Abstract 5355: Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion

Abstract

Abstract A number of studies have established the role of receptor tyrosine kinases (RTKs) and their downstream signaling targets, including PI3K/Akt/mTOR and Stat3, as important regulatory factors of epidermal proliferative capacity during both chemical (two-stage) and UV skin carcinogenesis. We have previously shown that total protein levels of Twist1 can be regulated by Stat3 in the two-stage skin carcinogenesis model and that nuclear localization of Twist1 was restricted to the proliferative compartment. Twist1, a protein commonly associated with the induction of EMT, has previously been shown to associate with cell cycle effectors, such as p53 and p21, as well as impact cell cycle progression. The cell cycle regulatory functions of Twist1 have been linked to p53 via phosphorylation of Twist1 at Ser42 by Akt. To investigate the role of Twist1 in cellular proliferation, primary keratinocytes were infected with lentiviral particles to create Twist1 knockdown or overexpressing cells, and were used, together with wild-type primary keratinocytes, to analyze the role of Twist1 during cell cycle progression. Western blot analysis of lysates from primary keratinocytes indicated that the expression of cell cycle regulatory proteins Cyclin E1, NF-κB, and c-Myc, coincided with the decrease or increase of Twist1. Protein levels of p21 exhibited an inverse correlation to Twist1 expression. Additionally, ChIP analysis identified Cyclin D1, Cyclin E1, c-Myc, and NF-κB as novel transcriptional targets of Twist1. To further investigate the role of Twist1, we have established transgenic mice with deletion of Twist1 in the basal compartment of the epidermis using BK5.Cre (i.e. BK5.Cre x Twistflox/flox mice). BK5.Cre x Twistflox/flox mice exhibited a significant reduction in epidermal thickness and labeling index (LI) compared to wild-type mice following treatment with TPA. In comparison to wild-type mice, acetone treated Bk5.Cre x Twistflox/flox mice also displayed a significant reduction of epidermal LI. Western blot analysis of epidermal lysates collected from BK5.Cre x Twistflox/flox mice treated with TPA demonstrated a reduction of Cyclin E1 and reduced phosphorylation of Rb at Ser795 compared to wild-type mice, further implicating the regulatory capacity of Twist1 during keratinocyte proliferation. Twist1 Ser42 phosphorylation also correlated with expression of the aforementioned cell cycle proteins. Collectively, these results, combined with other data to be presented, suggest that Twist1 plays a key role in keratinocyte proliferation during skin tumor promotion. Research supported by CA76520 and T32 ES007247. Citation Format: Jaya Srivastava, Everardo Macias, Kaoru Kiguchi, John DiGiovanni. Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5355. doi:10.1158/1538-7445.AM2014-5355