Mitochondria in Prostate Cancer Diversity

 描述（由申请人提供）：雄激素受体（AR）在前列腺的正常发育、前列腺癌发生和前列腺癌进展为晚期转移性疾病中起重要作用。传统的观点认为，AR只定位于细胞核，而细胞核AR调节前列腺癌发展所必需的基因。这倒是真的。然而，我们证明了一个以前未被认识到的功能，AR在线粒体。我们已经发现，AR 1）直接定位于线粒体和2）间接转录调节核基因，其产物定位于线粒体并执行线粒体功能。我们的研究揭示了i）AR定位于原代前列腺组织和细胞系中的线粒体中，ii）AR被输入到分离的线粒体中，以及iii）AR含有能够将外源蛋白（例如绿色荧光蛋白）靶向线粒体的线粒体定位信号（MLS）。AR间接控制多种核DNA（nDNA）编码的线粒体氧化磷酸化（mtOXPHOS）亚基的表达，包括NDUFB 8（复合物I）、SDHB（复合物II）、UQCRC 2（复合物III）、COXII亚基（复合物IV）和ATP 5A（复合物V）。AR还下调TFAM，GFM 1和GFM 2基因，这些基因控制线粒体DNA（mtDNA）含量。与此一致，在表达AR的PC 3-AR前列腺细胞中，mtDNA含量和mtDNA编码的考克斯II蛋白的表达显著降低。值得注意的是，我们证明了非裔美国人（AA）前列腺肿瘤中的mtDNA含量比白人美国人（CA）肿瘤中的mtDNA含量低6倍以上。正常前列腺线粒体DNA含量AA组低于CA组。为了确定前列腺癌多样性的潜在线粒体基础，我们对超过6000个AA和33，000个CA中的变体进行了全面的基于种族的生物信息学分析，并在AA的AR基因中发现了位于两个结构域的错义变体：包含MLS的N末端结构域和DNA结合结构域。值得注意的是，CA中的错义突变仅在含有AR的核定位信号（NLS）的铰链结构域中发现。AR变体S598 G在PC 3细胞中的表达比野生型减少更多TFAM的表达，TFAM控制mtDNA含量。我们假设AR错义变体/突变体仅存在于AA中，有助于线粒体功能的“获得”或“丧失”，从而导致AA中的前列腺癌多样性。目标1：确定AR错义变体/突变体和mtDNA含量对AA和CA中前列腺癌转移和复发的预后意义。目标2：评估线粒体AR错义变体/突变体作为线粒体功能直接调节剂的意义，这些线粒体功能影响mtOXPHOS超复合物的组成、组织、稳定性和活性以及细胞凋亡。目的3：评价核AR错义变异体/突变体作为线粒体功能间接调节因子的意义。目标4：使用小鼠异种移植模型来建立线粒体和核AA和CA特异性AR错义变体/突变体在前列腺肿瘤发生和转移中的意义。

 DESCRIPTION (provided by applicant): The androgen receptor (AR) plays an important role in normal development of the prostate gland, in prostate carcinogenesis, and in the progression of prostate cancer to advanced metastatic disease. Traditional thinking is that AR localizes exclusively to the nucleus and that nuclear AR regulates genes that are essential to prostate cancer development. This is true. However, we demonstrate a previously unrecognized function of AR in mitochondria. We have discovered that AR 1) directly localizes into the mitochondria and 2) indirectly transcriptionally regulates nuclear genes whose products localize into mitochondria and perform mitochondrial functions. Our studies reveal that i) AR localizes into mitochondria in primary prostate tissues and cell lines, ii) AR is imported into isolated mitochondria, and iii) AR contains a mitochondrial localization signal (MLS) capable of targeting foreign proteins, such as green fluorescent protein, into mitochondria. Indirectly, AR controls expression of a variety of nuclear DNA (nDNA)-encoded mitochondrial oxidative phosphorylation (mtOXPHOS) subunits, including NDUFB8 (Complex I), SDHB (Complex II), UQCRC2 (Complex III), COXII subunit (Complex IV), and ATP5A (Complex V). AR also down-regulates the TFAM, GFM1, and GFM2 genes, which control mitochondrial DNA (mtDNA) content. Consistent with this, the mtDNA content and the expression of mtDNA-encoded COX II protein is significantly reduced in PC3-AR prostate cells expressing AR. Notably, we demonstrate that the mtDNA content in prostate tumors of African-Americans (AA) is >6 times less than in tumors of Caucasian-Americans (CA). mtDNA content was also lower in normal prostates of AA than CA. To identify the underlying mitochondrial basis of prostate cancer diversity, we conducted comprehensive, race-based bioinformatics analyses of variants in more than 6000 AA and 33,000 CA and discovered, in the AR gene of AA, missense variants located in two domains: the N-terminal domain containing the MLS and the DNA-binding domain. Of note, missense mutations in CA were found only in the hinge domain containing the nuclear localization signal (NLS) of AR. Expression of AR variant S598G in PC3 cells reduced more than the wild type the expression of TFAM, which controls mtDNA content. We hypothesize that AR missense variants/mutants present, solely in AA, contribute to the "gain" or "loss" of mitochondrial function and thereby to prostate cancer diversity in AA. AIM 1: Determine the prognostic significance of AR missense variants/mutants and mtDNA content on prostate cancer metastasis and reoccurrence in AA and CA. AIM 2: Evaluate the significance of mitochondrial AR missense variants/mutants as direct regulators of mitochondrial functions that affect composition, organization, stability, and activity of mtOXPHOS super-complexes and apoptosis. AIM 3: Evaluate the significance of nuclear AR missense variants/mutants as indirect regulators of mitochondrial function. AIM 4: Use mouse xenograft model to establish the significance of mitochondrial and nuclear AA- and CA-specific AR missense variants/mutants on prostate tumorigenesis and metastasis.