Role of the TGF-beta signaling pathway in salivary gland inflammation and fibrosis: The primary role of salivary glands in humans is to produce an exocrine secretion, saliva, which provides (A) most of the innate host defenses for the upper GI tract, (B) specific protective and reparative functions for tooth enamel and the oral mucosa, and (C) essential molecules required for adequate mastication and processing of a food bolus. Any condition that results in decreased saliva production is undesirable and will have a negative impact on a patient's oral health and quality of life. There are 3 major causes of reduced saliva production: prescription medications, Sjogren's syndrome, and salivary gland fibrosis due to factors such as chronic inflammation or therapeutic radiation for head and neck cancer. The latter 2 conditions lead to irreversible damage to the salivary gland secretory tissue, and both are resistant to current therapeutic interventions. TGF-beta plays a key role in the onset and resolution of autoimmune diseases, chronic inflammation, and fibrosis. To study the role of TGF-beta1 in the salivary gland, we used Cre/loxP technology to either conditionally stimulate or inhibit the TGF-beta signaling pathway. The inhibition of TGF-beta signaling pathway in salivary glands mimicked Sjogren's disorder in female mice. These loss of function findings needed further analysis using a complementary gain-of-function approach. In order to study the effects of excess TGF-beta signaling in the salivary glands of a mouse, we needed to develop a transgenic line (Beta1glo) that could undergo targeted conditional overexpression of active TGF-beta1. Although our engineered transgene utilizes a ubiquitous promoter, the expression of TGF-beta 1 is dependent upon Cre-mediated genetic recombination to cause removal of an interceding floxed EGFP gene. These Beta1glo mice were then bred with the MMTV-Cre transgenic line. Initially, the resulting Beta1glo/MMTV-Cre mice died perinatally but showed signs of inhibited salivary gland branching morphogenesis. Later, mice from one of the transgenic Beta1glo lines were born and survived to adulthood, but displayed salivary gland hypofunction. In these Beta1glo/MMTV-Cre mice, the overexpression of TGF-beta1 in the salivary gland resulted in substantial fibrosis and acinar atrophy. A major portion of the normal salivary gland parenchyma was replaced by fibrous tissue, and the salivary glands showed signs of atrophy in both granular convoluted ducts (GCDs) and in the acini. This phenotype mimics the acute radiation damage to salivary glands seen in HNSCC patients who given radiation therapy. The symptomology seen in both the TGF-beta RIcoko and the Beta1glo/MMTV-Cre mice suggests that disruptions in TGF-beta signaling may be involved in pathological conditions of salivary glands such as fibrosis and Sjogren's syndrome. Molecular roles of TGF-beta signaling in head and neck squamous cell carcinogenesis: Head-and-neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer, with an annual incidence of more than 500,000 cases worldwide. In the United States alone, 47,560 new cases are diagnosed with HNSCC each year. Despite the improvements in diagnosis and comprehensive treatment, the overall 5 year survival rate of HNSCC is only about 50%, and this number has not changed in more than two decades. Tobacco and alcohol consumption, as well as viral agents, are the major risk factors involved in the development of HNSCC. These risk factors, together with genetic susceptibility, result in the accumulation of multiple genetic and epigenetic alterations in a multi-step process of cancer development. However, the underlying cellular and molecular mechanisms that contribute to the initiation and progression from normal epithelia to invasive squamous cell carcinoma have not been delineated. A better understanding of the molecular carcinogenesis of HNSCC may allow for early detection, margin evaluation, prognostication, and development of new strategies for treatment. There is accumulating evidence suggesting the involvement of the TGF-beta signaling pathway in head-and-neck carcinogenesis. TGF-beta is believed to have a dual but context-dependent role in carcinogenesis, acting either as a tumor suppressor or a tumor promoter. Mutations and polymorphisms of TGF-beta receptor I (RI) have been associated with human HNSCC. However, the precise role of TGF-beta signaling in HNSCC has not been delineated.
The aims of our studies are to understand the molecular role of the RI-mediated signaling pathway in the etiology of HNSCC, and to test whether a disruption of TGF-beta signaling at the receptor level leads to spontaneous tumor formation, or if it only increases susceptibility. In order to understand the precise role of the TGF-beta receptor I (RI) in neuronal cells, we generated a mouse model with conditional deletion of TGF-beta signaling in neurons by crossing RI floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 conditional knockout (cKO) mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. These mice can serve as a unique mouse model of SCC to evaluate tumorigenicity and effect of anticancer therapeutics. To further refine the development of these tumors in the head and neck area, we developed an inducible oral epithelium-specific knockout system by crossing RI floxed mice with K14-CreERtam mice. By applying tamoxifen (TM) to the mouse oral cavity to induce Cre expression, we were able to conditionally delete RI in the oral epithelia. No spontaneous tumors in the head and neck epithelia of the RIf/f;K14-CreERtam (RIcKO) mice were noted after 11 months of observation. However, upon tumor induction with 7, 12-dimethylbenzanthracene (DMBA), 20% of RIcKO mice developed HNSCC starting 16 weeks after treatment, while no tumors were observed in control littermates during the same time period. We were able to demonstrate that the TGF-beta signaling pathway was inactivated in the RIcKO mice treated with DMBA, and that there was a simultaneous increase in the expression of the cell proliferation marker Ki67 within the basal layer of the head and neck epithelia. The absence of spontaneous tumor formation in the head and neck epithelia of RIcKO mice suggests that, rather than initiation, loss of RI may play a more crucial role in tumor progression of mouse HNSCC. In addition to the loss of TGF-beta signaling, there was also a concomitant activation of Akt in the tumors of the DMBA-treated RIcKO mice, suggesting crosstalk between these two cellular signaling pathways.
Showing the most recent 10 out of 15 publications
