TGF-beta: Inflammation, Fibrosis, and Cancer The TGF-beta superfamily of multifunctional growth factors is involved in key processes such as cell proliferation, differentiation, embryonic development, carcinogenesis, immune dysfunction, inflammation, and wound healing. Three highly homologous isoforms of TGF-beta have been identified in mammals, and they share a common signaling pathway. Our primary focus has been to delineate the precise role of the TGF-beta signaling pathway in development, inflammation, and carcinogenesis. We initiated these studies in 1992 with TGF-beta1 knockout mice (Kulkarni et a., Proc. Natl. Acad. Sci. USA 90: 770-774, 1993). More than half of these mice died in utero, and those born alive developed multifocal and fatal inflammation, which prevented us from carrying out a detailed analysis of this isoforms cellular functions during various physiological processes. In order to overcome this problem, we have been testing strategies to generate conditional deletion, activation, or alteration of the TGF-beta1 signaling pathway. Role of 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 defense 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 oral health and the patients quality of life. Major causes of reduced saliva production include prescription medications, therapeutic radiation for head and neck cancer, and chronic inflammatory conditions such as Sjogrens syndrome. Radiation and chronic inflammation can ultimately result in fibrotic damage to the salivary gland. We then bred 3 of these lines to MMTV-Cre mice, the best transgenic mouse line capable of inducing recombination in the salivary gland. Initially, all the double positive pups (designated beta1glo/MC mice) that were born from this crossing died perinatally, probably due to the broad expression of the Cre recombinase in the MMTV line. However, a salivary gland phenotype that involved inhibited branching morphogenesis and aberrant development was observed. The dysplastic growth resulting from overexpression of TGF-beta1 also caused mislocalization of aquaporin-5, an important water channel in acinar cell development. Upon continued breeding with MMTV-Cre mice, one of our beta1glo transgenic lines eventually produced beta1glo/MC offspring that were capable of surviving into adulthood. With these adult beta1glo/MC mice, we had the capacity to examine salivary gland function. To induce salivation, the beta1glo/MC mice were injected with the drug pilocarpine. All the beta1glo/MC mice showed salivary gland hypofunction where no measurable amount of saliva could be recovered upon pilocarpine injection. Histological examination of the salivary glands revealed that the salivary gland hypofunction was a consequence of widespread fibrosis. Overexpression of TGF-beta1 was detected in the fibrotic 1glo/MC salivary glands, along with a concomitant increase in TGF-beta signaling by phosphorylation of the signaling molecule Smad2. PCR analysis also proved there was recombination of the transgene in the salivary glands of these mice. The widespread fibrosis in the salivary glands resulted in both acinar cell atrophy and loss of granular convoluted ducts in the beta1glo/MC mice. Aquaporin 5 expression and mucin staining were decreased in the 1glo/MC mice, supporting the observations of acinar cell atrophy. Overexpression of TGF-beta1 resulted in the replacement of the normal salivary gland parenchyma by collagens and other matrix proteins. In addition, smooth muscle actin, a marker for activated myofibroblasts, was elevated in the salivary glands of the beta1glo/MC mice along with increased expression of connective tissue growth factor, a pro-fibrotic secreted protein that can be induced by TGF-beta. The long-term effects of the chronic fibrosis resulted in immunological changes such as increased intracellular adhesion molecule-1 and IL-17 expression. Infiltrating mast cells were also seen throughout the fibrotic lesions of the beta1glo/MC mice. The mast cells detected in the salivary glands were most likely involved in promoting fibrosis rather than in any type I hypersensitivity reactions. No alterations, however, could be detected through the IL-13 signaling pathway, another pro-fibrotic cytokine. Molecular roles of TGF-beta signaling in head and neck squamous cell carcinogenesis (HNSCC) Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. The underlying cellular and molecular mechanisms that contribute to the initiation and progression of HNSCC have not been completely delineated. Tobacco, alcohol consumption and human papillomavirus (HPV) are the major risk factors associated with the development of HNSCC. These risk factors, together with genetic susceptibility, result in the accumulation of multiple genetic and epigenetic alterations in a multistep process of cancer development. In addition, the tumor microenvironment also contributes significantly to head and neck carcinogenesis. Recent advances in the understanding of the oncogenesis of HNSCC have revealed multiple deregulated signaling pathways. TGF-beta and PTEN/PI3K/Akt/mTOR pathways are among the most frequently altered signaling routes. Both pathways play central roles in numerous cellular processes including metabolism, cell growth, apoptosis (programmed cell death), survival, and differentiation, which ultimately contribute to HNSCC progression. The effects of TGF-beta signaling in carcinogenesis largely depend on the tissue of origin and the tumor type. In most types of human cancer, TGF-beta plays a paradoxical role in cancer development by acting as a tumor suppressor during the early stages, and as a tumor promoter during the later stages. Several reports have noted that mutations and polymorphisms of TGFBR1 and Smad signaling are associated with HNSCC, suggesting that TGF-beta functions as a potent tumor suppressor. However, it is not clear whether alterations in TGF-beta signaling act alone or in concert with those involving other pathways to promote a pro-oncogenic phenotype in advanced late-stage HNSCC. The PI3K/Akt pathway is important for suppressing apoptosis and promoting cell growth and proliferation. In HNSCC, hyperactivation of PI3K can be induced by mutations or by enhanced activity of its upstream activators, including the activation of Ras oncoproteins or inactivation of PTEN (phosphatase and tensin homolog deleted on chromosome 10). PTEN is a potent tumor suppressor gene and a negative regulator of the PI3K/Akt pathway. While PTEN mutations were identified in 0-16% of HNSCC, loss of PTEN expression was observed in 29% of tongue cancers, and loss of heterozygosity (LOH) of PTEN locus was identified in 40% of HNSCCs. Additionally, 47% of HNSCC cases showed at least one molecular alteration in the PI3K/Akt pathway, including PI3KCA and AKT2 amplification, p11-alpha overexpression, and PTEN protein downregulation. This suggests the critical role of the PTEN/PI3K/Akt signaling pathways in the carcinogenesis of HNSCC. The studies from our previous mouse model indicate that there may be negative crosstalk between the TGF-βtumor suppressor and the PI3K/Akt pathways.

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