SIK2 maintains breast cancer stemness by phosphorylating LRP6 and activating Wnt/ß-catenin signaling.

Breast cancer stem cells (BCSCs) are the main drivers of recurrence and metastasis. However, commonly used drugs rarely target BCSCs. Via screenings, we found that Salt-inducible kinase 2 (SIK2) participated in breast cancer (BC) stemness maintenance and zebrafish embryos development. SIK2 was upregulated in recurrence samples. Knockdown of SIK2 expression reduced the proportion of BCSCs and the tumor initiation of BC cells. Mechanistically, SIK2, phosphorylated by CK1α, directly phosphorylated LRP6 in a SIK2 kinase activity-dependent manner, leading to Wnt/ß-catenin signaling pathway activation. ARN-3236 and HG-9-91-01, inhibitors of SIK2, inhibited LRP6 phosphorylation and ß-catenin accumulation and disturbed stemness maintenance. In addition, the SIK2-activated Wnt/ß-catenin signaling led to induction of IDH1 expression, causing metabolic reprogramming in BC cells. These findings demonstrate a novel mechanism whereby Wnt/ß-catenin signaling pathway is regulated by different kinases in response to metabolic requirement of CSCs, and suggest that SIK2 inhibition may potentially be a strategy for eliminating BCSCs.

CDK11 negatively regulates Wnt/ß-catenin signaling in the endosomal compartment by affecting microtubule stability.

Objectives: Improper activation of Wnt/ß-catenin signaling has been implicated in human diseases. Beyond the well-studied glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1 (CK1), other kinases affecting Wnt/ß-catenin signaling remain to be defined. Methods:To identify the kinases that modulate Wnt/ß-catenin signaling, we applied a kinase small interfering RNA (siRNA) library screen approach. Luciferase assays, immunoblotting, and real-time polymerase chain reaction (PCR) were performed to confirm the regulation of the Wnt/ß-catenin signaling pathway by cyclin-dependent kinase 11 (CDK11) and to investigate the underlying mechanism. Confocal immunofluorescence, coimmunoprecipitation (co-IP), and scratch wound assays were used to demonstrate colocalization, detect protein interactions, and explore the function of CDK11. Results: CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/ß-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/ß-catenin signaling receptor complexes, in a manner dependent on intact adenomatosis polyposis coli (APC) protein. Further analysis showed that CDK11 modulation of Wnt/ß-catenin signaling engaged the endolysosomal machinery, and CDK11 knockdown enhanced the colocalization of Wnt/ß-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes. Mechanistically, CDK11 was found to function in Wnt/ß-catenin signaling by regulating microtubule stability. Depletion of CDK11 down-regulated acetyl-α-tubulin. Moreover, co-IP assays demonstrated that CDK11 interacts with the α-tubulin deacetylase SIRT2, whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/ß-catenin signaling receptor complexes. CDK11 was found to suppress cell migration through altered Wnt/ß-catenin signaling. Conclusions: CDK11 is a negative modulator of Wnt/ß-catenin signaling that stabilizes microtubules, thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.

NEK4 kinase regulates EMT to promote lung cancer metastasis.

Epithelial-to-mesenchymal transition (EMT) is a dynamic transitional state from the epithelial to mesenchymal phenotypes. Numerous studies have suggested that EMT and its intermediate states play important roles in tumor invasion and metastasis. To identify novel regulatory molecules of EMT, we screened a siRNA library targeting human 720 kinases in A549 lung adenocarcinoma cells harboring E-cadherin promoter-luciferase reporter vectors. NIMA-related kinase-4 (NEK4) was identified and characterized as a positive regulator of EMT in the screening. Suppression of NEK4 resulted in the inhibition of cell migration and invasion, accompanying with an increased expression of cell adhesion-related proteins such as E-cadherin and ZO1. Furthermore, NEK4 knockdown caused the decreased expression of the transcriptional factor Zeb1 and Smads proteins, which are known to play key roles in EMT regulation. Consistently, overexpression of NEK4 resulted in the decreased expression of E-cadherin and increased expression of Smad3. Using a mouse model with tail vein injection of NEK4 knockdown stable cell line, we found a lower rate of tumor formation and metastasis of the NEK4-knockdown cells in vivo. Thus, this study demonstrates NEK4 as a novel kinase involved in regulation of EMT and suggests that NEK4 may be further explored as a potential therapeutic target for lung cancer metastasis.

