PDZK1通过调控PDGFRβ通路影响肾癌发生发展及分子机制的研究

Renal cell carcinoma (RCC) comprises approximately 3% of all cancers, the seventh most common cancer in men and the ninth in women. The main histologic subtype, clear cell RCC (ccRCC), accounts for 70-80% of all cases. Localized RCC can be successfully cured by surgery in most cases. Unfortunately, about 30% of patients relapse after nephrectomy or present at diagnosis with metastatic disease (mRCC). Despite the therapeutic improvements made in the past decade, mRCC is still a largely incurable disease. The currently available therapeutic options for mRCC include drugs targeting tyrosine kinase inhibitors (TKIs) and inhibitors of the mammalian target of rapamycin (mTOR) serine-threonine kinase. Unfortunately, since renal cell cancer is an heterogenous disease based on its morphological and molecular features, 20 to 30% of mRCC patients do not respond at all to targeted agents while the remaining ultimately progress after an initial benefit. There is current need for molecular biomarkers of RCC for the development of a personalized treatment and predictive approach for patient stratification. Unfortunately, no correlation was found so far among biomarker expression/alteration and clinical response to single TKIs and mTOR inhibitors. In addition, there is no clear-cut evidence that the ccRCC or the non-ccRCC histotypes respond differently to specific target therapies. Taken together, there is a great need to develop the molecular mechanism of cancerogensis and progression of ccRCC, to identify new diagnostic and prognostic markers, as well as developing novel therapeutic strategies for treatment of advanced RCC..We recently reported that PDZK1 level was significantly decreased in clear cell renal cell carcinoma (ccRCC) cells, and PDZK1 could predict poor clinical outcome in patients with ccRCC. However, the functional significance of PDZK1 in renal neoplasms has not been defined, and therefore its potential prognostic and therapeutic significance in RCC remains unknown. The results of our preliminary studies suggested that PDZK1 could bind with the carboxyl terminus of PDGFRβ and rendered PDGFRβ phosphorylation. Also PDZK1 could inhibit the proliferation and invasion of ccRCC cells. Thus we propose our hypothesis: By direct interaction with the carboxyl terminus of PDGFRβ, PDZK1 block PDGFRβ phosphorylation and its downstream signaling activation. Thus abnormally low expression of PDZK1 in ccRCC enhances proliferation and invasion ability of cancer cells by overactivation of PDGFRβ and its downstream signalings. In this study, we will further confirm the interaction of PDZK1 and PDGFRβ with a variety of methods in ccRCC cells, including GST pull dwon, overlay, co-immunoprecipitation, and conforcal microcopy, etc., and will further investigate the effects of this interaction on the PDGFRβ signalings, and the proliferation and invasion of renal cancer cells, and drug sensitivity of PDZK1 on RCC cells treatment with multi-kinase inhibitors..Taken together, we set out to investigate the biological and clinical relevance of PDZK1 and its antitumorigenic properties in RCC, aiming to develop better treatments for this cancer in this study. PDZK1 will be a promising candidate to predict the clinical outcome in patients with RCC and to select patient subgroups, which might benefit from a more personalized medicine. To translate these findings into a clinically relevant tool, further prospective studies in larger cohorts are needed and should be a priority.

靶向药物是治疗转移性肾癌的一线药物，约30％的患者对该治疗无效，亟需加深对肾癌发生发展机制的研究，发现新的药靶和预测靶向药物疗效的标志物。我们最近报道PDZK1可评价肾癌预后，但其在肾癌发生发展中的作用及可否预测靶向治疗效果尚不清晰。我们初步发现PDZK1可抑制肾癌增殖迁移并与PDGFRβ结合抑制受体活化。据此推测肾癌中PDZK1下调激活PDGFRβ通路，导致肾癌的发生发展。本申请拟在分子、细胞、动物、临床等多个层次研究PDZK1与PDGFRβ的相互作用、对PDGFRβ通路的调控机制及对肾癌细胞增殖和侵袭转移的影响、对肾癌靶向药物敏感性的影响。本研究将揭示PDGFRβ通路调控的新机制，阐明PDZK1在肾癌发生发展中的作用机理。该结果将对前述PDZK1作为肾癌预后分子标志物的新发现提供重要的理论依据，也为PDZK1可否用于预测肾癌靶向药物疗效提供实验证据。本研究具有重要的理论价值和应用前景。

肾癌也称肾细胞癌（Renal Cell Carcinoma，RCC），占所有成人恶性肿瘤的 2-3％，其中 70-80%为肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)。原位 RCC 在大多数情况下可以通过手术成功治愈，然而文献报道约 30％的患者在肾切除术后出现复发或转移，同时肾癌起病隐匿，25-30%的患者首诊时就已经发生转移，属于转移性肾癌（metastatic renal cell carcinoma，mRCC）。mRCC对常规放疗、化疗均不敏感，其中位生存期仅为 13 个月，5 年存活率不足10%。近年研究发现VHL缺氧诱导因子-缺氧反应基因通路（VHL-HIF-HRG 通路）在肾癌发生发展过程中起极为重要的作用。70%以上的ccRCC中存在VHL失活，HIF-α表达水平显著增加，促使HIF-α与HIF-β形成活化的 HIF 二聚体，继而与缺氧反应基因（HRG）启动子区的缺氧反应元件结合，转录激活其下游的VEGF和PDGF等基因的表达，激活VEGFR、PDGFR等受体及其下游信号转导系统，如PI3K/Akt/mTOR和 Ras/Raf/MEK/ERK等通路，产生促进血管生成和细胞增殖和细胞迁移等作用，导致RCC的发生与发展。我们的研究发现，PDZK1可以通过其PDZ结构域与PDGFRβ羧基末端相互作用，从而抑制PDGFRβ磷酸化及其活性，进而抑制ERK通路的激活。此外，我们发现PDZK1对肾癌细胞增殖、克隆形成、细胞周期的影响是经PDZK1与PDGFRβ相互作用介导而实现的。PDZK1通过与PDGFRβ相互作用可抑制ERK通路的激活，从而抑制肾癌细胞的增殖。对肾癌组织样本的分析发现PDZK1在分期、预后差的患者肾癌组织中表达减少，同时磷酸化PDGFRβ明显增加。GSEA结果表明PDGFRβ通路激活、细胞增殖、ERK通路激活相关基因富集在PDZK1低表达肾癌组织中，说明PDZK1对肾癌细胞的调控是由于其抑制PDGFRβ磷酸化进而抑制ERK通路激活引起的。该研究结果不仅有助于阐明PDGFRβ在细胞内表达调控的分子机制，明确PDZK1在肾癌细胞增殖中的作用，同时也为寻找新型肾癌治疗药物的靶标提供一定的理论依据。