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。

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.