Molecular Roles of TGF-beta Signaling in Salivary Glands and Oral Cancer

TGF-β信号通路在唾液腺炎症和纤维化中的作用：人类唾液腺的主要作用是产生外分泌物唾液，其提供（A）上胃肠道的大部分先天宿主防御，（B）牙釉质和口腔粘膜的特异性保护和修复功能，和（C）充分咀嚼和食物团加工所需的必需分子。任何导致唾液产生减少的状况都是不希望的，并且会对口腔健康和患者的生活质量产生负面影响。唾液分泌减少的主要原因有三个：处方药，头颈癌的治疗性放射，以及干燥综合征。后两种病症导致唾液腺分泌组织的不可逆损伤，并且两者都对当前的治疗干预具有抗性。TGF-β在自身免疫性疾病、慢性炎症和纤维化的发病和消退中起关键作用。 本研究的目的是描述TGF-β信号在唾液腺炎症中的确切功能。与NIDCR基因治疗和治疗分支合作，我们通过使用MMTV-Cre小鼠条件性灭活TGF-β受体I的表达或通过将含有Cre的腺病毒载体通过下颌下腺的插管主要排泄管逆行输注递送至小鼠唾液腺来损害小鼠唾液腺中的TGF-β信号传导。有趣的是，在MMTV-Cre小鼠产生的TGF-β受体I条件性敲除（TbetaRIcoko）中，只有雌性小鼠在唾液腺、乳腺和心脏中发生了致命的多灶性炎症。雌性TbetaRIcoko小鼠还显示唾液腺中Th 1促炎细胞因子增加，并表现出外周T细胞的上调。 此外，这些小鼠的唾液腺中也显示出水通道蛋白-5的非典型分布，表明可能存在分泌障碍。 将编码Cre重组酶的腺病毒载体给予唾液腺仅在雌性TGF-β RI floxed小鼠的腺体中导致炎性病灶，但在野生型雄性和雌性小鼠或雄性TbetaRI floxed小鼠中不导致炎性病灶。 这些结果表明，雌性小鼠是唯一容易发展的炎症性疾病，因为受损的TGF-β信号在他们的唾液腺。TGF-β 1的过表达与许多人类疾病有关，如特发性肺纤维化、系统性硬化症和肝纤维化。TGF-β 1也参与瘢痕的形成和辐射诱导的纤维化。为了研究TGF-β在唾液腺纤维化中的作用，我们建立了一种小鼠模型，该模型将允许对TGF-β 1的活性和HA标记版本的表达进行时间和空间控制。用MMTV-Cre建立了8个小鼠品系，培育了2个品系。初步结果显示双阳性小鼠的围产期死亡率。 TGF-β信号在头颈部鳞状细胞癌发生（HNSCC）中的分子作用：TGF-β被认为在癌发生中具有双重但依赖于环境的作用，作为肿瘤抑制剂或肿瘤促进剂。TGF-受体I（TbetaRI）的突变和多态性与人类HNSCC相关。然而，TGF-β信号转导在HNSCC中的确切作用尚未阐明。我们研究的目的是了解TbetaRI介导的信号通路在HNSCC病因学中的分子作用，并测试在受体水平上TGF-β信号传导的破坏是否导致自发肿瘤形成或仅增加易感性。 我们之前已经报道了TGF-β受体I（TbetaRI）条件性敲除（COKO）小鼠模型，该模型通过将TGF-β受体I（TbetaRI）floxed小鼠与neuroadherent-H（NF-H）Cre小鼠杂交产生。35%的F1 COKO小鼠在眶周和/或肛周区域发生自发性鳞状细胞癌（SCC）。将这些肿瘤移植到无胸腺裸鼠中导致62%的致瘤性。这些小鼠可以作为一个独特的SCC小鼠模型，以评估肿瘤的发生和抗癌治疗的效果。我们还通过将TbetaRI floxed小鼠与K14-CreERtam小鼠杂交开发了一种诱导型口腔上皮特异性敲除系统。通过将他莫昔芬应用于小鼠口腔以诱导Cre表达，我们能够有条件地删除口腔上皮中的TbetaRI。观察11个月后，在TbetaRIf/f;K14-CreERtam（TbetaR 1cKO）小鼠的头颈部上皮中未观察到自发性肿瘤。然而，在用7，12-二甲基苯并蒽（DMBA）诱导肿瘤后，20%的TbetaR 1cKO小鼠在治疗后16周开始发展HNSCC，而在同一时间段内在对照同窝仔中没有观察到肿瘤。对这些肿瘤的分子分析正在进行中。我们将利用我们的小鼠模型来开发新的策略，用于在HNSCC的治疗性治疗中靶向TGF-β信号通路。 这两个项目之间的工作分配：唾液腺疾病：30%; HNSCC：70%

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 patient's quality of life. There are 3 major causes of reduced saliva production: prescription medications, therapeutic radiation for head and neck cancer, and Sjogrens syndrome. The latter 2 conditions lead to irreversible damage to 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. The aim of this study was to delineate the precise function of TGF-beta signaling in salivary gland inflammation. In collaboration with the NIDCR Gene Therapy and Therapeutics Branch, we impaired TGF-beta signaling in mouse salivary glands by conditionally inactivating expression of TGF-beta receptor I using either MMTV-Cre mice or by delivering adenoviral vector containing Cre to mouse salivary glands via retrograde infusion of the cannulated main excretory ducts of submandibular glands. Interestingly, among the TGF-beta receptor I conditional knockouts (TbetaRIcoko) generated with MMTV-Cre mice, only female mice developed lethal multifocal inflammation in salivary glands, mammary glands, and the heart. The female TbetaRIcoko