USP18 induction regulates immunometabolism to attenuate M1 signal-polarized macrophages and enhance IL-4-polarized macrophages in systemic lupus erythematosus.

Effective treatment of systemic lupus erythematosus (SLE) remains an unmet need. Different subsets of macrophages play differential roles in SLE and the modulation of macrophage polarization away from M1 status is beneficial for SLE therapeutics. Given the pathogenic roles of type I interferons (IFN-I) in SLE, this study investigated the effects and mechanisms of a mitochondria localization molecule ubiquitin specific peptidase 18 (USP18) preserving anti-IFN effects and isopeptidase activity on macrophage polarization. After observing USP18 induction in monocytes from SLE patients, we studied mouse bone marrow-derived macrophages and showed that USP18 deficiency increased M1signal (LPS + IFN-γ treatment)-induced macrophage polarization, and the effects involved the induction of glycolysis and mitochondrial respiration and the expression of several glycolysis-associated enzymes and molecules, such as hypoxia-inducible factor-1α. Moreover, the effects on mitochondrial activities, such as mitochondrial DNA release and mitochondrial reactive oxygen species production were observed. In contrast, the overexpression of USP18 inhibited M1signal-mediated and enhanced interleukin-4 (IL-4)-mediated polarization of macrophages and the related cellular events. Moreover, the levels of USP18 mRNA expression showed tendency of correlation with the expression of metabolic enzymes in monocytes from patients with SLE. We thus concluded that by preserving anti-IFN effect and downregulating M1 signaling, promoting USP18 activity may serve as a useful approach for SLE therapeutics.

USP18 enhances dengue virus replication by regulating mitochondrial DNA release.

Dengue virus (DENV) infection remains a challenging health threat worldwide. Ubiquitin-specific protease 18 (USP18), which preserves the anti-interferon (IFN) effect, is an ideal target through which DENV mediates its own immune evasion. However, much of the function and mechanism of USP18 in regulating DENV replication remains incompletely understood. In addition, whether USP18 regulates DENV replication merely by causing IFN hyporesponsiveness is not clear. In the present study, by using several different approaches to block IFN signaling, including IFN neutralizing antibodies (Abs), anti-IFN receptor Abs, Janus kinase inhibitors and IFN alpha and beta receptor subunit 1 (IFNAR1)knockout cells, we showed that USP18 may regulate DENV replication in IFN-associated and IFN-unassociated manners. Localized in mitochondria, USP18 regulated the release of mitochondrial DNA (mtDNA) to the cytosol to affect viral replication, and mechanisms such as mitochondrial reactive oxygen species (mtROS) production, changes in mitochondrial membrane potential, mobilization of calcium into mitochondria, 8-oxoguanine DNA glycosylase 1 (OGG1) expression, oxidation and fragmentation of mtDNA, and opening of the mitochondrial permeability transition pore (mPTP) were involved in USP18-regulated mtDNA release to the cytosol. We therefore identify mitochondrial machineries that are regulated by USP18 to affect DENV replication and its association with IFN effects.

Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways.

Mitochondria regulate the immune response after dengue virus (DENV) infection. Microarray analysis of genes identified the upregulation of mitochondrial cytidine/uridine monophosphate kinase 2 (CMPK2) by DENV infection. We used small interfering RNA-mediated knockdown (KD) and CRISPR-Cas9 knockout (KO) approaches, to investigate the role of CMPK2 in mouse and human cells. The results showed that CMPK2 was critical in DENV-induced antiviral cytokine release and mitochondrial oxidative stress and mitochondrial DNA release to the cytosol. The DENV-induced activation of Toll-like receptor (TLR)-9, inflammasome pathway, and cell migration was suppressed by CMPK2 depletion; however, viral production increased under CMPK2 deficiency. Examining mouse bone marrow-derived dendritic cells from interferon-alpha (IFN-α) receptor-KO mice and signal transducer and activator of transcription 1 (STAT1)-KO mice, we confirmed that CMPK2-mediated antiviral activity occurred in IFN-dependent and IFN-independent manners. In sum, CMPK2 is a critical factor in DENV-induced immune responses to determine innate immunity.

Mitochondrial protein CMPK2 regulates IFN alpha-enhanced foam cell formation, potentially contributing to premature atherosclerosis in SLE.

