Tumor metastasis, the process in which cancer cells detach from the primary tumor, migrate in the blood or lymph fluid, invade a distant tissue and prosper in this new niche, is considered the primary cause of mortality in cancer-related deaths. The Palladin (PALLD) gene is a structural protein widely expressed in mammalian tissues and has a pivotal role in cytoskeletal dynamics and motility in developing, normal and diseased tissues. Palladin has been linked to the progression of breast, pancreatic and renal cancers. In previous work carried out in our lab, we showed that primary tumor resection followed by palladin downregulation via local administration of microRNA-96 significantly decreased the number and size of lung metastasis in mice. The MET/HGF axis is a signaling pathway with a central part in embryonic development and cancer progression. In this study, using single cell morphokinetic analysis, we investigate in depth palladin`s contribution to cellular motility and characterize its part in the MET/HGF axis in breast cancer.
First, by analyzing gene coexpression, we determined that palladin is significantly correlated with MET/HGF signaling. Next, a palladin deficient aggressive breast cancer cell line was developed using CRISPR/Cas9. Using live-cell time-lapse fluorescent imaging, particle tracking and machine learning, we concluded that HGF induced motility is greatly hindered in the absence of palladin. Further analysis of migration patterns implicates palladin as a major contributor to collective cell motility. Total gene expression and proteomic analysis of PALLD knockout cells revealed 11 differentially expressed genes which reinforced palladin`s centrality to cellular motility. Finally, analysis of the TCGA-BRCA dataset validated that HGF expression is in correlation with decreased survival of patients with tumors expressing elevated levels of palladin.
Our work strengthens the link between palladin and cellular motility and metastasis in cancer. More specifically, we present evidence for the dependence of migration induced by the principal signaling pathway, the MET/HGF axis, on an adequate level of functional palladin. Additionally, by employing state of the art methodology, we offer an analysis of palladin`s precise contribution to cellular motility in the highest level of detail obtained so far. Overall, these results support our ongoing hypothesis: interference with palladin`s expression, translation or function will reduce the metastatic spread and aid in the treatment of aggressive breast tumors.