Molecular Mechanisms of Renal Cancer

描述（由申请人提供）：透明细胞肾细胞癌（Clear-cell renal cell carcinoma, ccRCC）是肾癌中最常见的恶性组织学类型，目前尚无有效的转移性治疗方法。ccRCC的特点是在大多数（60%-80%）肿瘤中早期丢失von Hippel-Lindau肿瘤抑制基因（VHL）。VHL缺失的一个主要致癌作用是诱导缺氧诱导因子（HIF）和HIF调节基因导致血管生成，血管生成通过提供细胞外来源的营养物质来支持肿瘤生长。在我们之前的工作中，我们发现VHL的缺失调节了通过自噬从细胞内获取营养的途径：VHL通过诱导miR-204的表达，靶向自噬调节因子LC3B，抑制致癌自噬。通过抑制HIF， VHL诱导LC3C的表达，LC3C是LC3B的同源物，具有肿瘤抑制活性。本研究的重点是TRPM3通道（瞬时受体电位M3 Ca2+渗透性非选择性阳离子通道），它是宿主基因在其内含子6中编码miR-204的蛋白产物。我们发现TRPM3在ccRCC中起促癌作用。TRPM3在ccRCC中过表达，调控两种自噬途径；是LC3B/LC3A自噬所必需的，抑制lc3c介导的自噬。与miR-204相反，TRPM3在蛋白积累水平上受到VHL的抑制。我们的假设是，TRPM3对自噬程序的影响在其促进RCC肿瘤生长的能力中起着至关重要的作用，并且TRPM3对自噬程序的影响是通过调节细胞内钙介导的。在Aim 1中，我们将研究VHL抑制TRPM3的分子机制，主要假设是VHL诱导miR-204靶向并抑制其宿主基因产物的表达。在Aim 2中，通过操纵受TRPM3影响的不同自噬调节因子的表达，我们将确定TRPM3致癌活性所必需的自噬途径。在Aim 3中，我们将操纵细胞外和细胞内Ca2+和Ca2+调节的激酶参与自噬的调节，并确定在TRPM3被敲除的VHL（-）或VHL（-）细胞中对自噬的影响。在Aim 4中，我们将研究TRPM3敲低的细胞中LC3C的转录诱导机制。通过这一建议，我们期望进一步确立VHL作为自噬的主要调节因子。

DESCRIPTION (provided by applicant): Clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant histological type of kidney cancer, for which there are no effective methods of treatment for metastatic disease. ccRCC is characterized by early loss of the von Hippel-Lindau tumor-suppressor gene (VHL) in a majority (60%-80%) of tumors. One major oncogenic effect of VHL loss is the induction of hypoxia-inducible factor (HIF) and HIF-regulated genes leading to angiogenesis, which supports tumor growth by providing nutrients from extracellular sources. In our previous work, we discovered that loss of VHL regulates pathways that secure nutrients from intracellular sources through autophagy: VHL, by inducing expression of miR-204, targets the autophagic regulator, LC3B, and inhibits oncogenic autophagy. By inhibiting HIF, VHL induces expression of LC3C, an LC3B ortholog, which has tumor suppressive activity. This proposal is focused on the TRPM3 channel (transient receptor potential M3 Ca2+-permeable, nonselective cation channel), which is the protein product of the host gene that encodes miR-204 in its intron 6. We discovered that TRPM3 plays a pro-oncogenic role in ccRCC. TRPM3 is overexpressed in ccRCC and regulates two autophagic pathways; it is necessary for LC3B/LC3A autophagy and inhibits LC3C-mediated autophagy. In contrast to miR-204, TRPM3 is repressed by VHL at the level of protein accumulation. Our hypothesis is that the effects of TRPM3 on autophagic programs play an essential role in its ability to promote RCC tumor growth, and that the effects of TRPM3 on autophagic programs are mediated through regulation of intracellular calcium. In Aim 1, we will investigate the molecular mechanism by which VHL represses TRPM3, with the leading hypothesis that VHL-induced miR-204 targets and represses expression of its host gene product. In Aim 2, by manipulating expression of different autophagic regulators affected by TRPM3, we will identify the autophagic pathways essential for the oncogenic activity of TRPM3. In Aim 3 we will manipulate extracellular and intracellular Ca2+ and Ca2+-regulated kinases implicated in the regulation of autophagy and determine the effects on autophagy in cells that are VHL(-) or VHL(-) with TRPM3 knocked down. In Aim 4 we will study the mechanisms of transcriptional induction of LC3C in cells with knockdown of TRPM3. With this proposal, we expect to further establish VHL as a major regulator of autophagy.