Isozyme specific effects of PKCs in thyroid cells

描述(申请人提供)：甲状腺肿瘤是最常见的内分泌恶性肿瘤。据估计，在这个国家进行的尸检中，高达90%的身体显示存在生长缓慢的甲状腺肿瘤。虽然分化良好的滤泡性和乳头状甲状腺肿瘤的患者预后良好，但间变性甲状腺肿瘤很快就会致命。激活RAS突变在人类甲状腺肿瘤中尤其普遍。RAS基因突变在良性腺瘤中被发现，在滤泡性癌和间变性癌中频率较高。Ras下游效应因子B-Raf的突变是乳头状甲状腺肿瘤中最常见的突变事件。这些观察结果支持RAS在甲状腺肿瘤的发生和发展中的作用。在甲状腺乳头状肿瘤中，有很大一部分表现出PKCepsilon基因的扩增和重排，导致PKCepsilon的N端片段的表达在结构上类似于V1结构域，V1结构域是一种选择性地抑制PKCepsilon易位的多肽。有趣的是，大多数乳头状甲状腺肿瘤显示PKCepsilon表达降低。此外，RET/PTC癌基因的表达诱导了选择性易位，随后PKCepsilon的表达下调。在滤泡性甲状腺肿瘤中，PKCalpha的表达增加，这种肿瘤也含有RAS突变。我们的假设是，单个PKC同工酶在RAS启动和维持甲状腺细胞转化过程中起着重要作用。我们的初步数据表明，PKCDelta选择性地复制了致癌RAS对细胞周期异常进展和细胞凋亡的急性影响；PKCepsilon是RAS诱导的形态变化所必需的；PKCs模拟了RAS对甲状腺分化的抑制作用；RAS转化的甲状腺细胞显示了PKC表达和活性的变化。本研究旨在探讨单个PKC同工酶在大鼠甲状腺细胞RAS转化的启动和维持中的作用。这将使用高度特异的分子试剂来完成，包括PKC同工酶的腺病毒、选择性的PKC多肽激活剂和抑制剂以及RNA干扰。这一分析将为RAS调控甲状腺细胞增殖、分化和存活的分子机制提供新的见解，并可能导致选择性地损害肿瘤细胞增殖和/或重新激活分化基因表达的新策略的开发。

DESCRIPTION (provided by applicant): Thyroid tumors are the most common endocrine malignancy. It has been estimated that up to 90% of the autopsies performed in this country reveal the presence of slow-growing thyroid tumors. While the prognosis for patients with well-differentiated follicular and papillary thyroid tumors is good, anaplastic thyroid tumors are rapidly fatal. Activating Ras mutations are particularly prevalent in human thyroid tumors. Ras mutations are found in benign adenomas and at a higher frequency in follicular and anaplastic carcinomas. Mutations in B-Raf, a downstream Ras effector, are the most frequent mutational event in papillary thyroid tumors. These observations support roles for Ras in the initiation and progression of thyroid tumors. A large proportion of papillary thyroid tumors exhibit amplification and rearrangement of the PKCepsilon gene, leading to the expression of an N-terminal fragment of PKCepsilon structurally similar to the V1 domain, a peptide that selectively inhibits PKCepsilon translocation. Interestingly, most papillary thyroid tumors exhibit decreased expression of PKCepsilon. Moreover, expression of the RET/PTC oncogene induced the selective translocation, followed by downregulation of PKCepsilon. PKCalpha expression is increased in follicular thyroid tumors, tumors that also harbor Ras mutations. It is our hypothesis that individual PKC isozymes play essential roles in the initiation and maintenance of thyroid cell transformation by Ras. Our preliminary data demonstrate that PKCdelta selectively reproduces the acute effects of oncogenic Ras on aberrant cell cycle progression and apoptosis; that PKCepsilon is required for Ras-induced morphological changes; that PKCs mimic the inhibitory effects of Ras on thyroid differentiation; and that Ras-transformed thyroid cells exhibit alterations in PKC expression and activity. The proposed studies investigate the roles of individual PKC isozymes in the initiation and maintenance of Ras transformation in rat thyroid cells. This will be accomplished using highly specific molecular reagents including adenoviruses for PKC isozymes, selective PKC peptide activators and inhibitors and RNA interference. This analysis will provide novel insight into the molecular mechanisms through which Ras dysregulates thyroid cell proliferation, differentiation and survival, and may give rise to the development of new strategies to selectively impair tumor cell proliferation and/or reactivate differentiated gene expression.