Abstract Proteins of the cofilin pathway are key regulators of actin dynamics. There are three cofilin isoforms encoded by human and mice. Of these, ADF-cofilin(ADF) and cofilin1(COF) are expressed in most epithelial tissues while muscle-cofilin is not. Recent data has indicated that components of the cofilin pathway(ADF, COF) are frequently mis-regulated in cancer cells, but there are conflicting reports on how these cofilins and the upstream regulatory LIM kinases contribute to the malignant phenotype by their over-expression, or suppression. The data suggest that it is the balance of the cofilin pathway regulators, and the cofilin pathway “output” which determines the invasiveness of tumor cells. The cofilin pathway must therefore be considered as a whole. Therefore, we will address the role of the cofilin pathway in skin homeostasis and cancer by deletion of ADF and COF in the skin. We generated K14-Cre-ER/ADF(-/-)/COF(fl/fl) mice, which are nullizygous for ADF and have floxed COF under control of keratin14 Cre recombinase, as ubiquitous cofilin deletion is embryonic lethal. After 4-OHT-induced COF deletion, we examined the effects on skin homeostasis and skin tumorigenesis. However, the homozygous ADF and COF knockout mice died within a few days following induction of Cre. There was a thickening of the epidermis due to hyperplasia of the spinous layer of cells. A marked increase in apoptosis of epithelial cells was also evident, predominantly affecting cells of the basal layer, resulting in ulceration of the skin and oral mucosa. These animals become acutely sick due to the oral ulceration leading to reduced food and water intake. Immunohistochemical analysis showed that the “stress inducible” keratin6, which is usually observed in hyperproliferative conditions, was aberrantly expressed in the epidermis of the homozygous ADF and COF knock out mice. Both in vitro and in vivo experiments indicated that E-cadherin was lost from cell-cell contacts in double ADF/COF knockout keratinocytes. In addition, the double ADF/COF knockout keratinocytes migrated in a mesenchymal manner and their movement was 4 times faster than wild type keratinocytes, which retained cell-cell contacts and moved in collective manner. All these data indicate that the output of the cofilin pathway is likely to be involved in cancer cell processes, including migration, proliferation, differentiation and apoptosis. We are currently generating tumor-derived cell lines from invasive squamous cell carcinomas in which both ADF and COF have been deleted and analyzing cancer-associated behaviour both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4307. doi:1538-7445.AM2012-4307