ROCK2/rasHa cooperation induce malignant conversion via p53 loss, elevated NF-κβ and tenascin C-associated rigidity but p21 inhibits ROCK2/NF-κβ-mediated progression

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)- activated ROCK2 [K14.ROCKer] were crossed to mice expressing epidermal activated ras Ha [HK1.ras1205]. At 8 weeks, 4HT-treated K14.ROCKer-HK1.ras1205 cohorts exhibited papillomas similar to HK1.ras1205 controls; however, K14.ROCKer-HK1.ras1205 histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma [wdSCC], exhibiting p53 loss, increased proliferation, and novel NF-κβ expression. By 12 weeks, K14.ROCKer-HK1.ras1205 wdSCCs exhibited increased NF-κβ and novel tenascin C, indicative of elevated rigidity; yet despite continued ROCK2 activities /p-Mypt1 inactivation, progression to SCC required loss of compensatory p21 expression. K14.ROCKer -HK1.ras1205 papillomatogenesis also required a wound-promotion stimulus, confirmed by breeding K14.ROCKer into promotion-insensitive HK1.ras1276 mice, suggesting a permissive K14.ROCKer-HK1.ras1205 papilloma context [wound-promoted/NF-κβ+ve/p53-ve/p21+ve] preceded K14.ROCKer-mediated [p-Mypt1/tenascin C/rigidity] malignant conversion. Malignancy depended on ROCKer/p-Mypt1 expression, as cessation of 4HT-treatment induced disorganised tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue ECM suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal-layer p21 which confined endogenous ROCK2/p-Mypt1/NF-κβ to supra-basal layers, and was paralleled by restored basal-layer p53. In later SCCs, 4HT-cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κβ expression and tenascin C-associated rigidity; with p-Mypt1 inactivation/actinomyosin-mediated contractility to facilitate invasion. However, p21-associated inhibition of early-stage malignant progression and the intense expression in papilloma outgrowths, identifies a novel, significant antagonism between p21 and ras Ha/ROCK2/NF-κβ signalling in skin 3 carcinogenesis. Collectively these data show that ROCK2 activation induces malignancy in rasHa-initiated/promoted papillomas in the context of p53 loss and novel NF-κβ expression;whilst increased tissue rigidity and cell motility/contractility help mediate tumour progression