to be the binding site of HiF1α under hypoxic condition. RNA sequencing of HaCaT (non-tumorigenic keratinocyte) and A5-RT3 cell, which is a metastatic derivative of HaCaT led to the identification of 4488 genes which are upregulated and 1403 genes which are downregulated in A5-RT3 compared to HaCaT cells. WIP, N-WASP and

Role of WIP in metastasis and study of transcriptional regulation of N-Wasp