METHODS: Matrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis.
RESULTS: Treatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects.
CONCLUSIONS: Our findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.
方法:基质胶侵袭试验,明胶降解试验,细胞散射试验,采用MTT法和集落形成法研究侵袭性癌细胞的增殖和迁移能力。免疫印迹,免疫细胞化学,和免疫沉淀分析用于确定蛋白质表达和蛋白质-蛋白质相互作用。采用4T1侵袭性小鼠癌模型评估肿瘤生长和肺转移。
结果:吉西他滨(核苷类似物抗癌剂)对胰腺癌(Panc-1,MiaPaca-2)和乳腺癌(MDA-MB-231)细胞的治疗可扩增MDM-2表达并增加EMT特性。相反,NGD16增强了肿瘤抑制因子Par-4的表达,并抑制了这些细胞的侵袭和迁移能力。此外,Par-4的诱导有效地减少了MDM-2以及pro-EMT标记,然而,增加了上皮标志物的表达。此外,siRNA介导的Par-4沉默表明NGD16以Par-4依赖性方式发挥其EMT抑制作用。机械上,Par-4激活通过破坏MDM-2-p53相互作用激发p53,恢复了癌细胞的上皮特征。此外,通过siRNA对MDM-2的部分敲除显著地显示了NGD16的抗增殖和抗侵袭作用。最后,NGD16有效抑制小鼠乳腺癌模型中的肿瘤生长和肺转移,而没有任何不良作用。
结论:我们的发现揭示了Par-4是一个关键的治疗靶点,NGD16(Par-4的药理学调节剂)是抑制EMT和相关化疗耐药的潜在工具,可以在临床上用于治疗侵袭性癌症。