Expression of PFKP was the highest among PFK isoforms in NCI-60 cell lines (Supporting Fig. 6B), further supporting that cancer-specific expression of PFKP is regulated by miR-520a/b/e and TARDBP. We next assessed the clinical relevance of TARDBP in HCC. Expression of TARDBP is significantly associated with prognosis when estimated by receiver operating characteristic (ROC) analysis. Areas under the curve (AUCs) of TARDBP expression over 3-year overall survival (OS) were 0.6 (95% confidence interval [CI]: 0.53-0.66; P = 0.007) (Fig. 6A). When patients were stratified according to expression level of TARDBP, patients with high TARDBP expression

showed significantly shorter survival (P = 3.8 × 10−4; Fig. 6B). Association of TARDBP with prognosis is further supported by its significant correlation with the 65-gene risk score (r = Opaganib chemical structure 0.5; P = 2.2 × 10−16) (Fig. 6C) that was previously developed for prediction of recurrence.31 Significant positive correlation between expression of TARDBP and PFKP in HCC patients is also concordant with their roles as positive regulators for cell growth (Fig. 6D). The critical roles of TARDBP and its downstream targets, the miR-520

family, in cell growth and the significant correlation of TARDBP with patient survival strongly suggested that TARDBP and its downstream targets would be potential therapeutic targets for cancer treatment. To test this, we carried out a mouse xenograft experiment with CP-868596 SK-Hep1 cells and siRNA specific to TARDBP. Compared to treatment with control siRNA, treatment with siTARDBP resulted in a significant reduction MCE in tumor weight (Fig. 7A), recapitulating the effects of silencing TARDBP in vitro. Efficient silencing of TARDBP by siRNA was confirmed by immunostaining of TARDBP and its downstream target, PFKP, and further validated by qRT-PCR (Fig. 7B,C and Supporting Fig. 7). As expected, cell proliferation, as examined by Ki67 immunostaining, was significantly decreased in tumors

treated with siTARDBP (Fig. 7B). In addition, lactate and ATP levels were also significantly decreased (Fig. 7C) and expression of miR-520b and miR-520e (Fig. 7D) was significantly increased in siTARDBP-treated mice, compared to control. These results clearly demonstrate the importance of TARDBP in tumor growth and the potential of TARDBP as a therapeutic target. In the current work, we have presented a mechanistic link from TARDBP to PFKP, the rate-limiting enzyme of glycolysis, and we also have provided evidence suggesting that this pathway is associated with poor prognosis of HCC. A notable finding was the identification of the miR-520 family as an intermediary regulator of this pathway. Although TARDBP was originally identified as a transcription repressor binding to the human immunodeficiency virus transactivation response region,1 downstream targets and molecular mechanisms related to its transcription repressor activity have not been properly explored.