ROCK2/rasHa co-operation induces malignant conversion via p53 loss, elevated NF-κB and tenascin C-associated rigidity, but p21 inhibits ROCK2/NF-κB-mediated progression

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)-activated ROCK2 (K14. ROCKer) were crossed with mice expressing epidermal-activated ras(Ha) (HK1.ras(1205)). At 8 weeks, 4HT-treated K14. ROCKer/HK1.ras(1205) cohorts exhibited papillomas similar to HK1.ras1205 controls; however, K14. ROCKer/HK1.ras(1205) histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma (wdSCC), exhibiting p53 loss, increased proliferation and novel NF-kB expression. By 12 weeks, K14. ROCKer/HK1. ras(1205) wdSCCs exhibited increased NF-kB 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 promotioninsensitive HK1. ras(1276) mice, suggesting a permissive K14. ROCKer/ HK1. ras1205 papilloma context (wound-promoted/NF-.B+/p53 -/ p21(+)) preceded K14. ROCKer-mediated (p-Mypt1/ tenascinC/rigidity) malignant conversion. Malignancy depended on ROCKer/ pMypt1 expression, as cessation of 4HT treatment induced disorganized tissue architecture and p21-associated differentiation in wdSCCs; yet tenascin C retention in connective tissue extracellular matrix suggests the rigidity laid down for conversion persists. Novel papilloma outgrowths appeared expressing intense, basal layer p21 that confined endogenous ROCK2/p-Mypt1/NF-k\B 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-kB 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 rasHa/ROCK2/NF-kB signalling in skin carcinogenesis. Collectively, these data show that ROCK2 activation induces malignancy in rasHa-initiated/promoted papillomas in the context of p53 loss and novel NF-kB expression, whereas increased tissue rigidity and cell motility/contractility help mediate tumour progression.