Abstract 2864: Regulation and targeting of Fyn in cutaneous squamous cell carcinoma

Abstract

Abstract Over 700,000 cases of cutaneous squamous cell carcinoma (cSCC) occur in the United States every year. Despite this high rate of diagnosis no therapies currently exist that target the molecular mechanism of tumorigenesis. Between 20 and 60% of these tumors contain mutated Ras; however targeting Ras with therapeutics has been unsuccessful in clinical trials. Expression of active H-Ras (G12V) in HaCaT cells (immortalized, non-transformed keratinocytes) results in upregulated expression of Fyn, a Src family kinase that is active in many malignancies and is involved in the elevated invasion/migration of Ras-transformed HaCaT cells. The specific objectives of this project include exploring the mechanism through which Ras activation increases Fyn levels and determining how high levels of Fyn promote cancer cell migration and survival. Using RT-qPCR we verified that Fyn expression is selectively elevated in cells with increased Ras activity, but levels of other Src family kinases (eg Src, Lyn, Yes, Blk, Lck) are not altered. To explore the transcriptional regulation of Fyn in Ras-transformed HaCaT cells we subcloned segments (−2 kb to −50 bp) of the human Fyn promoter in front of firefly luciferase and transfected the reporter constructs into HaCaT and HaCaT-Ras cells (transformed by constitutively active H-Ras). We identified a region −100 to −50 base pairs upstream of the transcription start site that is necessary for Fyn upregulation in HaCaT-Ras cells. The −80 to −65 region of the Fyn promoter contains putative GATA-1, GATA-2, GATA-3, CP2 or TATA transcription factor binding sites. We have also targeted Fyn in Ras-transformed cells using the Src family kinase inhibitor Dasatinib. Already used to treat chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia, Dasatinib is also in clinical trials to treat solid tumors. Treatment with varying concentrations of Dasatinib did not reveal greater changes in cell viability, cell cycle progression, apoptosis or autophagy in transformed HaCaT-Ras cells as compared to HaCaT cells. However, upon Dasatinib exposure HaCaT-Ras cells change dramatically in morphology, enhancing cell-cell contacts and reverting from a stellate appearance to the rounded morphology of untransformed HaCaT cells. Due to previous work verifying Fyn's role in promoting the invasion and migration of transformed keratinocytes, we propose that Fyn inhibition in keratinocytes with increased Ras activity will restore cell to cell contacts and provide a potential therapeutic target in the treatment of cSCCs. 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 2864. doi:1538-7445.AM2012-2864