TGF-ALPHA PROCESSING IN ORAL CANCER

癌细胞受到内源性生长因子的自分泌受体刺激，这种刺激有助于恶性转化和癌症发展。口腔癌细胞常表现为EGFR的增加。临床相关性以及体外和体内研究强烈表明，TGF-α对EGFR刺激的增加有助于致癌作用。TGF-α被制成跨膜生长因子，其经历受调节的胞外域切割或“脱落”以释放可溶性和可扩散的TGF-α。转基因实验表明，这种跨膜TGF-α的切割是其刺激癌发展的能力所必需的。TGF-α胞外域脱落由TACE介导，TACE是一种跨膜金属蛋白酶，最初发现其能够介导TNF-α切割。激活TACE和随后的TGF-α切割的机制直到最近才被了解。我们最近已经表明，激活酪氨酸激酶受体的生长因子诱导TGF-α以及TNF-α和L-选择素的胞外域脱落。生长因子诱导的TGF-α胞外域脱落是通过激活Erk MAP激酶途径介导的，并且不需要新的蛋白质合成。我们还表明，TACE的胞质结构域响应于生长因子刺激而磷酸化，并且TGF-α胞外域的胞质结构域裂解。该提案现在建立在这些发现的基础上，旨在表征导致TGF-α脱落激活的信号传导机制及其在口腔癌发展中的作用。我们将该建议细分为四个目标。在目标1中，我们提出了TACE的生长因子诱导的磷酸化及其在TACE激活和胞外域脱落中的作用。在目标2中，我们提出了两种不同的方法来识别，克隆和表征激酶，其磷酸化TACE并以这种方式激活TACE介导的脱落以响应生长因子刺激。在目标3中，我们提出鉴定和功能表征与TACE相互作用的蛋白质，并以这种方式调节TACE活化和胞外域脱落。最后，在目标4中，我们将评估TGF-α及其胞外域切割的作用，作为TACE激活和胞外域脱落的结果。最后，在目标4中，我们将评估TGF-α及其胞外域切割的作用，作为TACE激活在体内口腔鳞状细胞癌发生和肿瘤发展中的结果。

Cancer cells are subject to autocrine receptor stimulation by endogenous growth factors, and this stimulation contributes to malignant transformation and cancer development. Oral carcinoma cells often show increased (EGFR). Clinical correlation and in vitro and in vivo studies strongly suggest that increased EGFR stimulation by TGF-alpha contributes to carcinogenesis. TGF-alpha is made as a transmembrane growth factor, which undergoes regulated ectodomain cleavage or "shedding" to release soluble and diffusible TGF-alpha. Transgenic experiments suggest that this cleavage of transmembrane TGF-alpha is required for its ability to stimulate carcinoma development. TGF-alpha ectodomain shedding is mediated by TACE, a transmembrane metalloprotease, which was originally discovered for its ability to mediate TNF-alpha cleavage. The mechanisms that activate TACE and consequent TGF-alpha cleavage were unknown until recently We have recently shown that growth factors, which activate tyrosine kinase receptors, induce ectodomain shedding of TGF-alpha as well as TNF- alpha and L-selectin Growth factor-induced TGF-alpha ectodomain shedding is mediated through activation of the Erk MAP kinase pathway and does not require new protein synthesis. We have also shown that the cytoplasmic domain of TACE is phosphorylated in response to growth factor stimulation and that the cytoplasmic domain of TGF-alpha ectodomain cleavage. This proposal now builds on these findings and is aimed at characterizing the signaling mechanism(s) that lead to activation of TGF-alpha shedding and its role in oral carcinoma development. We have subdivided the proposal in four Aims. In Aim 1, we propose to characterize the growth factor-induced phosphorylation of TACE and its role in TACE activation and ectodomain shedding. In Aim 2, we propose two different approaches to identify, clone and characterize the kinase, which phosphorylates TACE and in this way activates TACE mediated shedding in response to growth factor stimulation. In Aim 3, we propose to identify and functionally characterize proteins that interact with TACE and, in this way regulate TACE activation and ectodomain shedding. Finally, in Aim 4, we will evaluate the role of TGF-alpha and its ectodomain cleavage, as a result of TACE activation and ectodomain shedding. Finally, in Aim 4, we will evaluate the role of TGF-alpha and its ectodomain cleavage, as a result of TACE activation in carcinogenesis and tumor development of oral squamous carcinoma in vivo.