CXCL13: a mediator of prostate cancer progression

 描述(申请人提供)：前列腺癌是美国男性癌症相关死亡的第二大原因。大量证据表明，蛋白激酶C epsilon(PKC)与癌症进展有关。PKC在包括前列腺癌在内的上皮性癌症中显著上调，这可能反映了它参与了疾病的病因和进展。我们发现，前列腺特异性PKC转基因小鼠(PB-PKC)发生癌前病变(PIN)，值得注意的是，它们在Pten单倍体缺陷背景下发生侵袭性前列腺癌。理解这种协同性的分子基础是高度相关的，因为PI3K途径的负调节因子肿瘤抑制因子Pten的缺失是前列腺癌的常见变化。我们建立了大量的细胞模型，概括了PKC的过表达和Pten的丢失，这些模型在体外显示出增强的生长、运动和侵袭力，并获得了在裸鼠体内的致瘤潜力。此外，基因图谱和生物信息学分析表明，PKC过表达/Pten缺失细胞表现出转移性前列腺癌特有的基因表达模式。我们发现，在这个微阵列分析中最受欢迎的是趋化因子CXCL13。值得注意的是，PKC与Pten缺失协同作用，强烈诱导CXCL13mRNA的表达和释放，而CXCL13或其受体(CXCR5)的RNAi缺失会削弱PKC高表达/Pten缺陷细胞的增殖和迁移能力。初步数据还表明，PKC和PI3K是CXCR5的下游效应因子，这表明可能存在一个正向扩增环，可能起到恶性循环的作用。在特定的目标1中，我们将使用可诱导沉默的方法来确定CXCL13：CXCR5轴在前列腺癌发生中的相关性。基质CXCL13的潜在贡献将通过免疫活性小鼠和参与CXCL13产生的淋巴细胞的耗尽来检验。此外，基因敲除的CXCL13和CXCR5小鼠将与PB-PKC小鼠杂交，作为一种方法来评估CXCL13：CXCR5轴在前列腺病变发生和关键有丝分裂/存活通路激活中的需求。在特定的目标2中，我们将研究CXCL13：CXCR5在前列腺腔内注射转移中的作用，并评估该途径在转移到骨转移中的作用。机制研究将继续探索Rac，一种小的G蛋白，及其交换因子(Rac-GEF)在由PKC-PI3K-CXCL13-CXCR5途径驱动的运动/侵袭表型中的潜在相关性。在具体目标3中，我们将重点研究PKC和PI3K诱导CXCL13产生的机制，具体剖析Akt、ERK、STAT3、NF-B和特定的p110 PI3K在控制CXCL13产生中的作用。我们还将研究PKC作为一个潜在的CXCR5下游效应因子。最后，在特定的目标4中，我们将使用大量的前列腺癌标本建立PKC/Pten-CXCL13在人类前列腺癌中的相关性，并评估与疾病进展和复发的相关性。除了机制上的影响，拟议的研究可能会对个性化前列腺癌治疗的预后和治疗产生重大影响。

 DESCRIPTION (provided by applicant): Prostate cancer is the second leading cause of cancer-related deaths among men in the US. A large body of evidence links protein kinase C epsilon (PKC) to cancer progression. PKC is markedly up-regulated in epithelial cancers, including prostate cancer, potentially reflecting its involvement in the etiology and progression o the disease. We found that prostate-specific PKC transgenic mice (PB-PKC) develop preneoplastic lesions (PINs), and remarkably, they develop invasive prostatic adenocarcinomas in a Pten haplodeficient background. Understanding the molecular basis of this cooperativity is highly relevant, as loss of the tumor suppressor Pten, a negative regulator of the PI3K pathway, is a common alteration of prostate cancer. We generated a number of cellular models recapitulating PKC overexpression and Pten loss, which display enhanced growth, motility, and invasiveness in vitro, and acquire tumorigenic potential in nude mice. Moreover, gene profiling and bioinformatics analysis revealed that PKC overexpressing/Pten depleted cells display a pattern of gene expression distinctive of human metastatic prostate cancer. We found that the "top hit" in this microarray analysis is the chemokine CXCL13. Notably, PKC cooperates with Pten loss to strongly induce CXCL13 mRNA levels and its release, and RNAi depletion of CXCL13 or its receptor (CXCR5) impairs the ability of PKC overexpressing/Pten-deficient cells to proliferate and migrate. Preliminary data also suggest that PKC and PI3K are downstream effectors of CXCR5, suggesting a positive amplification loop that may act as a vicious cycle. In Specific Aim 1, we will establish the relevance of the CXCL13:CXCR5 axis in prostate tumorigenesis using an inducible silencing approach. A potential contribution of stromal CXCL13 will be examined using immunocompetent mice and depletion of lymphocytes involved in CXCL13 production. In addition, knock-out CXCL13 and CXCR5 mice will be crossed with PB- PKC mice as an approach to assess the requirement of the CXCL13:CXCR5 axis in the development of prostate lesions and the activation of key mitogenic/survival pathways. In Specific Aim 2, we will examine the role of CXCL13:CXCR5 in metastasis using intraprostatic injections and also assess the role of this pathway in metastatic dissemination to bone. Mechanistic studies will be pursued to explore the potential relevance of Rac, a small G-protein, and its exchange factors (Rac-GEFs) in the motile/invasive phenotype driven by the PKC-PI3K-CXCL13-CXCR5 pathway. In Specific Aim 3, we will focus on the mechanisms involved in CXCL13 induction by PKC and PI3K, specifically dissecting the roles of Akt, Erk, Stat3, NF-B and specific p110 PI3Ks in controlling CXCL13 production. We will also investigate PKC as a potential CXCR5 downstream effector. Lastly, in Specific Aim 4 we will establish the relevance of the PKC/Pten-CXCL13 association in human prostate tumors using a large collection of prostate cancer specimens, and assess correlations with disease progression and recurrence. In addition to the mechanistic implications, the proposed studies may have major prognostic and therapeutic impact for personalized prostate cancer treatment.