(B) Changes in specific protein markers in HuH-7 and HuH-7R tumors tissues. rearrangement through Cdc42 activation. Mutation analyses revealed that phosphorylation at S102 affects the migratory and invasive potential of HuH-7R cells. Our collective findings suggest that sorafenib promotes YB-1 phosphorylation through effect from the EGFR/PI3K/AKT pathway, leading to significant enhancement of hepatocellular carcinoma (HCC) cell metastasis. Elucidation of the specific mechanisms of action of YB-1 may aid in the development of effective strategies to suppress metastasis and overcome resistance. < 0.001). Upon combining the two datasets, we observed overlap of 148 differentially expressed molecules, which were further subjected to gene ontology (GO) analyses using database for annotation, visualization, and integrated discovery (DAVID) (Figure 1A). Functional enrichment analysis revealed the top three significant functions (< 0.05) of the differentially expressed molecules as cytoskeleton (= 40, < 0.001), cellCcell adhesion (= 12, < 0.001) and microtubule-based movement (= 7, < 0.001) (Figure S1). This finding supports the involvement of the differentially expressed molecules in regulation of cytoskeletal structures and the implication of effects of these molecules on cell migration or invasion. Subsequently, the differentially expressed molecules were clustered through Protein-protein interaction (PPI) network construction using STRING. A total of 47 Rabbit Polyclonal to XRCC3 molecules with combination scores 0.95 were enrolled for PPI network construction. Twenty genes with roles in cellular movement-related functions were identified in the center cluster of the PPI network, and 27 other differentially expressed molecules were determined as potential regulatory genes of those in the center cluster (Figure S2). To further identify significant regulatory factors among the 27 differentially expressed molecules, functional enrichment analysis was performed with GO using DAVID. The top enriched biological process was gene expression Regorafenib Hydrochloride (= 11, < 0.001) (Supplemental Table S3). Among the 11 differentially expressed molecules with potential roles in gene expression, transcription was the top enriched function (= 3, value = 0.11) (Supplemental Table S4), including YB-1, pituitary tumor transforming gene 1 (PTTG1), and proliferation-associated protein 2G4 (PA2G4). YB-1 has previously been identified as a metastasis-related molecule . Accordingly, we focused on YB-1 as a potential gene Regorafenib Hydrochloride expression regulatory molecule with cellular movement-related functions in drug-resistant HCC. High protein expression and the phosphorylation at Ser102 of YB-1 in HuH-7R cells were validated via immunoblotting (Figure 1B). Compared to HCC patients with low YB-1 expression, those expressing high levels of YB-1 had significantly shorter median OS (3.9 vs. 6.9 years, Regorafenib Hydrochloride < 0.001), as determine from analysis of the TCGA dataset (total sample size, = 343) (Figure 1C). Our data suggest that high YB-1 expression is associated with poor OS and may be linked to malignant progression of HCC. Accordingly, we further investigated the role of YB-1 and its potential correlation with sorafenib resistance in HuH-7R cells. Open in Regorafenib Hydrochloride a separate window Figure 1 Bioinformatic identification and validation of Y-box binding protein-1 (YB-1) as a potential biomarker in HuH-7R cells. (A) Strategy for identification of sorafenib resistance-related differentially expressed molecules in hepatocellular carcinoma (HCC). (B) Phosphorylation levels of YB-1 and total YB-1 expression in HuH-7 and HuH-7R cells validated by immunoblotting, upper panel. Relative changes of the expression levels were quantified using GAPDH as normalization control, lower panel. (* < 0.05; ** < 0.01). (C) Kaplan-Meir OS curves of the YB-1 gene among 343 HCC patients in the TCGA dataset. The value was calculated with the log-rank test (*** < 0.001). 2.2. YB-1 Phosphorylation Is Regulated by Different Signaling Pathways Induced by Sorafenib in HuH-7 Cells and HuH-7R Cells Data from our bioinformatic analyses suggest that YB-1 phosphorylation is involved in facilitating the sorafenib resistance in HuH-7R cells. To characterize the different mechanistic responses of parental HuH-7 and HuH-7R cells to sorafenib treatment, we utilized several inhibitors for the mitogen-activated protein kinase (MAPK) signaling pathway (sorafenib, U0126.