Targeting STK32C suppresses colon cancer progression with combinatorial potential with 5-fluoruracil via HSP90 and PI3K/AKT/mTOR signaling axis

Background: In this work, the oncogenic pathogenesis of STK32C was explored in colorectal cancer cells in association with HSP90 and PI3K/AKT/mTOR signaling. Methods: TCGA analysis, tissue microarray, next generation sequencing analysis, RNA interference, wound-healing, transwell, MTT, clonogenic, TUNEL and flow cytometry assays, RT-qPCR and Western blotting were employed in HCT116 and SW620 cells transfected with STK32C depletion or overexpression plasmid. Nude mouse tumor model was used to confirm in vitro data. Close interaction between STK32C and HSP90 was investigated by Homology analysis, Co-immunoprecipitation, Coomassie blue staining, GST pulldown assay and immunofluorescence. CompuSyn and SynergyFinder softwares were employed for synergy analysis with 5-Fu Results: STK32C was highly expressed in HCT116, HT29, SW480, and SW620 cells compared to CCD-18Co fibroblasts with poor prognosis. STK32C depletion exerted cytotoxic, anti-migratory, and anti-invasive effects, inhibited the expression of pro-PARP, pro-Caspase-3, PI3K, p-AKT, p-mTOR, HSP90, and Bcl-2 and increased Bax, sub-G1 population, Annexin V and TUNEL-positive cells in HCT116 and SW620 cells. Also, STK32C depletion enhanced the antitumor activity of 5-FU in HCT116 cells. STK32C binds directly to N-terminal domain of HSP90 by homology, colocalization and binding. Consistently, HSP90 N-terminal inhibitor Ganetespib reduced STK32C and p-AKT1, while the HSP90 C-terminal inhibitor EGCG or AKT inhibitor LY294002 did not affect STK32C. Furthermore, STK32C depletion reduced the growth of HCT116 cells in BALB/c mice with decreased expression of STK32C, HSP90, PCNA, and AKT and activation of caspase 3. Conclusions: Targeting STK32C inhibits colorectal cancer progression with combinatorial potential with 5FU via HSP90 and PI3K/AKT/mTOR signaling.背景：本研究以结直肠癌细胞为研究对象，探讨STK32C(STK32C)的致癌发病机制及其与热休克蛋白90(HSP90)、磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/mTOR)信号通路的关联。 方法：本研究采用TCGA分析(TCGA analysis)、组织微阵列(tissue microarray)、下一代测序分析(next generation sequencing analysis)、RNA干扰(RNA interference)、划痕愈合实验(wound-healing assay)、Transwell小室实验(transwell assay)、MTT比色法(MTT assay)、克隆形成实验(clonogenic assay)、TUNEL测定法(TUNEL assay)、流式细胞术(flow cytometry)、实时定量聚合酶链反应(RT-qPCR)及蛋白质印迹法(Western blotting)，对转染STK32C敲低或过表达质粒的HCT116与SW620细胞进行实验；同时利用裸鼠肿瘤模型(nude mouse tumor model)验证体外实验结果。通过同源性分析、免疫共沉淀(Co-immunoprecipitation)、考马斯亮蓝染色(Coomassie blue staining)、GST pull-down实验(GST pulldown assay)及免疫荧光(immunofluorescence)，探究STK32C与HSP90的相互作用；采用CompuSyn软件(CompuSyn)与SynergyFinder软件(SynergyFinder)对5-氟尿嘧啶(5-FU)进行协同效应分析。 结果：相较于CCD-18Co正常成纤维细胞(CCD-18Co fibroblasts)，STK32C在HCT116、HT29、SW480及SW620细胞中呈高表达，且该高表达与结直肠癌患者的不良预后相关。敲低STK32C可发挥细胞毒性、抗迁移及抗侵袭作用，抑制HCT116与SW620细胞中多聚ADP核糖聚合酶前体(pro-PARP)、半胱氨酸天冬氨酸蛋白酶3前体(pro-Caspase-3)、PI3K、磷酸化AKT(p-AKT)、磷酸化mTOR(p-mTOR)、HSP90及B细胞淋巴瘤-2(Bcl-2)的表达，并上调Bax蛋白、亚G1期细胞比例、膜联蛋白V(Annexin V)阳性细胞及TUNEL阳性细胞数。此外，敲低STK32C可增强5-FU在HCT116细胞中的抗肿瘤活性。通过同源性分析、共定位及结合实验证实，STK32C可直接结合HSP90的N端结构域。与之一致的是，HSP90 N端抑制剂Ganetespib可降低STK32C与磷酸化AKT1(p-AKT1)的表达，而HSP90 C端抑制剂表没食子儿茶素没食子酸酯(EGCG)或AKT抑制剂LY294002对STK32C的表达无影响。进一步实验显示，敲低STK32C可抑制BALB/c裸鼠体内HCT116细胞的肿瘤生长，同时降低瘤组织中STK32C、HSP90、增殖细胞核抗原(PCNA)及AKT的表达，并激活半胱氨酸天冬氨酸蛋白酶3(Caspase 3)。 结论：靶向STK32C可通过HSP90及PI3K/AKT/mTOR信号通路抑制结直肠癌进展，且与5-FU具有联合应用潜力。