Molecular Mechanisms of Renal Cancer

描述(申请人提供)：肾透明细胞癌(CcRCC)是肾癌中最常见和最恶性的组织类型，目前还没有有效的治疗方法来治疗转移性疾病。CcRCC的特点是在大多数肿瘤(60%-80%)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效应是诱导低氧诱导因子(HIF)和HIF调节的基因导致血管生成，通过提供细胞外来源的营养物质来支持肿瘤的生长。在我们以前的工作中，我们发现VHL的丢失调节了通过自噬从细胞内获得营养的途径：VHL通过诱导miR-204的表达，靶向自噬调节因子LC3B，并抑制致癌的自噬。VHL通过抑制HIF，诱导具有肿瘤抑制活性的Lc3B同源基因Lc3c的表达。本研究主要针对编码miR-204基因内含子6的宿主基因的蛋白产物TRPM3通道(瞬时受体电位M3钙离子通透性非选择性阳离子通道)进行了研究。TRPM3在肾小管细胞癌中高表达，调节两条自噬途径；它是LC_3B/LC_3A自噬所必需的，并抑制LC_3C介导的自噬。与miR-204不同，TRPM3是在蛋白质积累水平上被VHL抑制的。我们的假设是，TRPM3对自噬程序的影响在其促进肾细胞癌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来实现的。在目标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。在目标2中，通过操纵TRPM3影响的不同自噬调节因子的表达，我们将识别对TRPM3的致癌活性至关重要的自噬途径。在目标3中，我们将操纵与自噬调节有关的细胞外和细胞内钙和钙调节激酶，并确定TRPM3被敲除的VHL(-)或VHL(-)细胞对自噬的影响。在目标4中，我们将研究在TRPM3被敲除的细胞中LC3C转录诱导的机制。有了这项建议，我们预计将进一步确立VHL作为自噬的主要监管机构。

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.