核纤层蛋白 laminA/C 促进前列腺癌侵袭转移的分子机制及作为潜在治疗靶点的研究

Prostate cancer remains the second most common cause of cancer-related death in Western countries. A previous proteomics study suggested the nuclear membrane protein laminA/C to be a maker to discriminate low and high Gleason score tumors and to identify high risk cancers. To characterize its function in prostate cancer cells, we performed a detailed expression analysis in prostate cancer tissue and explored the consequences of down or up-regulation of laminA/C in prostate cancer cells. Our results confirm an increased laminA/C protein expression in high risk cancers and show association of expression with tumor cell formations at the invasion fronts of tumors and in invasion "spearheading" tumor cell clusters. In the prostate tumor cell lines LNCaP, DU145, and PC3 shRNA knockdown or overexpression of laminA/C resulted in inhibition or stimulation, respectively, of cell growth, colony formation, migration and invasion. Further mechanism studies suggested that the laminA/C-related malignant behavior is regulated through modulation of the phosphoinositide 3-kinase (PI3K)/AKT/PTEN signaling pathway. In this study, we found two interesting problems, first, the expression of laminA/C was heterogeneous with low immunostaining of central tumor areas and higher intensity staining at tumor boundary areas especially in subcapsular extensions and in a region of tumor growth into the adjacent lipoid tissue, second, survival of prostate cancer cell lines with stable knockdown laminA/C was dramatically reduced than that of benign prostatic hyperplasia epithial cell lines with stable knockdown laminA/C, on the other hand, apoptosis rate of them was increased compared with that of benign prostatic hyperplasia epithial cell lines. The mechanism of the heterogeneous expression pattern of lamninA/C is unknown till now. So we hypothesize that laminA/C is a characteristic biomarker to distinguish aggressive prostate cancer cell differentiation and a molecular target for treatment of invasive prostate cancer. We plan to establish and analyze benign basal epithelium, low-invasive or high-invasive prostate cancer cell lines and invasive prostate cancer animal models to unravel the undelaying mechanism. A candidate pathway that might be involved is epithelial to stromal transition (EMT). From this point of view, we will further explore laminA/C genomic differences or miroRNA regulation difference in the different benign and tumor models using DNA　sequence and molecular biology technology; At the same time, we will explore if laminA/C is related to biomarkers and pathway of EMT on the cell model by gene overexpression or knockdown techniques; At last, we will explore the laminA/C siRNA treatment effect in an invasive prostate cancer animal model. In summary, we will supply enough experimental datas to support our views for laminA/C as a new molecular biomaker in the progression of prostate cancer and potentially novel target for invasive prostate cancer therapeutics.

前列腺癌死亡率居男性癌症死亡率的第二位，高度异质性是其区别于其它肿瘤的重要特点。最近研究提出lamin A 可能是识别肿瘤分化和判断预后的新的肿瘤标志物。前期研究发现laminA/C 可增加前列腺癌细胞迁移及侵袭力；下调时可增加前列腺癌细胞的凋亡率；在前列腺癌组织中表达显著差异。目前，尚不知道lamin A/C表达差异性的意义及介导凋亡的机制。我们推测它可能是一种侵袭性前列腺癌的标志物及治疗靶标，可通过上皮间质转换机制调控癌细胞的迁移及侵袭。本项目拟采用免疫组织化学技术研究组织芯片中laminA/C与上皮及间质细胞标志物的关联性；建立laminA/C表达差异的细胞模型，研究上皮间质转换中信号通路及miRNA调控的差异；建立侵袭性动物模型，研究靶向前列腺癌中laminA/C 的疗效及机制。进而阐明laminA/C表达差异性的意义，揭示其可作为新的肿瘤标志物及治疗靶标提供可靠的实验依据。

本项目的研究重点在于评价lamin A/C是识别前列腺癌分化和判断预后的新的肿瘤标志物。Lamin A/C在Gleason分级不同的前列腺癌组织中表达具有显著差异性，推测它可能是一种侵袭性前列腺癌的标志物及治疗靶标，可通过上皮间质转换（epithelial to mesenchymal transition，EMT）机制调控癌细胞的迁移及侵袭。首先本研究采用免疫组织化学技术研究了不同Gleason分级的前列腺癌组织芯片中laminA/C 与6种EMT标志物（E-cadherin、β-catenin、vimentin、N-cadherin、slug、snail）的表达模式及关联性；并且利用免疫荧光技术在高侵袭力的前列腺癌细胞系（PC-3M-1E8）和低侵袭力的前列腺癌细胞系（PC-3M-2B4）中验证了这种表达模式。结果显示：LaminA/C的表达模式与EMT标志物的表达模式无明显相关性；然而laminA/C的过表达及敲除可显著改变6种EMT标志物的表达。其次，研究了laminA/C表达不相同的PC-3M-2B4和PC-3M-1E8的miRNA表达谱，共筛选出80个有意义上调的miRNA及97个有意义下调的miRNA。KEGG信号通路聚类分析提示，侵袭力差异的细胞系中存在生长因子信号通路、细胞黏附及代谢方式等明显差异，报告基因技术证实在PC-3M-1E8中明显下调的hsa-miR-146b-5p可直接作用于laminA/C的3’UTR，从而导致laminA/C的表达下调，可能是引起laminA/C表达差异的主要原因。进一步对laminA/C基因的深度测序中，识别出PC-3M-1E8中lamin A/C 的7号外显子区的有意突变位点 (p.L387LI)，推测它可能是引起laminA/C 表达差异的又一原因。最后，建立PC-3M-1E8裸鼠异体移植动物模型，局部注射靶向laminA/C siRNA，HE 染色结果提示laminA/C siRNA可引起肿瘤组织局部坏死，局部注射成功率受限于肿瘤瘤体的致密性。综合以上实验结果，得出结论lamin A/C可准确识别前列腺癌的分化，作为判断预后的新的肿瘤标志物，同时是EMT过程发生的根本，采用siRNA制剂靶向lamin A/C的局部肿瘤组织治疗策略有一定的局限性。