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Abstract Background KH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood. Methods KSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC. Results Our results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3’UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses. Conclusion The current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

Abstract Background Due to the difficulties in early diagnosing and treating hepatocellular carcinoma (HCC), prognoses for patients remained poor in the past decade. In this study, we established a screening model to discover novel prognostic biomarkers in HCC patients. Methods Candidate biomarkers were screened by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of five HCC normal (N)/tumor (T) paired tissues and preliminarily verified them through several in silico database analyses. Expression levels and functional roles of candidate biomarkers were respectively evaluated by immunohistochemical staining in N/T paired tissue (n = 120) and MTS, colony formation, and transwell migration/invasion assays in HCC cell lines. Associations of clinicopathological features and prognoses with candidate biomarkers in HCC patients were analyzed from GEO and TCGA datasets and our recruited cohort. Results We found that the transmembrane P24 trafficking protein 9 (TMED9) protein was elevated in HCC tissues according to a global proteomic analysis. Higher messenger (m)RNA and protein levels of TMED9 were observed in HCC tissues compared to normal liver tissues or pre-neoplastic lesions. The TMED9 mRNA expression level was significantly associated with an advanced stage and a poor prognosis of overall survival (OS, p = 0.00084) in HCC patients. Moreover, the TMED9 protein expression level was positively correlated with vascular invasion (p = 0.026), OS (p = 0.044), and disease-free survival (p = 0.015) in our recruited Taiwanese cohort. In vitro, manipulation of TMED9 expression in HCC cells significantly affected cell migratory, invasive, proliferative, and colony-forming abilities. Conclusions Ours is the first work to identify an oncogenic role of TMED9 in HCC cells and may provide insights into the application of TMED9 as a novel predictor of clinical outcomes and a potential therapeutic target in patients with HCC.

Abstract Background Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the cure rates for AML patients remain low, and the notorious adverse effects of chemotherapeutic drugs drastically reduce the life quality of patients. Penfluridol, a long-acting oral antipsychotic drug, has an outstanding safety record and exerts oncostatic effects on various solid tumors. Until now, the effect of penfluridol on AML remains unknown. Methods AML cell lines harboring wild-type (WT) Fms-like tyrosine kinase 3 (FLT3) and internal tandem duplication (ITD)-mutated FLT3 were used to evaluate the cytotoxic effects of penfluridol by an MTS assay. A flow cytometric analysis and immunofluorescence staining were employed to determine the cell-death phenotype, cell cycle profile, and reactive oxygen species (ROS) and acidic vesicular organelle (AVO) formation. Western blotting and chemical inhibitors were used to explore the underlying mechanisms involved in penfluridol-mediated cell death. Results We observed that penfluridol concentration-dependently suppressed the cell viability of AML cells with FLT3-WT (HL-60 and U937) and FLT3-ITD (MV4–11). We found that penfluridol treatment not only induced apoptosis as evidenced by increases of nuclear fragmentation, the sub-G1 populations, poly (ADP ribose) polymerase (PARP) cleavage, and caspase-3 activation, but also triggered autophagic responses, such as the light chain 3 (LC3) turnover and AVO formation. Interestingly, blocking autophagy by the pharmacological inhibitors, 3-methyladenine and chloroquine, dramatically enhanced penfluridol-induced apoptosis, indicating the cytoprotective role of autophagy in penfluridol-treated AML cells. Mechanistically, penfluridol-induced apoptosis occurred through activating protein phosphatase 2A (PP2A) to suppress Akt and mitogen-activated protein kinase (MAPK) activities. Moreover, penfluridol’s augmentation of intracellular ROS levels was critical for the penfluridol-induced autophagic response. In the clinic, we observed that patients with AML expressing high PP2A had favorable prognoses. Conclusions These findings provide a rationale for penfluridol being used as a PP2A activator for AML treatment, and the combination of penfluridol with an autophagy inhibitor may be a novel strategy for AML harboring FLT3-WT and FLT3-ITD.