AKAP12通过与PIP3的相互作用调控PI3K/Akt信号通路与其抑制结直肠癌侵袭转移的研究

In our previous study, A kinase anchor protein 12 (AKAP12) decreasing expression induced by hypermethylation was detected in the colorectal cancer. Re-expression of AKAP12 could inhibit colorectal cells growth, proliferation, invasion, migration and transformation capacity and increase the apoptosis. DNA microarray results showed the difference expression of the genes, which were related with PI3K/Akt pathway, in the fibroblast cells between the AKAP12 knockout mice and the wildtype. We also found the interaction between the AKAP12 and PIP3, and AKAP12 could inhibit the Akt phosphorylation. In this study, we will designate AKAP12 as an antagonist of the PI3K/AKT signaling pathway and identify its molecular mechanism in the inhibition of colorectal cancer metastasis. PCR-based mutagenesis, GST pulldown assay and coimmunoprecipitation will be employed to locate the binding fragment of AKAP12 with PIP3, and further confirm this AKAP12-PIP3 interaction could suppress the PIP3-induced AKT activation. The role of AKAP12 in cell growth, apoptosis, invasion and metastasis will be investigated by overexpression of AKAP12 in the PTEN deficient colorectal cancer cell line. This study will clarify the inhibitory mechanism of AKAP12 on the colorectal cancer metastasis from a PI3K/Akt pathway perspective. Moreover, the tumor suppressor function of AKAP12 in the PI3K pathway will also provide colorectal cancer with a promising treatment target.

我们前期工作中发现AKAP12在结直肠癌中存在启动子甲基化导致的表达下降，且发现其表达能抑制肿瘤转移，诱导凋亡。预实验中用芯片分析AKAP12基因敲除的老鼠中分离纤维母细胞与野生型老鼠的纤维母细胞之间的基因差异，显示AKAP12与PI3K/Akt通路密切相关，发现AKAP12与PIP3有相互作用并抑制Akt磷酸化。本课题将研究AKAP12通过拮抗PI3K/Akt信号抑制结直肠癌转移及其机制，通过基于PCR的突变技术、GST pulldown和免疫沉淀鉴定AKAP12与PIP3结合的片段，且证明这两者的相互作用可抑制PIP3对Akt的激活；通过PTEN缺失的结肠癌细胞中过表达AKAP12研究其对PI3K通路抑制和对癌细胞的生长、凋亡、侵袭和转移的影响。本项目不仅可从P13K/Akt通路角度阐明AKAP12抑制结直肠癌转移的机制，AKAP12抑制PI3K通路也为结肠腺癌的治疗提供新的靶点。

AKAP12 / Gravin（A激酶锚定蛋白12）在多种肿瘤中发挥着肿瘤抑制作用。但HDAC3和AKAP12之间的相关性及潜在机制仍不清楚。采用免疫组织化学，qRT-PCR和Western blot方法检测96例结直肠癌及癌旁组织以及结肠癌SW480细胞中HDAC3和AKAP12的表达。用细胞计数试剂盒-8（CCK-8）测定法，集落形成测定法，流式细胞术，细胞周期和transwell试验检测HDAC3和AKAP12对CRC细胞的增殖，凋亡和转移的影响。在69例（79.2％）肿瘤组织中检测到AKAP12表达减少或丧失，而HDAC3在96例肿瘤组织中有50例（52.1％）表达上调。 在HDAC3抑制剂曲古抑菌素A（TSA）和RGFP966处理后，AKAP12的mRNA和蛋白质水平显著增加。 从机理上看，HDAC3在AKAP12的内含子1区域内直接结合被或是抑制AKAP12表达的机制之一。 本研究发现AKAP12沉默或AKAP12 / HDAC3共沉默，有利于CRC细胞的增殖，集落形成，细胞周期和迁移，单独si-HDAC3达到相反的效果。 AKAP12敲除后抗凋亡Bcl-2蛋白的表达升高，PI3K / AKT信号传导的活性增加，都有利于AKAP12调控的集落形成和迁移的原因之一。这些结果表明，AKAP12可能是结合HDAC3治疗CRC的潜在预后指标和治疗靶点。