mice also showed an increase in Th1 proinflammatory cytokines in salivary glands and exhibited an upregulation of peripheral T cells. In addition, these mice also displayed an atypical distribution of aquaporin-5 in their salivary glands, suggesting likely secretory impairment. Administration of an adenoviral vector encoding Cre recombinase to the salivary glands resulted in inflammatory foci only in the glands of female TGF-beta RI floxed mice, but not in wild-type male and female mice or male TbetaRI floxed mice. These results suggest that female mice are uniquely susceptible to developing inflammatory disorders because of impaired TGF-beta signaling in their salivary glands. Overexpression of TGF-beta1 has been implicated in many human disorders such as idiopathic pulmonary fibrosis, systemic sclerosis, and hepatic fibrosis. TGF-beta1 is also involved in the formation of scars and in radiation-induced fibrosis. To study the roles of TGF-beta in salivary gland fibrosis, we generated a mouse model that will allow temporal and spatial control of the expression of an active and HA-tagged version of TGF-beta1. We have established 8 mouse lines and bred 2 lines with MMTV-Cre. Preliminary results show perinatal lethality in the double-positive mice. Molecular roles of TGF-beta signaling in head and neck squamous cell carcinogenesis (HNSCC): 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- receptor I (TbetaRI) 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 roles of the TbetaRI-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 only increases the susceptibility. We have earlier reported the TGF-beta receptor I (TbetaRI) conditional knockout (COKO) mouse model generated by crossing TGF-beta receptor I (TbetaRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 COKO mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anticancer therapeutics. We also developed an inducible oral epithelium-specific knockout system by crossing TbetaRI floxed mice with K14-CreERtam mice. By applying tamoxifen to the mouse oral cavity to induce Cre expression, we were able to conditionally delete TbetaRI in the oral epithelia. No spontaneous tumors in the head and neck epithelia of the TbetaRIf/f;K14-CreERtam (TbetaR1cKO) mice were noted after 11 months of observation. However, upon tumor induction with 7,12-dimethylbenzanthracene (DMBA), 20% of TbetaR1cKO mice developed HNSCC starting 16 weeks after treatment, while no tumors were observed in control littermates during the same time period. The molecular analysis of these tumors is now underway. We will utilize our mouse models to develop new strategies for targeting the TGF-beta signaling pathway in the therapeutic treatment of HNSCC. Distribution of efforts between these two projects: Salivary Gland Disorders: 30%; HNSCC: 70%