Xiao-Jing Wang

TGF-β and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4^–/– mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3^–/– mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2^–/– tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2^–/– mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss–associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss–associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss–associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss–associated EMT during skin carcinogenesis.

Mouse models of human cancer play an important role in understanding the mechanisms of carcinogenesis and have accelerated the search for finding new molecular targets for cancer therapy. However, genetically engineered mouse models for head and neck squamous cell carcinoma (HNSCC) have only recently overcome major technical obstacles and begun to be explored. Here we review the current progress in the development of mouse models for human HNSCC, with emphasis on conditional transgenic and knockout mouse models. These new models faithfully recapitulate human HNSCC at both the pathologic and molecular levels. These animal models will not only be useful to define the roles of specific genes in HNSCC development and progression but will also provide a unique tool for developing and testing new therapeutic approaches. © 2006 Wiley Periodicals, Inc. Head Neck, 2006

The prognosis of head-and-neck squamous cell carcinoma (HNSCC) has not been improved in the past 20 years. Validation of HNSCC biomarkers for targeted therapy has been hindered by a lack of animal models mimicking human HNSCC at both the pathological and molecular levels. Here we report that overexpression of K-ras or H-ras and loss of transforming growth factor-β type II receptor (TGFβRII) are common events in human HNSCC. Activation of either K-ras or H-ras in combination with TGFβRII deletion from mouse head-and-neck epithelia caused HNSCC with complete penetrance, some of which progressed to metastases. These tumors displayed pathology indistinguishable from human HNSCCs and exhibited multiple molecular alterations commonly found in human HNSCCs. Additionally, elevated endogenous TGFβ1 in these lesions contributed to inflammation and angiogenesis. Our data suggest that targeting common oncogenic pathways in tumor epithelia together with blocking the effect of TGFβ1 on tumor stroma may provide a novel therapeutic strategy for HNSCC.

Smad4 is the common mediator for TGF signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyperplasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGF and bone morphogenetic protein (BMP) signaling pathways, including p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome 10 (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGF/BMP pathway, which interacts with the Pten signaling pathway.

In the present study, we demonstrated that human skin cancers frequently overexpress TGF-β₁ but exhibit decreased expression of the TGF-β type II receptor (TGF-βRII). To understand how this combination affects cancer prognosis, we generated a transgenic mouse model that allowed inducible expression of TGF-β₁ in keratinocytes expressing a dominant negative TGF-βRII (ΔβRII) in the epidermis. Without ΔβRII expression, TGF-β₁ transgene induction in late-stage, chemically induced papillomas failed to inhibit tumor growth but increased metastasis and epithelial-to-mesenchymal transition (EMT), i.e., formation of spindle cell carcinomas. Interestingly, ΔβRII expression abrogated TGF-β₁–mediated EMT and was accompanied by restoration of membrane-associated E-cadherin/catenin complex in TGF-β₁/ΔβRII compound tumors. Furthermore, expression of molecules thought to mediate TGF-β₁–induced EMT was attenuated in TGF-β₁/ΔβRII–transgenic tumors. However, TGF-β₁/ΔβRII–transgenic tumors progressed to metastasis without losing expression of the membrane-associated E-cadherin/catenin complex and at a rate higher than those observed in nontransgenic, TGF-β₁–transgenic, or ΔβRII-transgenic mice. Abrogation of Smad activation by ΔβRII correlated with the blockade of EMT. However, ΔβRII did not alter TGF-β₁–mediated expression of RhoA/Rac and MAPK, which contributed to increased metastasis. Our study provides evidence that TGF-β₁ induces EMT and invasion via distinct mechanisms. TGF-β₁–mediated EMT requires functional TGF-βRII, whereas TGF-β₁–mediated tumor invasion cooperates with reduced TGF-βRII signaling in tumor epithelia.

It has been shown that Smad3 exerts both tumor-suppressive and -promoting roles. To evaluate the role of Smad3 in skin carcinogenesis in vivo, we applied a chemical skin carcinogenesis protocol to Smad3 knockout mice (Smad3^−/− and Smad3^+/−) and wild-type littermates (Smad3^+/+). Smad3^−/− mice exhibited reduced papilloma formation in comparison with Smad3^+/+ mice and did not develop any squamous cell carcinomas. Further analysis revealed that Smad3 knockout mice were resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced epidermal hyperproliferation. Concurrently, increased apoptosis was observed in TPA-treated Smad3^−/− skin and papillomas when compared with those of wild-type mice. Expression levels of activator protein-1 family members (c-jun, junB, junD, and c-fos) and transforming growth factor (TGF)-α were significantly lower in TPA-treated Smad3^−/− skin, cultured keratinocytes, and papillomas, as compared with Smad3^+/+ controls. Smad3^−/− papillomas also exhibited reduced leukocyte infiltration, particularly a reduction of tumor-associated macrophage infiltration, in comparison with Smad3^+/+ papillomas. All of these molecular and cellular alterations also occurred to a lesser extent in Smad3^+/− mice as compared with Smad3^+/+ mice, suggesting a Smad3 gene dosage effect. Given that TGF-β1 is a well-documented TPA-responsive gene and also has a potent chemotactic effect on macrophages, our study suggests that Smad3 may be required for TPA-mediated tumor promotion through inducing TGF-β1–responsive genes, which are required for tumor promotion, and through mediating TGF-β1–induced macrophage infiltration.

In the present study, we show that transforming growth factor β1 (TGF-β1) was frequently overexpressed in human head and neck squamous cell carcinomas (HNSCCs) and adjacent tissues in comparison with normal head and neck tissues. To determine the role of TGF-β1 overexpression in HNSCC carcinogenesis, we generated transgenic mice in which TGF-β1 transgene expression can be induced in head and neck epithelia. TGF-β1 transgene induction in head and neck epithelia, at levels similar to those in human HNSCCs, caused severe inflammation and angiogenesis. Consequently, TGF-β1-transgenic epithelia exhibited hyperproliferation. These phenotypes correlated with enhanced Smad signaling in transgenic epithelia and stroma. Our study suggests that TGF-β1 overexpression at early stages of HNSCC formation provides a tumor promoting microenvironment.

Transforming growth factor β1 (TGFβ1), a potent keratinocyte growth inhibitor, has been shown to be overexpressed in keratinocytes in certain inflammatory skin diseases and has been thought to counteract the effects of other growth factors at the site of inflammation. Surprisingly, our transgenic mice expressing wild‐type TGFβ1 in the epidermis using a keratin 5 promoter (K5.TGFβ1wt) developed inflammatory skin lesions, with gross appearance of psoriasis‐like plaques, generalized scaly erythema, and Koebner's phenomenon. These lesions were characterized by epidermal hyperproliferation, massive infiltration of neutrophils, T lymphocytes, and macrophages to the epidermis and superficial dermis, subcorneal microabscesses, basement membrane degradation, and angiogenesis. K5.TGFβ1wt skin exhibited multiple molecular changes that typically occur in human Th1 inflammatory skin disorders, such as psoriasis. Further analyses revealed enhanced Smad signaling in transgenic epidermis and dermis. Our study suggests that certain pathological condition‐induced TGFβ1 overexpression in the skin may synergize with or induce molecules required for the development of Th1 inflammatory skin disorders.