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的发展。口腔癌细胞常表现为EGFR增高。临床相关性和体内外研究强烈提示，tgf - α刺激EGFR增加有助于致癌。tgf - α是一种跨膜生长因子，经过调节的外膜结构域裂解或“脱落”，释放出可溶性和可扩散的tgf - α。转基因实验表明，跨膜tgf - α的这种分裂是其刺激肿瘤发展的能力所必需的。tgf - α外畴脱落是由TACE介导的，TACE是一种跨膜金属蛋白酶，最初因其介导tnf - α裂解的能力而被发现。直到最近，激活TACE和随后的tgf - α裂解的机制尚不清楚。我们最近发现，激活酪氨酸激酶受体的生长因子，诱导tgf - α以及TNF- α和l -选择素的外畴脱落，生长因子诱导的tgf - α外畴脱落是通过激活Erk MAP激酶途径介导的，不需要新的蛋白质合成。我们还发现，TACE的胞质结构域在受到生长因子刺激时被磷酸化，tgf - α胞质结构域被外切。本研究以这些发现为基础，旨在描述导致tgf - α脱落激活的信号机制及其在口腔癌发展中的作用。我们把提案细分为四个目标。在Aim 1中，我们提出表征生长因子诱导的TACE磷酸化及其在TACE激活和外胞域脱落中的作用。在Aim 2中，我们提出了两种不同的方法来鉴定、克隆和表征该激酶，该激酶磷酸化TACE，并通过这种方式激活TACE介导的脱落，以响应生长因子刺激。在Aim 3中，我们提出鉴定和功能表征与TACE相互作用的蛋白质，并以此方式调节TACE激活和外胞域脱落。最后，在Aim 4中，我们将评估tgf - α及其外畴切割的作用，这是TACE激活和外畴脱落的结果。最后，在Aim 4中，我们将评估tgf - α及其外域切割在口腔鳞状癌的体内癌变和肿瘤发展中的作用，这是TACE激活的结果。

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.