Abstract 930: Inducible ROCK 2/rasHa cooperation requires wound promotion to achieve malignancy in transgenic mouse skin carcinogenesis, whereas inducible ROCK 2/PTEN loss fails to achieve benign papilloma
Authors: Masre Siti F. , Samuel Michael S. , Olson Michael F. , Greenhalgh David A. 
Published: May 2015

Abstract
To study tumour progression mechanism in vivo, ROCK 2 signalling deregulation and co-operation with activated rasHa or PTEN loss was investigated in transgenic mouse skin carcinogenesis. Transgenic mice that expressed a 4-hydroxytamoxifen (4HT)-activated human ROCK 2-estrogen receptor fusion transgene from a keratin 14 promoter [K14.ROCKer] were crossed to mice expressing activated rasHa exclusively in epidermal transit amplifying keratinocytes [HK1.ras-ROCKer] or mice where cutaneous PTEN loss was achieved in basal layers, hair follicles and stem cell keratinocytes via topical RU486 treatment of bi-genic K14.creP/Δ5PTENflx genotypes [K14.Δ5PTEN-ROCKer]. Initial 4HT-treatments of K14.ROCKer mice [3/wk; 26 wks] induced epidermal and follicular hyperplasia but no papillomas; whilst treatment of HK1.ras line 1205 gave ear tag papillomas [10-12 wks] typically smaller than vehicle controls, but no malignant conversion. In contrast, 4-HT treated HK1.ras-Rocker papillomas [10-12 wks] exhibited areas of carcinoma in situ and well-differentiated squamous cell carcinoma [wdSCC]. With time [16-20wks] wdSCC areas increased, concomitant with loss of p53, increased p-AKT and altered differentiation marker expression; however histotypes remained wdSCC despite continued 4HT-induced Rock 2 activity; as confirmed by downstream MYPT-1 phosphorylation. Furthermore, papillomas regressed whenever 4-HT treated HK1.ras-Rocker mice lost ear tags, suggesting that continued promotion from wounding during papillomatogenesis was critical to achieving the appropriate [late-stage] papilloma context that facilitated Rock 2-mediated conversion. To test this idea, K14.ROCKer mice were crossed to HK1.ras line 1276, which is insensitive to wound promotion, and such bi-genic mice did not exhibit papillomas; while in reverse, demonstrating the need for initiation, classic TPA-promoted K14.ROCKer mice were also devoid of papillomas. In addition, as previously rasHa/PTENnull mice exhibited papillomas prone to conversion on acquisition of an additional oncogene [e.g. fos], K14.ROCKer mice were bred into a K14.cre.Δ5PTEN strain and bi-genic mice treated with Ru486 [3wks] and 4HT [&gt;26wks]. Again, in the absence of rasHa no papillomas were observed and mice exhibited increased hyperplasia and altered differentiation consistent with disruption of epidermal physiology. These data suggest that Rock 2 deregulation plays major roles in malignant conversion and progression, and in cooperation with rasHa activation or PTEN loss, the mechanism requires additional promotion/initiation events during papillomatogenesis to create the context of late-stage papilloma where Rock 2 activities become causal; an idea being tested in tri-genic HK1.ras/Δ5PTEN-Rocker genotypes.
Citation Format: Siti F. Masre, Michael S. Samuel, Michael F. Olson, David A. Greenhalgh. Inducible ROCK 2/rasHa cooperation requires wound promotion to achieve malignancy in transgenic mouse skin carcinogenesis, whereas inducible ROCK 2/PTEN loss fails to achieve benign papilloma. [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 930. doi:10.1158/1538-7445.AM2014-930