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

TGF-β 信号传导在唾液腺和口腔癌中的分子作用

• 批准号: 8148649
• 负责人: Ashok Kulkarni
• 金额: $ 55.74万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148649
• 关键词: Molecular; Role; Salivary Glands; Signal Transduction; Transforming Growth Factor beta; malignant mouth neoplasm

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.

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，即唾液，它提供（A）上胃肠道的大部分先天宿主防御，（B）对牙釉质和口腔粘膜的特定保护和修复功能，以及（C）充分咀嚼和加工食物丸所需的必需分子。任何导致唾液分泌减少的情况都是不受欢迎的，并且会对患者的口腔健康和生活质量产生负面影响。唾液分泌减少有 3 个主要原因：处方药、干燥综合征以及慢性炎症或头颈癌放射治疗等因素导致的唾液腺纤维化。后两种情况会导致唾液腺分泌组织发生不可逆的损伤，并且两者都对当前的治疗干预有抵抗力。 TGF-β 在自身免疫性疾病、慢性炎症和纤维化的发生和消退中发挥着关键作用。为了研究 TGF-β1 在唾液腺中的作用，我们使用 Cre/loxP 技术来有条件地刺激或抑制 TGF-β 信号通路。唾液腺中 TGF-β 信号通路的抑制类似于雌性小鼠的干燥病。 这些功能丧失的发现需要使用补充功能获得方法进行进一步分析。为了研究小鼠唾液腺中过量 TGF-β 信号传导的影响，我们需要开发一种转基因品系 (Beta1glo)，它可以有针对性地条件性过度表达活性 TGF-β1。 尽管我们的工程转基因利用了普遍存在的启动子，但 TGF-β1 的表达依赖于 Cre 介导的基因重组，从而导致去除中间的 floxed EGFP 基因。然后将这些 Beta1glo 小鼠与 MMTV-Cre 转基因品系进行繁殖。 最初，所得的 Beta1glo/MMTV-Cre 小鼠在围产期死亡，但显示出唾液腺分支形态发生受到抑制的迹象。 后来，来自一种转基因 Beta1glo 品系的小鼠出生并存活至成年，但表现出唾液腺功能减退。 在这些 Beta1glo/MMTV-Cre 小鼠中，唾液腺中 TGF-β1 的过度表达导致大量纤维化和腺泡萎缩。 正常唾液腺实质的主要部分被纤维组织取代，并且唾液腺在颗粒回​​管（GCD）和腺泡中都显示出萎缩的迹象。这种表型模仿了接受放射治疗的 HNSCC 患者唾液腺的急性放射损伤。在 TGF-β RIcoko 和 Beta1glo/MMTV-Cre 小鼠中观察到的症状表明，TGF-β 信号传导的破坏可能与唾液腺的病理状况有关，例如纤维化和干燥综合征。 TGF-β信号传导在头颈鳞状细胞癌发生中的分子作用： 头颈鳞状细胞癌 (HNSCC) 是最常见的人类癌症类型之一，全球每年发病率超过 500,000 例。仅在美国，每年就有 47,560 例新病例被诊断为 HNSCC。尽管诊断和综合治疗有所进步，但HNSCC的总体5年生存率仅为50%左右，并且这个数字二十多年来没有变化。吸烟和饮酒以及病毒制剂是 HNSCC 发生的主要危险因素。这些风险因素与遗传易感性一起导致癌症发展的多步骤过程中多种遗传和表观遗传改变的积累。然而，导致从正常上皮细胞向侵袭性鳞状细胞癌的起始和进展的潜在细胞和分子机制尚未被阐明。更好地了解 HNSCC 的分子致癌机制可能有助于早期检测、边缘评估、预测和开发新的治疗策略。 越来越多的证据表明 TGF-β 信号通路参与头颈癌发生。 TGF-β被认为在致癌过程中具有双重但依赖于环境的作用，既可以作为肿瘤抑制因子，也可以作为肿瘤促进因子。 TGF-β 受体 I (RI) 的突变和多态性与人类 HNSCC 相关。然而，TGF-β 信号传导在 HNSCC 中的确切作用尚未阐明。我们研究的目的是了解 RI 介导的信号通路在 HNSCC 病因学中的分子作用，并测试受体水平上 TGF-β 信号传导的破坏是否会导致自发肿瘤形成，或者是否只会增加易感性。为了了解 TGF-β 受体 I (RI) 在神经元细胞中的精确作用，我们通过将 RI floxed 小鼠与神经丝-H (NF-H) Cre 小鼠杂交，建立了神经元中条件性删除 TGF-β 信号传导的小鼠模型。 35% 的 F1 条件敲除 (cKO) 小鼠在眶周和/或肛周区域发生自发性鳞状细胞癌 (SCC)。这些小鼠可以作为独特的鳞状细胞癌小鼠模型来评估致瘤性和抗癌治疗的效果。为了进一步完善头颈部肿瘤的发展，我们通过将 RI floxed 小鼠与 K14-CreERtam 小鼠杂交，开发了一种可诱导的口腔上皮特异性敲除系统。通过将他莫昔芬 (TM) 应用于小鼠口腔诱导 Cre 表达，我们能够有条件地删除口腔上皮细胞中的 RI。观察 11 个月后，RIf/f;K14-CreERtam (RIcKO) 小鼠的头颈部上皮细胞中没有发现自发性肿瘤。然而，在用 7, 12-二甲基苯并蒽 (DMBA) 诱导肿瘤后，20% 的 RIcKO 小鼠在治疗后 16 周开始出现 HNSCC，而同期对照同窝小鼠中没有观察到肿瘤。 我们能够证明，用 DMBA 治疗的 RIcKO 小鼠中 TGF-β 信号通路失活，并且头颈上皮基底层内细胞增殖标记物 Ki67 的表达同时增加。 RIcKO 小鼠的头颈部上皮细胞中没有自发形成肿瘤，这表明 RI 的丧失可能在小鼠 HNSCC 的肿瘤进展中发挥更重要的作用，而不是引发。除了 TGF-β 信号传导丧失外，DMBA 治疗的 RIcKO 小鼠肿瘤中还伴随着 Akt 激活，表明这两种细胞信号传导途径之间存在串扰。