Unimpaired skin carcinogenesis in Desmoglein 3 knockout mice

Abstract

The contribution of adherens junction inactivation, typically by downregulation or mutation of the transmembrane core component E-cadherin, to cancer progression is well recognized. In contrast, the role of the desmosomal cadherin components of the related cell-cell adhesion junction, the desmosome, in cancer development has not been well explored. Here, we use mouse models to probe the functional role of desmosomal cadherins in carcinogenesis. Because mice lacking the desmosomal cadherin Desmoglein 3 (Dsg3) have revealed a crucial role for Dsg3 in cell-cell adhesion in stratified epithelia, we investigate the consequence of Dsg3 loss in two models of skin carcinogenesis. First, using Dsg3-/- keratinocytes, we show that these cells display adhesion defects in vitro and compromised tumor growth in allograft assays, suggesting that Dsg3 enables tumor formation in certain settings. In contrast, using an autochthonous model for SCC development in response to chronic UVB treatment, we discover a surprising lack of enhanced tumorigenesis in Dsg3-/- mice relative to controls, unlike mice lacking the desmosomal component Perp. Accordingly, there is no defect in the apoptotic response to UVB or enhanced immune cell infiltration upon Dsg3 loss that could promote tumorigenesis. Thus, Dsg3 does not display a clear function as a tumor suppressor in these mouse skin cancer models. Continued unraveling of the roles of Dsg3 and other desmosomal constituents in carcinogenesis in different contexts will be important for ultimately improving cancer diagnosis, prognostication, and treatment.

Figure 1. Dsg3 deficiency in keratinocytes does not dramatically affect localization or solubility of other desmosomal components, but does compromise cell-cell adhesion.

A) Immunofluorescence analysis to examine localization of various desmosomal components in Dsg3+/+, Dsg3+/−, Dsg3−/− mouse keratinocyte monolayers after 48 hours of treatment with 2 mM Ca²⁺. DAPI is used as a nuclear marker (abbreviations: Dsg3 =  Desmoglein 3, Dsg1+2 =  Desmoglein 1 and 2, Dsg1 =  Desmoglein1, Ecad =  E-cadherin, Pkp3 =  Plakophilin 3, Pg =  Plakoglobin, Dsp1+2 =  Desmoplakin 1 and 2). B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of Dsg3+/+ and Dsg3−/− mouse keratinocyte monolayers after 48 hours of treatment with 2 mM Ca²⁺. 1 and 2 denote two different keratinocyte samples. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as a loading control for the urea fraction. C) Graph indicating the average percentage (+/− SD) of fragments released from Dsg3−/− keratinocyte monolayers after mechanical stress relative to wild-type controls in a mechanical dissociation assay. Experiments were performed in triplicate. p<0.0001, Student’s t-test.

Figure 2. Dsg3 facilitates transformed keratinocyte allograft tumor growth.

A) Experimental design for allograft tumor assays. B) Western blot analysis of p53−/−;Dsg3+/− and p53−/−;Dsg3−/− mouse keratinocytes transduced with HRasV12 lentiviruses confirms efficient HRasV12 expression relative to uninfected Dsg3+/− mouse keratinocytes. C) Graph displaying the volume of each tumor formed five weeks after implantation of HRasV12;p53−/−;Dsg3+/− (n = 16) and HRasV12;p53−/−;Dsg3−/− (n = 16) keratinocytes. p = 0.018, Student’s t-test. D) Representative hematoxylin and eosin (H and E)-stained sections of HRasV12;p53−/−;Dsg3+/− and HRasV12;p53−/−;Dsg3−/− tumors.

Figure 3. Dsg3 does not affect tumor incidence, tumor volume, or tumor grade in UVB-induced skin carcinogenesis.

A) Representative hematoxylin and eosin (H and E)-stained sections of Dsg3+/+ and Dsg3−/− mouse tongue epithelium. Arrow indicates the presence of blisters in the Dsg3−/− tongue epithelium. B) Photograph of an adult Dsg3−/− mouse, showing hair loss typical of Dsg3 deficiency. C) Schematic diagram illustrating the experimental design and timeline for the UVB-induced SCC model used. D) Kaplan-Meier analysis of tumor-free survival of Dsg3+/+ and Dsg3−/− mice subjected to chronic UVB treatment. p = 0.17, log rank test. E) Graph indicating the tumor volume in each Dsg3+/+ (24 tumors collected) and Dsg3−/− (8 tumors collected) mouse after 52 weeks of UVB treatment. p = 0.65, Student’s t-test. F) Histological analysis by H and E staining, showing the percentages of sarcomas and squamous cell carcinomas among the tumors observed in mice of each genotype. A representative H and E-stained sarcoma is shown. G) Analysis of the percentages of SCCs of different grades in Dsg3−/− and Dsg3+/+ mice. Photographs show representative H and E-stained SCCs with different levels of differentiation (highly, moderately, and poorly differentiated).

Figure 4. Dsg3 deficiency does not affect UVB-induced apoptosis or immune cell infiltration in vivo.

A) Graph indicating the average number of cleaved Caspase 3 (CC3) positive cells +/− SD per cm of epidermis in Dsg3+/+ and Dsg3−/− mice 24 hours after 2.5 kJ/m² UVB. n = 3 for each condition. B) Representative images of cleaved Caspase 3 immunohistochemistry in the epidermis of mice analyzed in A). Arrows indicate the apoptotic cells. C) Graph indicating the average number of mast cells +/− SD in the skin of Dsg3+/+ and Dsg3−/− mice after 52 weeks of UVB treatment. n = 12 for Dsg3+/+ mice and n = 5 for Dsg3−/− mice. D) Representative toluidine blue staining of the skin of Dsg3+/+ and Dsg3−/− mice after 52 weeks of UVB treatment. Arrows indicate mast cells stained by toluidine blue. Insets show higher magnification images of toluidine blue-stained skins, highlighting mast cells.

Figure 5. Dsg3 loss in the skin results in Dsg1 and Dsg2 upregulation.

Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of mouse skin lysates from Dsg3+/+ and Dsg3−/− mice. 1 and 2 denote samples from two mice. GAPDH serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as the loading control for the urea fraction.