Tumor Suppressing Pathways in Renal Cancer

描述(由申请人提供)： 肾透明细胞癌(RCC)是肾癌中最常见和最恶性的组织学类型，目前尚无有效的转移性疾病治疗方法。它的特点是在绝大多数(60%-80%)的肿瘤中早期丢失von Hippel-Lindau肿瘤抑制基因(VHL)。MicroRNAs(MiRs)是一种短小的非编码RNA，它与mRNAs上的特定元件结合，抑制具有调节功能的肿瘤靶蛋白的翻译。我们发现，与正常肾脏相比，Mir204在人肾细胞癌中的表达普遍降低，并且表达降低的程度与肿瘤分级和肿瘤进展密切相关。与此一致，我们发现miR204具有抑瘤活性；它在体外对VHL(-)肾癌细胞具有细胞毒作用，并抑制这些细胞在裸鼠体内形成的肿瘤。VHL正向调节miR204的表达，保护非致瘤细胞免受miR204的细胞毒作用。我们进一步证实了miR204抑制宏自噬。与此一致，我们发现MAP1LC3B(LC3B)蛋白是一个直接的mir204靶标，它是宏观自噬的调节因子。我们还确定了其他几个在调节宏自噬方面具有潜在作用的mir204靶点。宏自噬是一种特定的程序，细胞通过它分解细胞内的细胞器以消除它们，并获得营养。因此，巨细胞自噬是支持癌细胞存活的一种内在营养来源。我们的一般假设是，在肾癌过程中VHL的丢失促进了mir204的丢失和特定靶点的去抑制，从而激活了依赖于LC3B的宏观自噬，这使癌细胞能够获得有助于其生存和肿瘤生长的细胞内营养物质。这使得RCC VHL(-)细胞沉迷于巨自噬活动，因此当巨自噬被外源mir204抑制时，容易受到合成死亡的影响，细胞死亡是由饥饿引起的。相反，VHL的存在刺激内源性mir204的表达，导致抑制宏自噬程序，使细胞对外源性mir204的活性不敏感。为了验证这一假说，我们将定义依赖mir204和LC3B的巨型自噬在肾癌形成中的作用(目标1)，定义其他特定的mir204靶标在巨型自噬和肿瘤形成中的作用(目标2)；确定导致肾癌中mir204表达减少的分子机制(目标3)；以及确定人肾癌肿瘤和正常肾脏样本中mir204靶标和一般自噬调节因子的水平(目标4)。影响：在这里，我们提供了以前未知的信号通路的证据，该通路是由肾癌中VHL和miR204的缺失激活的，该通路调节癌细胞的生存。了解这一途径将为开发治疗恶性肾细胞癌的新的治疗方法奠定基础。 公共卫生相关性： 肾癌对军人，特别是40岁以上的军人和退伍军人来说是一个严重的健康问题。肾癌占美国所有成人恶性肿瘤的2%至3%，2009年报告的新增病例为57760例，死亡病例为12980例。1995-2004年国防医学流行病学数据库分析，肾癌的发病率专门针对军人，在第4个十年之后，与未调整的发病率每10万人年1.5例相比，相当大幅度地增加到8.5例。此外，肾癌在男性中的发病率几乎是女性的两倍，大约80%的军事人员是男性。与不吸烟的人相比，吸烟导致患肾癌的风险增加47%，而且相对风险与每天吸烟的数量呈正相关。吸烟对军人来说是一个重要的健康问题。目前估计，30%的现役军人和74.2%(0.7%)的退伍军人是吸烟者或在生活中的某个时候吸烟，而从未服役的人中这一比例为48.4%(0.5%)。

DESCRIPTION (provided by applicant): Renal clear-cell carcinoma (RCC) is the most prevalent and malignant histological type of kidney cancer, with no effective methods of treatment for metastatic disease. It is characterized by an early loss of the von Hippel- Lindau tumor-suppressor gene (VHL) in a large majority (60%-80%) of tumors. MicroRNAs (miRs) are short noncoding RNAs that bind to specific elements on mRNAs to repress translation of target proteins with regulatory functions in cancer. We have discovered that expression of mir204 is universally decreased in human RCC tumors, as compared with normal kidneys, and that the degree of reduced expression strongly correlates with tumor grade and cancer progression. Consistent with this, we found that miR204 has tumor- suppressing activity; it is cytotoxic to VHL(-) RCC cells in vitro and inhibits growth of tumors formed by these cells in nude mice. VHL positively regulates expression of miR204, and protects non-tumorigenic cells from cytotoxic activity of miR204. We further established that miR204 inhibits macroautophagy. Consistent with this, we found that MAP1LC3B (LC3B) protein, a regulator of macroautophagy, is a direct mir204 target. We also identified several other mir204 targets with potential roles in the regulation of macroautophagy. Macroautophagy is a specific program by which cells break down intracellular organelles to eliminate them, and also to obtain nutrients. Thus, macroautophagy serves as an internal source of nutrients to support the survival of cancer cells. Our general hypothesis is that loss of VHL during RCC promotes loss of mir204 and derepression of specific targets, thus activating LC3B-dependent macroautophagy, which gives cancer cells access to intracellular nutrients that contribute to their survival and to tumor growth. This renders RCC VHL(-) cells addicted to macroautophagic activity, and thus subject to synthetic lethality when macroautophagy is inhibited by exogenous mir204, with cell death caused by starvation. In contrast, the presence of VHL stimulates expression of endogenous mir204 and leads to suppression of the macroautophagic program, rendering the cells insensitive to the activity of exogenous mir204. To test this hypothesis, we will define the roles of mir204 and LC3B-dependent macroautophagy in RCC tumor formation (Aim 1), define the roles of other specific mir204 targets in macroautophagy and tumor formation (Aim 2); identify the molecular mechanisms leading to the decrease in mir204 expression in RCC (Aim 3); and determine levels of mir204 targets and general autophagic regulators in human RCC tumor and normal kidney samples (Aim 4). Impact: Here we provide evidence for a previously unknown signaling pathway activated by the losses of VHL and miR204 in RCC, which regulates survival of cancer cells. Understanding this pathway will lay the groundwork for the development of novel therapeutic approaches for the treatment of malignant RCC. PUBLIC HEALTH RELEVANCE: Relevance to VA Statement Renal cancer is a serious health concern for military personnel, particularly those beyond 40 years of age, and for military veterans. Renal cancer accounts for 2% to 3% of all adult malignancies in the US, with 57,760 new cases and 12,980 deaths reported in 2009. The Defense Medical Epidemiology Database for 1995-2004 analyzed that the incidence of renal cancer specifically for military members after the 4th decade of life it is quite dramatically increased to 8.5 as compared to 1.5 cases per 100,000 person-years of the unadjusted incidence. In addition, renal cancer occurs almost twofold more frequently in males than in females and approximately 80% of military personnel are males. Cigarette smoking leads to a 47% increase in risk for renal cancer relative to nonsmokers, and the relative risk correlates positively with the number of cigarettes smoked per day. Smoking represents an important health issue for military personnel. It is currently estimated that 30% of active military personnel and 74.2% ( 0.7%) of veterans are smokers or have smoked at some point in there lives, as compared to 48.4% (0.5%) of those who never served.