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

Abstract

To study ROCK2 activation in carcinogenesis, mice expressing 4-hydroxytamoxifen (4HT)-activated ROCK2 (K14.ROCK^er) were crossed with mice expressing epidermal-activated ras^Ha (HK1.ras¹²⁰⁵). At 8 weeks, 4HT-treated K14.ROCK^er/HK1.ras¹²⁰⁵ cohorts exhibited papillomas similar to HK1.ras¹²⁰⁵ controls; however, K14.ROCK^er/HK1.ras¹²⁰⁵ histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma (wdSCC), exhibiting p53 loss, increased proliferation and novel NF-κB expression. By 12 weeks, K14.ROCK^er/HK1.ras¹²⁰⁵ wdSCCs exhibited increased NF-κB 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.ROCK^er/HK1.ras¹²⁰⁵ papillomatogenesis also required a wound promotion stimulus, confirmed by breeding K14.ROCK^er into promotion-insensitive HK1.ras¹²⁷⁶ mice, suggesting a permissive K14.ROCK^er/HK1.ras¹²⁰⁵ papilloma context (wound-promoted/NF-κB⁺/p53^-/p21⁺) preceded K14.ROCK^er-mediated (p-Mypt1/tenascin C/rigidity) malignant conversion. Malignancy depended on ROCK^er/p-Mypt1 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-κB 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-κB 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-κB signalling in skin carcinogenesis. Collectively, these data show that ROCK2 activation induces malignancy in ras^Ha-initiated/promoted papillomas in the context of p53 loss and novel NF-κB expression, whereas increased tissue rigidity and cell motility/contractility help mediate tumour progression.