Mitochondria in Prostate Cancer Diversity

 描述(申请人提供)：雄激素受体(AR)在前列腺癌的正常发育、前列腺癌的发生以及前列腺癌向晚期转移性疾病的进展中起着重要的作用。传统的观点认为，AR仅定位于细胞核，而核AR调节对前列腺癌发展至关重要的基因。这是真的。然而，我们在线粒体中展示了一种以前未被认识的AR功能。我们发现，AR 1)直接定位于线粒体，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。我们的研究表明：1)AR定位于原代前列腺组织和细胞系的线粒体，2)AR被输入到分离的线粒体中，3)AR含有一个线粒体定位信号(MLS)，能够靶向外源蛋白，如绿色荧光蛋白，进入线粒体。AR间接调控核DNA(NDNA)编码的线粒体氧化磷酸化(MtOXPHOS)亚基的表达，包括NDUFB8(复合体I)、SDHB(复合体II)、UQCRC2(复合体III)、COXII亚基(复合体IV)和ATP5A(复合体V)。AR还下调控制线粒体DNA(MtDNA)含量的TFAM、GFM1和GFM2基因。与此一致的是，在表达AR的PC3-AR前列腺细胞中，mtDNA含量和mtDNA编码的COX II蛋白的表达显著减少。值得注意的是，我们证明非裔美国人(AA)的前列腺癌(AA)的mtDNA含量比高加索裔美国人(CA)的肿瘤低6倍。AA组正常前列腺组织线粒体DNA含量也低于CA组。为了确定前列腺癌多样性的潜在线粒体基础，我们对6000多个AA和33,000个CA的变异进行了全面的、基于种族的生物信息学分析，发现在AA的AR基因中，错义变异位于两个结构域：包含MLS的N-末端结构域和DNA结合域。值得注意的是，CA中的错义突变仅在含有AR核定位信号(NLS)的铰链区被发现。与野生型相比，AR变异体S598G在PC3细胞中的表达显著降低了控制线粒体DNA含量的TFAM的表达。我们假设，AR错义变体/突变体仅存在于AA中，导致线粒体功能的“增加”或“丧失”，从而导致AA中前列腺癌的多样性。目的：探讨AR错义突变和线粒体DNA含量对AA和CA前列腺癌转移和复发的预后意义。目的：评价线粒体AR错义变异体/突变体对线粒体功能的直接调节作用，从而影响mtOXPHOS超复合体的组成、组织、稳定性、活性和细胞凋亡。目的：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。目的：利用小鼠异种移植模型，探讨线粒体和细胞核中AA和CA特异性AR错义变异体/突变体在前列腺癌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.