GROWTH CONTROL OF NORMAL AND MALIGNANT KERATINOCYTES

自从这笔资金于1985年首次获得资助以来，我们的目标一直是了解分子和细胞 呼吸道鳞状细胞癌(SCC)的病理学 分子诊断、预后和治疗手段以改善这些患者的预后 疾病。自从我们发现大多数鳞状细胞癌细胞系对转化生长因子-I_1耐药以来 (TGFl_)介导的细胞周期停滞，我们使用了分子和免疫组织化学相结合的方法 显示TGFI受体表达缺失和/或突变失活的方法，或 下游中介Smad2和Smad4导致有限子集中的TGFI响应性的全局损失 SCC。这些研究确定了转化生长因子S作为肿瘤抑制因子的作用。然而，对于大多数病例来说， 逃逸TGF1介导的生长控制的机制尚不清楚。 我们的第一个目标是确定导致Smad2丢失的潜在分子机制 在人类干细胞亚群中发现激活或Smad4缺乏。基于Smad4在 介导Smad4‘S抑瘤作用的第二个目标是鉴定Smad4’S的转录 其中哪些靶点介导了Smad4‘S的抑瘤作用。 这笔拨款的第二部分测试了这样的假设，即在癌症发展过程中，TGFI_‘S生长停滞 功能可能与其诱导上皮细胞向间充质细胞转分化的能力脱钩 (EMT)。事实上，在这些条件下，转化生长因子信号的结构性激活可能会增强侵袭性和 转移。然而，人们对决定TGFI_Response特异性的机制知之甚少。我们的 目的是验证TGFI信号强度、激活动力学、核质的假说 两个R-SMAD，Smad2和-3的穿梭和去磷酸化，以及它们与 Smad4和其他细胞蛋白是TGFI_细胞反应特异性的主要决定因素，并且 它们的破坏导致肿瘤中的生长抑制功能与EMT的解偶联。我们独一无二 定位于解决这些问题，因为我们拥有磷酸化Smad2和-3抗体 生产，我们获得高选择性和有效的T_R-I激酶抑制剂，以及大规模组织 用于快速验证关键实验结果的人体鳞状细胞癌样本阵列。

Since this grant was first funded in 1985, it has been our goal to understand the molecular and cellular pathology of squamous cell cancers (SCC) of the aerodigestive tract, with the intent to develop novel molecular diagnostic, prognostic as well as therapeutic tools to improve the outcome of patients with these diseases. Since our discovery that most SCC cell lines are refractory to Transforming Growth Factor-I_ (TGFl_)-mediated cell cycle arrest, we have used a combination of molecular and immunohistochemical approaches to show that loss of expression and/or mutational inactivation of TGFI_ receptors, or the downstream mediators Smad2 and Smad4, results in global loss of TGFI_ responsiveness in a limited subset of SCCs. These studies firmly established TGF_'s role as a tumor suppressor. However, for most cases, the mechanisms of escape from TGFl_-mediated growth control remain unclear. Our first objective is to identify the underlying molecular mechanisms that account for loss of Smad2 activation or Smad4 deficiency found in a subset of human SCCs. Based on the key role Smad4 plays in mediating TGF_'s tumor suppressive function, our second objective is to identify Smad4's transcriptional targets and which of these mediate Smad4's tumor suppressive function. The second part of this grant tests the hypothesis that, during cancer development, TGFI_'s growth arrest function can become uncoupled from its ability to induce epithelial-to-mesenchymal transdifferentiation (EMT). In fact, under these conditions, constitutive activation of TGF_ signaling may enhance invasion and metastasis. However, little is known about the mechanisms that determine TGFI_ response specificity. Our objective is to test the hypothesis that TGFI_ signal strength, the kinetics of activation, nucleo-cytoplasmic shuttling and dephosphorylation of the two R-Smads, Smad2 and -3, and their differential associations with Smad4 and other cellular proteins are major determinants of TGFI_ cellular response specificity, and that their disruption results in uncoupling of the growth arrest function from EMT seen in cancer. We are uniquely positioned to address these questions because of the phospho-Smad2 and -3 antibodies that we have produced, our access to highly selective and potent inhibitors of the T_R-I kinase, and large scale tissue arrays of human SCC specimens to rapidly validate key experimental findings.