(PQ5) Relevance of VDAC2 heterogeneity for hepatic tumor growth and targeting

项目摘要/摘要 肝癌在世界范围内的发病率越来越高，已成为癌症相关死亡的第二大原因； 然而，目前知识中的一个重要缺口仍然是分子指纹和组织 可能允许其特异性靶向的肝癌/肝癌细胞。我们发现，表达水平 在线粒体凋亡途径中起中心作用的两种蛋白质VDAC2和Bak在正常人群中水平较低 肝细胞，并在肝肿瘤进展过程中增加。此外，我们还发现，亲- 凋亡的BH3-Only Bcl-2家族蛋白，Bid更有效地诱导线粒体凋亡 与正常肝相比，肝癌的发病率更高。这种差异可以通过VDAC2静默来消除 VDAC2在正常肝细胞中的表达，但前提是Bak没有被敲除。这个 人类数据库支持VDAC2-Bak异质性的潜在医学意义 VDAC2和Bak蛋白在肝癌中的表达我们还记录了亚细胞和细胞- Tbid-Bak途径中的细胞差异。最后，我们至少在细胞培养中证明了 肝癌细胞可通过TBID-Bak途径选择性杀伤(使用其激活剂和 其抑制物Mcl-1，而正常肝细胞则幸免于难。我们在这里，假设 肝脏中VDAC2和/或Bak丰度的异质性对肝癌/ 肝癌(1)生长和(2)Mcl-1抑制剂药物与细胞联合靶向 渗透性碳氢化合物固定的BID BH3多肽。细胞内的异质性被认为是(1) 单个线粒体，(2)在单个细胞中的每种细胞类型中，以及(3)在不同的细胞类型中。为了测试 我们的假设我们已经建立了基因靶向、小鼠肿瘤发生和 生化和显微成像方法。在这项研究中，我们将重点放在(1)评估异质性 VDAC2、Bak和Bak介导的OMM在肝癌细胞和正常肝细胞中的通透性 它们对VDAC2表达和线粒体融合的依赖性；(2)确定是否增加 Bak上调VDAC2表达对肝癌/肝癌进展的影响；(3)检测 如果用Mcl-1抑制剂(S-63845)和细胞通透性碳氢化合物固定的BID处理BAK激活 多肽可用于在不损害正常肝细胞的情况下杀死肝源性肿瘤；以及(4) 肝肿瘤细胞中VDAC2依赖蛋白的测定及其异质性评价 肝脏及其与肝癌/肝癌生长的相关性。通过解决这些问题，我们的研究将 为线粒体异质性在肝脏肿瘤发生中的作用提供线索并测试一种新的 肿瘤选择性靶向方法。

PROJECT SUMMARY/ABSTRACT Liver cancers with increasing frequency in the world have become the second cause of cancer related death; however an important gap in current knowledge remains the molecular fingerprint and organization of hepatocarcinoma/hepatoma cells which might allow their specific targeting. We found that the expression level of two proteins, VDAC2 and Bak, that are central to a mitochondrial apoptosis pathway, is low in normal hepatocytes and is increased during hepatic tumor progression. Furthermore, we also found that the pro- apoptotic BH3-only Bcl-2 family protein, Bid induces mitochondrial apoptosis more efficiently in hepatocarcinoma than in normal liver. This difference can be eliminated by VDAC2 silencing in hepatocarcinoma cells, and by VDAC2 expression in normal hepatocytes but only if Bak isn’t knocked out. The potential medical significance of the VDAC2-Bak heterogeneity is supported by human databases that show upregulation of VDAC2 and Bak proteins in liver cancer. We have also documented both subcellular and cell- to-cell differences in the tBid-Bak pathway. Finally, we have shown at least in cell culture, that hepatocarcinoma cells can be selectively killed through the tBid-Bak pathway (using its activators and suppressors of its inhibitor, Mcl-1 while normal hepatocytes are spared. We here, postulate that the heterogeneity in VDAC2 and/or Bak abundance in the liver are important for hepatoma/ hepatocarcinoma (1) growth and (2) targeting by the combination of an Mcl-1 inhibitor drug and a cell permeable hydrocarbon stapled Bid BH3 peptide. Heterogeneity is considered (1) within the cells among individual mitochondria, (2) in each cell type among single cells, and (3) among the different cell types. To test our hypothesis we have established a combination of genetic targeting, tumorigenesis in mouse, and biochemical and microscopic imaging approaches. In the study we will focus on (1) assessing heterogeneity of VDAC2, Bak and Bak-mediated OMM permeabilization in hepatocarcinoma cells and normal hepatocytes and their dependence on VDAC2 expression and mitochondrial fusion; (2) determining whether increased expression of VDAC2 through upregulation of Bak affects hepatoma/hepatocarcinoma progression; (3) testing if activation of Bak by treatment with Mcl-1 inhibitor (S-63845) and a cell permeable hydrocarbon stapled Bid peptide can be used to kill hepatocyte-derived tumors without damaging normal hepatocytes; and (4) determining additional VDAC2-dependent proteins in hepatic tumor cells and to evaluate their heterogeneity in the liver and their relevance for hepatoma/hepatocarcinoma growth. By addressing these points, our study will provide clues to the contribution of mitochondrial heterogeneity to hepatic tumorigenesis and test a novel tumor-selective targeting approach.