Concerted loss of TGFβ-mediated proliferation control and E-cadherin disrupts epithelial homeostasis and causes oral squamous cell carcinoma

Abstract

Although the etiology of squamous cell carcinomas of the oral mucosa is well understood, the cellular origin and the exact molecular mechanisms leading to their formation are not. Previously, we observed the coordinated loss of E-cadherin (CDH1) and transforming growth factor beta receptor II (TGFBR2) in esophageal squamous tumors. To investigate if the coordinated loss of Cdh1 and Tgfbr2 is sufficient to induce tumorigenesis in vivo, we developed two mouse models targeting ablation of both genes constitutively or inducibly in the oral-esophageal epithelium. We show that the loss of both Cdh1 and Tgfbr2 in both models is sufficient to induce squamous cell carcinomas with animals succumbing to the invasive disease by 18 months of age. Advanced tumors have the ability to invade regional lymph nodes and to establish distant pulmonary metastasis. The mouse tumors showed molecular characteristics of human tumors such as overexpression of Cyclin D1. We addressed the question whether TGFβ signaling may target known stem cell markers and thereby influence tumorigenesis. From our mouse and human models, we conclude that TGFβ signaling regulates key aspects of stemness and quiescence in vitro and in vivo. This provides a new explanation for the importance of TGFβ in mucosal homeostasis.

Fig. 1.

Altered TGFBR2 and CDH1 in patient prognosis and mouse model. (A) Overall survival of HNSCC patients when stratifying by TGFBR2 status. Patients with homozygous deletion or mutation of TGFBR2 showed lower survival compared with patients without a homozygous deletion or mutation of TGFBR2. (P = 0.71). (B) Overall survival of HNSCC patients when stratifying by CDH1 reverse phase protein array expression. Patients with downregulation of CDH1 protein expression showed lower survival compared with patients with unaltered CDH1 protein expression. (P = 0.58). (C) Contingency analysis of TGFBR2 and/or CDH1 expression and disease-free survival. Patients with altered TGFBR2 and/or altered CDH1 had a higher proportion of disease recurrence than patients with unaltered TGFBR2 or CDH1 (RR = 1.27, 95% CI = 0.83, 1.96). (D) Contingency analysis of TGFBR2 and/or CDH1 expression and overall survival. Patients with altered TGFBR2 and/or altered CDH1 had no proportional difference in overall survival compared with patients with unaltered TGFBR2 or CDH1 (RR = 1.06, 95% CI = 0.71, 1.60). (E) Cdh1/Tgfbr2 double knockout (dcKO) mice present with lesions that can involve the facial skin, in this case the whisker pad, due to their invasive nature (arrows). (F) Histological analysis of a OSCCs originating in the oral cavity extending to the buccal mucosa and involving the surface of the skin. (G) Low magnification of a representative tongue with focal tumor lesion on the ventral side (rectangle). (H) Normal mucosa of control animal. (I) Invasive SCC of the tongue (J higher magnification) invading into the muscle (black arrows). Scale bar, 50 micron.

Fig. 2.

Tumors exhibit disruption of adherens junctions and increased proliferation. Focal invasive areas (dashed white circles) of a tongue SCC are negative for E-cadherin (A, red) compared with the expression pattern of the normal tongue epithelium (B). (C) Nuclear pSmad2 (green) signal is lost in invasive areas (dashed white circles). (D) Normal mouse mucosa has positive pSmad2 signal throughout. B-catenin, red, is lost in these invasive cells (dashed white circles (E)), while (G) K14, green, stains positive. The signal is increased compared with the normal tongue epithelium (H) of a non-induced control animal. The tumor cells invading into the center of the tongue are strongly positive for Ki67 (I) and p63 (K) compared with normal tongue, (J) and (L), respectively. Scale bars, 50 micron.

Fig. 3.

OSCCs in Cdh1/Tgfbr2 knockout mice are able to metastasize regionally and distantly. Tumor cell nests could be detected with keratin 14, K14, staining in regional lymph nodes (A, hematoxylin and eosin; B and C, K14 immunofluorescence). Tumors can spread to the lung as seen on lung sections stained with hematoxylin and eosin (D), and anti-K14 antibody in (E, F higher magnification). Lung metastases are Ki67- and p63-positive (G–H). Scale bars, 50 micron.

Fig. 4.

Upregulation of Cyclin D1 (Ccdn1), c-Myc and Yap1 in mouse OSCCs. Mouse tumors are positive for Ccdn1 (A, B; red, DAPI blue), Myc (D, E; green, DAPI blue), and Yap1 (G, H; red, DAPI blue). Ccdn1 and Myc are up-regulation compared with normal mucosa (C and F, respectively). Yap1 (G, H) is localized to the nucleus in tumor tissues compared with cytoplasmic localization in normal oral mucosa (I). Scale bars, 50 micron.

Fig. 5.

Changes in putative stem cell marker expression during tumor progression. The expression of ITGB1 (A, green) was restricted to the basal layer in the normal human epithelium compared with human OSCC tissues (B), organotypic reconstructs with functional loss of E-cadherin and TGFBR2 (C) or mouse OSCCs (D). Similarly, PML (green) outlines the basal cell layer in normal human oral mucosa (E), but its signal increases in human (F) and mouse OSCCs (H) as well as loses it restriction to the basal layer in organotypic reconstructs (G). The expression of MECP2 mirrors PML expression and localization (I–L). Scale bars, 50 micron. Western blot analysis (M) of two paired mouse tissues, normal N versus tumor T shows the upregulation of Pdpn, Myc and Sox2. Upregulation of PDPN (red) in human OSCC is shown by immunofluorescence compared with normal human tongue (N, Ki67 is green). Western blots shown in this figure were cropped.