ROCK2 Activation Induces Malignant Conversion in rasHa-mediated Transgenic Mouse Skin Carcinogenesis Via p53 Loss, Elevated Nuclear Factor-κβ and Tenascin C-Associated Rigidity, but p21 Inhibits Early-Stage Progression

To study mechanisms of tumour progression, 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.ras1205]. 4HT-treatments of K14.ROCKer mice [3/wk; 26 wks] induced epidermal and follicular hyperplasia but no papillomas; whilst untreated K14.ROCKer -HK1.ras1205 cohorts exhibited papillomas similar to HK1.ras1205 controls [16wks]. In contrast by 8 weeks, 4HTtreated K14.ROCKer -HK1.ras1205 histotypes comprised a mixed papilloma/well-differentiated squamous cell carcinoma [wdSCC] that exhibited p53 loss, beginning in papilloma basal layers leading to increased proliferation. In addition papilloma histotypes also exhibited novel, ROCKer - associated NF-κβ expression in basal layer keratinocytes, prior to malignant conversion. By 12 weeks, K14.ROCKer -HK1.ras1205 wdSCCs exhibited further increases in NF-κβ expression together with the appearance of tenascin C expression, an extracellular matrix molecule indicative of elevated rigidity; yet despite continued ROCK2 activities, 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] malignant conversion [p-Mypt1/ actinomyosin-mediated mechanotransduction-tenascin C/rigidity]. Malignancy depended on ROCKer expression, as cessation of 4HTtreatment induced a p21-associated differentiation in wdSCC and appearance of novel papilloma outgrowths expressing intense, basal-layer p21 which confined endogenous ROCK2/p-Mypt1/NF-κβ to supra-basal layers, and restored basal-layer p53. In later SCCs, 4HT-cessation became irrelevant as endogenous ROCK2 expression increased, driving progression via p21 loss, elevated NF-κβ and tenascin C-associated rigidity, with p-Mypt1/actinomyosin-mediated contractility to facilitate invasion. Thus, ROCK2 activation induces malignancy in rasHa -initiated/promoted papillomas in the context of p53 loss, increased proliferation, and novel NF-κβ expression; whilst increased rigidity was associated with conversion and progression. However, p21 inhibition of early-stage malignant progression and intense expression in papilloma outgrowths identifies a significant antagonism between p21 and rasHa/ROCK2/NF-κβ signalling in skin carcinogenesis