BACKGROUND: Premature atherosclerosis occurs in patients with SLE; however, the mechanisms remain unclear. Both mitochondrial machinery and proinflammatory cytokine interferon alpha (IFN-α) potentially contribute to atherogenic processes in SLE. Here, we explore the roles of the mitochondrial protein cytidine/uridine monophosphate kinase 2 (CMPK2) in IFN-α-mediated pro-atherogenic events. METHODS: Foam cell measurements were performed by oil red O staining, Dil-oxLDL uptake and the BODIPY approach. The mRNA and protein levels were measured by qPCR and Western blotting, respectively. Isolation of CD4+ T cells and monocytes was performed with monoclonal antibodies conjugated with microbeads. Manipulation of protein expression was conducted by either small interference RNA (siRNA) knockdown or CRISPR/Cas9 knockout. The expression of mitochondrial reactive oxygen species (mtROS) was determined by flow cytometry and confocal microscopy. RESULTS: IFN-α enhanced oxLDL-induced foam cell formation and Dil-oxLDL uptake by macrophages. In addition to IFN-α, several triggers of atherosclerosis, including thrombin and IFN-γ, can induce CMPK2 expression, which was elevated in CD4+ T cells and CD14+ monocytes isolated from SLE patients compared to those isolated from controls. The analysis of cellular subfractions revealed that CMPK2 was present in both mitochondrial and cytosolic fractions. IFN-α-induced CMPK2 expression was inhibited by Janus kinase (JAK)1/2 and tyrosine kinase 2 (Tyk2) inhibitors. Both the knockdown and knockout of CMPK2 attenuated IFN-α-mediated foam cell formation, which involved the reduction of scavenger receptor class A (SR-A) expression. CMPK2 also regulated IFN-α-enhanced mtROS production and inflammasome activation. CONCLUSIONS: The study suggests that CMPK2 plays contributing roles in the pro-atherogenic effects of IFN-α.

Author Correction: PAK1 confers chemoresistance and poor outcome in non-small cell lung cancer via ß-catenin-mediated stemness.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120-mediated connexin43 phosphorylation.

The ω-3 fatty acids exert as an antioxidant via the G protein-coupled receptor 120 (GPR120). Icosapent ethyl, a purified eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connexin43 expression and whether the GPR120 signalling is involved in the protection. Male Wistar rats after ligating coronary artery were assigned to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative-nitrosative stress. In concert, myocardial connexin43 levels revealed a significant decrease in vehicle-treated infarcted rats compared with sham. These changes of oxidative-nitrosative stress and connexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative-nitrosative stress and connexin43 phosphorylation were abolished by administering AH-7614, an inhibitor of GPR120. SIN-1 abolished the Cx43 phosphorylation of icosapent ethyl without affecting GPR120 levels. Taken together, chronic use of icosapent ethyl after infarction is associated with up-regulation of connexin43 phosphorylation through a GPR120-dependent antioxidant pathway and thus plays a beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Hepatitis B virus X protein promotes tumor invasion and poor prognosis in hepatocellular carcinoma via phosphorylation of paxillin at Serine 178 by activation of the c-Jun NH2-terminal kinase.

Hepatitis B virus X protein (HBx) plays critical roles in hepatocellular tumorigenesis by activating different signaling pathways, including the c-Jun NH2-terminal kinase (JNK) pathway. Phosphorylation of paxillin (PXN) promotes cell migration via activation of the JNK signaling pathway, but PXN overexpression is not associated with poor outcome in patients with hepatocellular carcinoma (HCC). HBx gene manipulation and Western blotting indicated that phosphorylation of PXN at Serine 178 (pS178-PXN) by HBx may promote invasiveness in HCC cells via HBx-mediated JNK activation. Immunohistochemical analysis indicated a positive correlation between pS178-PXN and HBx expression levels in tumor specimens. The overall survival (OS) and relapse-free survival (RFS) were poorer in patients with high-pS178-PXN expressing or high-HBx expressing tumors than in patients with low-pS178-PXN expressing or low-HBx expressing tumors. In conclusion, phosphorylation of PXN at Serine 178 by HBx-mediated JNK activation may therefore play a critical role in tumor invasiveness and poor prognosis in patients with HBV-infected hepatocellular tumors. The expression levels of pS178-PXN may be a reliable prognostic biomarker to predict the clinical outcomes in patients with HBV-associated HCC.

Association of nuclear localization of SHP2 and YAP1 with unfavorable prognosis in non-small cell lung cancer.

Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is ubiquitously expressed in cytoplasmic localization, which in turn confers tumor malignancy and poor prognosis in various human cancers. YAP1 interacts with SHP2 to promote translocation of SHP2 to nucleus, which consequently promotes Wnt target activation. However, the oncogenic role of the nuclear localization of SHP2 in human cancers remains unclear. We hypothesized that nuclear SHP2 localization, in combination with nuclear YAP1 expression, could be associated with poor overall survival (OS) and relapse free survival (RFS) due to an increase in cyclin D1 and c-Myc mRNA expression following activation of Wnt/ß-catenin signaling. Immunohistochemical analysis of SHP2 and YAP1 protein expression in 102 tumors resected from patients with NSCLC revealed that nuclear SHP2 expression was well correlated with nuclear YAP1 expression (P < 0.001). Evaluation of cyclin D1 and c-Myc mRNA levels by the real-time reverse-phase polymerase chain reaction (RT-PCR) revealed that patients with high cyclin D1 and high c-Myc mRNA expressing tumors more commonly showed high nuclear YAP1 and high nuclear SHP2 (high/high) rather than the high/low, low/high, or low/low combinations (P < 0.001 for cyclin D1 and c-Myc). Kaplan-Meier and Cox-regression models showed OS and RFS to be poorer in patients in the high/high subgroup than in the low/low subgroup (OS: HR = 2.85, 95% CI, 1.52-5.35, P = 0.001; RFS: HR = 2.55, 95% CI, 1.37-4.72, P = 0.003). No prognostic significance was observed for the other two subgroups (low/high and high/low) when compared to the low/low subgroup in this study population. Therefore, we suggest that the prognostic value of SHP2 could reflect the nuclear localization of SHP2 and its interaction with nuclear YAP1, which led to subsequent upregulation of cyclin D1 and c-Myc mRNA expression via activation of the Wnt/ß-catenin signaling pathway.

