TGF-ALPHA PROCESSING IN ORAL CANCER

癌细胞受到内源性生长因子的自分泌受体的刺激，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增强型(EGFR)。临床相关性以及体外和体内研究强烈表明，转化生长因子-α增加的EGFR刺激有助于癌症的发生。转化生长因子-α是一种跨膜生长因子，通过调节胞外区域的裂解或“脱落”来释放可溶的和可扩散的转化生长因子-α。转基因实验表明，这种跨膜转化生长因子-α的切割是其刺激肿瘤发展所必需的。转化生长因子-α胞外结构域的脱落是由TACE介导的，它是一种跨膜金属蛋白酶，最初被发现是因为它能够介导肿瘤坏死因子-α的裂解。我们最近发现，激活酪氨酸激酶受体的生长因子可以诱导转化生长因子-α的胞外脱落，而肿瘤坏死因子-α和L-选择素生长因子诱导的转化生长因子-α胞外脱落是通过激活ERK-MAP激酶通路而实现的，不需要新的蛋白质合成。我们还表明，TACE的胞浆结构域在生长因子刺激下被磷酸化，而转化生长因子-α胞外结构域被切割。这项建议现在建立在这些发现的基础上，旨在表征导致转化生长因子-α脱落激活的信号机制(S)及其在口腔癌发展中的作用。我们将该提案细分为四个目标。在目标1中，我们建议表征生长因子诱导的TACE的磷酸化及其在TACE激活和胞外结构域脱落中的作用。在目标2中，我们提出了两种不同的方法来鉴定、克隆和鉴定该激酶，它使TACE磷酸化，并通过这种方式激活TACE介导的脱落，以响应生长因子的刺激。在目标3中，我们建议鉴定和鉴定与TACE相互作用的蛋白质，并通过这种方式调节TACE的激活和胞外结构域的脱落。最后，在目标4中，我们将评估由于TACE激活和胞外结构域脱落而导致的转化生长因子-α及其胞外结构域切割的作用。最后，在目标4中，我们将评估由于TACE激活而导致的转化生长因子-α及其胞外结构域裂解在口腔鳞癌发生和发展中的作用。

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.