Conditional Knockout of N-WASP Enhanced the Formation of Keratinizing Squamous Cell Carcinoma Induced by KRas(G12D)

SIMPLE SUMMARY: The expression of N-WASP (Neural Wiskott-Aldrich Syndrome Protein) is found to be reduced in certain human cancers, and this has been associated with poor prognoses for patients. To characterize the role of N-WASP in the tumorigenesis of squamous cell carcinoma (SCC), we generated mice which expressed a mutant oncogene with (N-WASP(HetG12D)) or without (N-WASP(KOG12D)) the expression of N-WASP upon Tamoxifen (TAM) injection. Both sets of mice had similar weights and did not display any abnormalities prior to the TAM injections. The N-WASP(KOG12D) mice displayed significantly greater weight loss and hair loss along the dorsal skin post-injection when compared to the N-WASP(HetG12D) mice. The N-WASP(KOG12D) mice developed tumors 2 weeks after their exposure to TAM, with 73% of these mice succumbing to the disease within 6 weeks of the injection. Subsequently, the remaining mice in this group died within 7 to 8 weeks post injection. On the contrary, the N-WASP(HetG12D) mice developed tumors only after 7 weeks following the injection and survived for up to 116 days. Our findings reveal that N-WASP deficiency activates signaling pathways which enhance cell proliferation. An N-WASP deficiency led to the acceleration of mutant-oncogene-induced SCC formation in mice, suggesting a tumor suppressor role for N-WASP in skin cancer. Thus, N-WASP could potentially serve as a novel marker for the development of skin cancer and the pathogenesis of SCC. ABSTRACT: Squamous cell carcinoma (SCC) is one of the most common forms of skin cancer in humans, and Neural Wiskott-Aldrich Syndrome Protein (N-WASP) plays a crucial role in epidermal homeostasis. To elucidate the role of N-WASP in skin cancer, we generated mice which expressed constitutively active KRas (KRas(G12D)) in keratinocytes with either homozygous (N-WASP(KOG12D)) or heterozygous (N-WASP(HetG12D)) N-WASP knockout upon Tamoxifen (TAM) injection. Both the N-WASP(KOG12D) and N-WASP(HetG12D) mice had similar body weights and no congenital malformations prior to the injection of TAM. Within 2 weeks of the injections, the N-WASP(KOG12D) mice exhibited significant reductions in weight coupled with visible tumors at numerous sites, unlike the N-WASP(HetG12D) mice, which had no visible tumors. We found that both sets of mice had oily, sticky skin and wet eyes 3 weeks after their exposure to TAM, indicating the overproduction of sebum/meibum. At 37 days post TAM injection, several notable observations were made. Tumors collected from the N-WASP(KOG12D) mice had small- to large-sized keratin pearls that were not observed in the N-WASP(HetG12D) mice. A Western blot and immunostaining analysis both highlighted significantly higher levels of expression of SCC markers, such as the cytokeratins 8, 17, 18, and 19 and TP63, in the tumors of the N-WASP(KOG12D) mice compared to those of the latter group. Furthermore, we noted increases in the expression levels of EGFR, P-ERK, GLUT1, P-mTOR, and P-4EBP in the N-WASP(KOG12D) mice, suggesting that the deletion of N-WASP in the keratinocytes enhanced KRas signaling and glucose uptake, resulting in aggressive tumor formation. Interestingly, a thickening of the epidermal layer within the esophagus and tongue was only observed in the N-WASP(KOG12D) mice. Immunostaining for PCNA emphasized a significantly higher number of PCNA-positive cells in the skin of the N-WASP(KOG12D) mice compared to their counterparts, implying that epidermal thickening and enhanced tumorigenesis are due to an increased proliferation of keratinocytes. Through our results, we have established that N-WASP plays a tumor-suppressive role in skin cancer.