PSMA激活Ras/Raf-1/MEK/ERK通路上调癌干因子SALL4促进前列腺癌快速增殖的机制研究

PSMA is a high specificity biomarker located in prostate cancer. Previous studies have showed the expression of PSMA is correlated with malignance of prostate cancer. But until now, the concrete mechanism is not clear. Our preliminary work had observed that silencing the PSMA can inhibit the rapid proliferation and metastasis of prostate cancer in vitro. In addition, we also found that the folate hydrolase activity of PSMA and the ERK pathway had involved in this processes. And this process is accompanied with the inhibition of calcium inflow. Sum up all the data and researches, we propose that the folate hydrolase activity of PSMA may activate Ras/Raf-1/MEK/ERK pathway by mediating the inflow of calcium. At the meantime, our team also detected the new transcription factor-SALL4 associated with tumor proliferation and found that the expression of P-ERK was in accordance with SALL4 expression. Together with an AP-1 binding site existed in the SALL4 gene promoter, we further conclude that P-ERK can activate the AP-1 factor in the cytoplasm and then the AP-1 enter into the nucleus, binding to the SALL4 gene promoter and promoting the expression of SALL4. As a result, the tumor can proliferate and metastasize rapidly. Our project is to knock down the folate hydrolase activity of PSMA by technique of mutating the N638A of PSMA on the basis of our previous studies, detect the cell bio-behaviors. In order to explore the mechanism of PSMA up-regulating SALL4, we put emphasis on detecting inflow of calcium, the expression of the Ras/Raf-1/MEK/ERK pathway and the nucleus factor SALL4. Finally, we will testify our hypothesis in vivo.

PSMA是前列腺癌高特异性瘤标，以往研究表明其与前列腺癌恶性行为密切相关，但具体机制不清。我们前期实验证实沉默PSMA可抑制前列腺癌快速增殖，并与其叶酸水解酶活性及下游分子ERK密切相关，同时伴有钙内流受抑，这提示PSMA的叶酸水解酶活性可介导钙内流激活细胞内Ras/Raf-1/MEK/ERK途径。同时我们检测基因启动子具有AP-1位点的癌干因子SALL4，发现SALL4与磷酸化ERK的表达具有一致性，因此我们推测：PSMA激活ERK通路后,可活化AP-1并使其进入胞核启动SALL4基因的表达,从而促进前列腺癌快速增殖。本项目拟在前期实验的基础上,利用定向诱变的技术使PSMA叶酸水解酶功能失活，检测前列腺癌细胞生物学行为变化；通过检测钙内流、Ras/Raf-1/MEK/ERK通路及其下游重要因子SALL4阐明前列腺癌细胞中PSMA调控SALL4的具体机制，并结合动物实验验证我们的猜测。

前列腺特异性膜抗原（PSMA）不单是一种高特异性瘤标，其在前列腺癌细胞的增殖、转移和侵袭中还具有促进作用，但其分子机理目前还不清楚。本项目通过RNAi技术沉默LNCAP细胞中PSMA基因后，在有/无LY294002条件下检测细胞功能和Akt信号通路的结果，发现与对照组相比，处理组p-AKt（Ser473）表达量显著降低，细胞功能检测发现细胞的增殖、转移及存活能力均下降。最后证实PSMA可以通过PI3K/AKT通路对前列腺癌细胞的增殖、转移、存活进行调控。另外，过表达PSMA和构建定向诱变移除N638A糖基的突变PSMA重组质粒后，我们发现过表达PSMA后可激活Ras/Raf-1/MEK/ERK相关信号通路，而定向突变N638叶酸水解酶糖基化位点后未见Ras/Raf-1/MEK/ERK信号通路活化。同时，我们利用免疫组化技术检测SALL4在前列腺癌中的表达，我们发现SALL4蛋白表达水平与Gleason评分、前列腺癌临床分期、预后及组织PSA表达密切相关，而与患者年龄、术前PSA、前列腺体积及组织雄激素受体表达无明显相关性。此外，抑制SHARPIN、加入NF-κB通路抑制剂、抑制Survivn及Livin均可抑制前列腺癌细胞增殖、迁移及侵袭能力，促进细胞凋亡，同时运用Western blot及RT-PCR技术，在体内及体外水平证实了SHARPIN通过NF-κB信号通路及下游的Survivin及livin分子促进前列腺癌的增殖、转移。另外，过表达实验也进一步证明SHARPIN可增强前列腺癌的细胞增殖及侵袭能力，抑制凋亡，其机制可能与NF-κB/.ERK/Akt信号通路及其下游的Bax、Bcl-2、Survivin、Caspase-3分子改变有关。最后，我们构建前列腺癌CRPC细胞LNCAP-AI，通过抑制SHARPIN、加入坏死性凋亡诱导剂TNF-α+ Z-VAD和抑制剂Nec-1，通过免疫荧光技术检测坏死性凋亡标记蛋白Rip1和Rip3蛋白表达水平，我们发现SHARPIN可能参与到LNCAP-AI细胞的发生发展过程，沉默SHARPIN 可以激活Rip1及其下游因子Rip3，从而使LNCaP-AI 细胞的坏死性凋亡比例升高。这些研究将为理解PSMA、SALL4及SHARPIN的功能及其调控前列腺癌增殖、存活、转移的分子途径提供理论依据，为前列腺癌治疗提供干预的新靶点。