Exogenous hydrogen sulfide protects against global cerebral ischemia/reperfusion injury via its anti-oxidative, anti-inflammatory and anti-apoptotic effects in rats.

The present study was undertaken to study the effects of exogenous hydrogen sulfide (H(2)S) on global cerebral ischemia-reperfusion(I/R) and the underlying mechanisms. After a 24h I/R, administration of NaHS, an exogenous donor for H(2)S, at the dose of 0.2 or 0.4 µmol/kg significantly decreased the apoplexy index, neurological symptom scoring, and brain infarcted area as compared to the I/R group in a dose dependent manner. At the same time, NaHS-treated rats displayed significant reduction of MDA content, and striking increase of SOD activity in the brain tissues compared with I/R group. The up-regulated mRNA levels of p47(phox) and gp91(phox) subunits of NADPH oxidase were also suppressed by NaHS treatment. In this study, the pro-inflammatory markers TNF-α and MCP-1 in I/R group were markedly increased by 24h I/R, which were significantly attenuated by NaHS in a dose-dependent manner. In contrast, the anti-inflammatory factor IL-10 was markedly induced by NaHS administration. In addition, the expression of the anti-apoptotic protein Bcl-2 was significantly decreased in I/R group compared with the sham-operated group. This reduction was significantly blunted in NaHS-treated group. On the contrary, the pro-apoptotic protein Bax content in brain tissues of I/R group was markedly elevated compared with sham-operated animals. And such an induction of Bax content was significantly ameliorated by NaHS. Taken together, our results suggest that hydrogen sulfide has potent protective effect against a severe cerebral injury induced by a global I/R possibly through the inhibition of oxidative stress, inflammation and apoptosis.

Use of blood urea nitrogen, creatinine, interleukin-6, granulocyte-macrophage colony stimulating factor in combination to predict the severity and outcome of abdominal sepsis in rats.

OBJECTIVES: Accurate and timely diagnosis and prognosis of sepsis remain challenging. A combination of markers, as opposed to single ones, may improve the prognosis, and therefore survival. This study compared the effectiveness of routinely used biomarkers of sepsis alone and in combination for the prediction of outcome in rats with abdominal sepsis. METHODS: Rats were subjected to sepsis induced by cecal ligation and puncture (CLP). Seventeen biomarkers were detected 12 h after the CLP. Correlation between the biomarkers and outcome of rats was analyzed; the correlated biomarkers were analyzed by logistic regression analysis and the area under the receiver operating characteristic (ROC) curve was computed to compare their performance in the prognosis of sepsis. RESULTS: A total of 49 rats were eligible for analysis. Body temperature (T), blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT), creatine kinase (CK), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) levels after the CLP were negatively correlated with the survival outcome, while platelet count (PLT), high-mobility group box protein 1 (HMGB1) and granulocyte-macrophage colony stimulating factor (GM-CCF) were positively correlated with the survival outcome (P < 0.05). Levels of BUN, Cr, IL-6, and GM-CSF after the CLP were independent predictors of outcome according to conditional logistic regression. The sensitivity and specificity of the four selected biomarkers in combination for predicting sepsis outcome were better than single ones (P < 0.05). CONCLUSION: A combination of different biomarkers improves the diagnostic accuracy and is more effective in the prognosis of sepsis in rats. Use of BUN, Cr, IL-6, GM-CSF in combination to predict the severity and outcome in rats with abdominal sepsis exhibited acceptable diagnostic characteristics.