Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK-/- C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK-/- cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

Na+/H+-Exchanger Family as Novel Prognostic Biomarkers in Colorectal Cancer.

BACKGROUND: The acidic characteristics of the tumor microenvironment (TME) are attributed to cancer cells' needs of metabolism which produce a large amount of H+. In order not to affect its own life activities, it needs to release H+ into the intercellular space through an efficient Na+/H+ exchanger. On account of the intestine whose physiological function is highly dependent on intestinal pH value, NHE family members may play a critical role in the occurrence and development of colorectal cancer (CRC). METHODS: TCGA, GEPIA2, ONCOMINE, UALCAN, STRING, TIMER, Cytoscape, TargetScan, ENCORI, LncBase v.2, DNMIVD, HPA, and CellMinerTM databases were used in our study. RESULTS: The mRNA expressions of SLC9A1, SLC9A2, SLC9A3, and SLC9A9 were evidently lower in COAD than in normal samples; however, the mRNA expressions of SLC9A5, SLC9A8, and SLC9B2 were higher. Besides, mRNA expressions of NHE family were extremely associated with clinicopathological features, tumor immune microenvironment and stemness score, DNA methylation, and patient prognosis in COAD. Moreover, we conjectured that NHE family may play a role through MAPK or ErbB signaling pathway according to the results of GO/KEGG enrichment analysis. At last, we found that NHE family members were key factors of various kinds of cancers. CONCLUSION: Our study indicated that NHE family represented new diagnostic and therapeutic targets for CRC, which could have important significance for the clinical treatment of CRC.

Bcl-3 promotes Wnt signaling by maintaining the acetylation of ß-catenin at lysine 49 in colorectal cancer.

Wnt/ß-catenin signaling plays a critical role in colorectal cancer (CRC) tumorigenesis and the homeostasis of colorectal cancer stem cells (CSCs), but its molecular mechanism remains unclear. B-cell lymphoma 3 (Bcl-3), a member of the IκB family, is overexpressed in CRC and promotes tumorigenicity. Here, we report a novel function of Bcl-3 in maintaining colorectal CSC homeostasis by activating Wnt/ß-catenin signaling. Silencing Bcl-3 suppresses the self-renewal capacity of colorectal CSCs and sensitizes CRC cells to chemotherapeutic drugs through a decrease in Wnt/ß-catenin signaling. Moreover, our data show that Bcl-3 is a crucial component of Wnt/ß-catenin signaling and is essential for ß-catenin transcriptional activity in CRC cells. Interestingly, Wnt3a increases the level and nuclear translocation of Bcl-3, which binds directly to ß-catenin and enhances the acetylation of ß-catenin at lysine 49 (Ac-K49-ß-catenin) and transcriptional activity. Bcl-3 depletion decreases the Ac-K49-ß-catenin level by increasing the level of histone deacetylase 1 to remove acetyl groups from ß-catenin, thus interrupting Wnt/ß-catenin activity. In CRC clinical specimens, Bcl-3 expression negatively correlates with the overall survival of CRC patients. A significantly positive correlation was found between the expression of Bcl-3 and Ac-K49-ß-catenin. Collectively, our data reveal that Bcl-3 plays a crucial role in CRC chemoresistance and colorectal CSC maintenance via its modulation of the Ac-K49-ß-catenin, which serves as a promising therapeutic target for CRC.

Cilia loss sensitizes cells to transformation by activating the mevalonate pathway.

Although cilia loss and cell transformation are frequently observed in the early stage of tumorigenesis, the roles of cilia in cell transformation are unknown. In this study, disrupted ciliogenesis was observed in cancer cells and pancreatic cancer tissues, which facilitated oncogene-induced transformation of normal pancreatic cells (HPDE6C7) and NIH3T3 cells through activating the mevalonate (MVA) pathway. Disruption of ciliogenesis up-regulated MVA enzymes through ß catenin-T cell factor (TCF) signaling, which synchronized with sterol regulatory element binding transcription factor 2 (SREBP2), and the regulation of MVA by ß-catenin-TCF signaling was recapitulated in a mouse model of pancreatic ductal adenocarcinoma (PDAC) and human PDAC samples. Moreover, disruption of ciliogenesis by depleting Tg737 dramatically promoted tumorigenesis in the PDAC mouse model, driven by KrasG12D , which was inhibited by statin, an inhibitor of the MVA pathway. Collectively, this study emphasizes the crucial roles of cilia in governing the early steps of the transformation by activating the MVA pathway, suggesting that statin has therapeutic potential for pancreatic cancer treatment.

Raf kinase inhibitor protein (RKIP) blocks signal transducer and activator of transcription 3 (STAT3) activation in breast and prostate cancer.

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.