Conditional Knockout of N-WASP Enhanced the Formation of Keratinizing Squamous Cell Carcinoma Induced by KRasG12D

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.

Keywords: N-WASP; keratin pearl; skin cancer.

Figure 1

The ablation of N-WASP in mice expressing KRas^G12D enhanced tumorigenesis, increased the tumor burden, and decreased survival. (A) Tumors appeared on the dorsal skin, lips, face, anus, and palm/soles of the N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection. The graph represents the number of tumors within the dorsal skin sections of the N-WASP^KOG12D and N-WASP^HetG12D mice; (B) H&E-stained dorsal skin sections of N-WASP^KOG12D mice at 12 (n = 5), 21 (n = 5), 30 (n = 5), and 37 (n = 11) days post TAM injection. (C) The graph represents the survival curve of the N-WASP^KOG12D (n = 15) and N-WASP^HetG12D (n = 12) mice following the TAM injection. (D) Average body weight of N-WASP^KOG12D and N-WASP^HetG12D mice after TAM injection (n = 5). Results: means ± SDs. *** p < 0.001; ** p < 0.01.

Figure 2

Ablation of N-WASP and expression of KRas^G12D caused defects in the formation of sebaceous glands. (A) H&E-stained dorsal skin sections of N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection (n = 11) (Red circle is sebaceous gland). Quantification of sebaceous glands (SG) in N-WASP^KOG12D, N-WASP^HetG12D, and N-WASP^KO mice at 37 days post TAM injection (n = 5); (B) Oil-Red-O-stained dorsal skin sections of N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection (n = 3). The graph represents the intensity of the Oil Red O staining of the dorsal skin sections of the N-WASP^KOG12D and N-WASP^HetG12D mice. Results: means ± SDs. *** p < 0.001.

Figure 3

The ablation of N-WASP and the expression of KRas^G12D led to the formation of keratin cysts with high levels of expression of keratins. (A) H&E-stained dorsal skin sections of the N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection (n = 11); (B,C) Immunostaining of TP63 and cytokeratin 5 in paraffin-embedded skin tissues of the N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection (n = 3); (D,E) Using Trizol, the total RNA was isolated from the skin tumor samples of the N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection, converted into cDNA, and used to performed a qPCR with Slpi and Sprr2d in the N-WASP^KOG12D and N-WASP^HetG12D mice after 7 weeks post TAM injection (n = 3). The expression level of GAPDH was used for normalization. Results: means ± SDs. * p < 0.05.

Figure 4

The ablation of N-WASP in KRas^G12D-expressing cells lead to the formation of Squamous Cell Carcinoma (SCC). (A) H&E-stained dorsal skin sections of N-WASP^WTG12D, N-WASP^HetG12D, and N-WASP^KOG12D mice at 37 days post TAM injection (n = 11); (B) PCNA immunostaining of paraffin-embedded tumor dorsal skin sections of N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection and the quantification of PCNA-positive cells (n = 3). The positive staining of PCNA in N-WASP^KOG12D and N-WASP^HetG12D skin sections were manually counted at random fields, as shown in the figure. Results: means ± SDs. *** p < 0.001; ** p < 0.01.

Figure 5

The ablation of N-WASP and expression of KRas^G12D induced epidermal thickening in the tongue, esophagus, and palm/soles. H&E-stained samples from the palm/soles (A), tongue (B), and esophagus (C) sections of the N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post TAM injection (n = 11). Quantification of epidermal thickening in the tongue and esophagus samples of N-WASP^KOG12D and N-WASP^HetG12D mice at 37 days post injection (n = 5). Results: means ± SDs. *** p < 0.001.

Figure 6

The loss of N-WASP in mice with KRas^G12D-induced tumors activates the EGFR and KRas signaling pathway. Protein lysates from epidermis samples of the N-WASP^KOG12D and N-WASP^HetG12D mice (at 37 days post TAM injection) were subjected to a Western blot analysis, using antibodies against Cyclin D1, EGFR, P-ERK1/2 (A), Glut1 (B), and P-4EPB. (C) GAPDH was used as a loading control (n = 3). Results: means ± SEs. *** p < 0.001 and * p < 0.05. uncropped WB images were shown in Figure S8.