PSMD4 is a novel therapeutic target in chemoresistant colorectal cancer activated by cytoplasmic localization of Nrf2.

Nuclear Nrf2 (nNrf2) binding to the antioxidant response element may promote chemoresistance in colorectal cancer. However, the shuttling of Nrf2 between cytoplasm and nucleus in colon cancer cells has revealed the possibility that cytoplasmic location of Nrf2 (cNrf2) may play a specific role in chemoresistance. Transfection of a nuclear location sequence (NLS)-wild-type or NLS-mutated Nrf2 expression vector into a stable shNrf2 HCT116 clone using the MTT assay to examine whether chemoresistance induced by cNrf2 may be greater than nNrf2. Different specific inhibitors and small hairpin (sh)RNAs of targeting genes were used to verify the mechanistic action of cNrf2 in chemoresistance and further confirmed by an animal model. The association of cNrf2 with chemotherapeutic response in patients with colorectal cancer was statistically analyzed. The MTT assay indicated that cNrf2 may play a more important role than nNrf2 in conferring 5-fluorouracil (5-FU) and oxaliplatin resistance in HCT116 cells. Mechanistically, cNrf2-induced PSMD4 expression was responsible for chemoresistance in the NLS-mutated Nrf2-tranfected shNrf2HCT116 clone via the NF-κB/AKT/ß-catenin/ZEB1 cascades. The tumor burden induced by the NLS-mutated Nrf2-transfected shNrf2HCT116 clone was completely suppressed by treatment with 5-FU in combination with carfilzomib. A higher prevalence of unfavorable chemotherapeutic response in colorectal cancer patients with cNrf2, PSMD4-positive, p-p65-positive, and nuclear ß-catenin tumors was observed when compared to their counterparts. cNrf2 may play a more important role than nNrf2 in the chemoresistance of colorectal cancer. Activation of the NF-κB/AKT/ß-catenin/ZEB1 cascade by PSMD4 may be responsible for cNrf2-mediated chemoresistance. CONDENSED ABSTRACT: CNrf2 may play a more important role than nNrf2 in conferring 5-FU and oxaliplatin resistance. This observation in patients seemed to support the findings of the cell and animal models and suggested that PSMD4 may be responsible cNrf2-mediated chemoresistance via the NF-κB/AKT/ß-catenin /ZEB1 cascades.

A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop.

Purpose: MicroRNA-630 plays dual roles in apoptosis and drug resistance in human cancers. However, the role of miR-630 in resistance to tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma remains to be elucidated. Methods: Manipulation of miR-630 and its targeted gene YAP1 and/or combination of inhibitor treatments was performed to explore whether low miR-630 could confer TKI resistance due to de-targeting YAP1, and this could decrease proapoptotic protein Bad expression through the miR-630/YAP1/ERK feedback loop. A retrospective study was conducted to examine whether the expression of miR-630 and YAP1 could be associated with TKI therapeutic response in patients with lung adenocarcinoma. Results: Low miR-630 expression may confer TKI resistance via increased SP1 binding to the miR-630 promoter due to ERK activation by YAP1 de-targeting. Persistent activation of ERK signaling via the miR-630/YAP1/ERK feedback loop may be responsible for TKI resistance in EGFR-mutated cells. Moreover, a decrease in Bad expression by its phosphorylation at Serine 75 through ERK activation conferred low miR-630-mediated TKI resistance by modulating the apoptotic pathway. Xenographic tumors induced by miR-630-knockdown PC9 and PC9GR cells in nude mice were nearly suppressed by the combination of gefitinib with the YAP1 inhibitor verteporfin or an MEK/ERK inhibitor AZD6244. Patients with low miR-630 and high YAP1 expressing tumors had a higher prevalence of unfavorable responses to TKI therapy and poorer outcomes when compared with their counterparts. Conclusion: MiR-630 may be a potential biomarker for the prediction of TKI therapeutic response and outcome in patients with lung adenocarcinoma.

MicroRNA-630 may confer favorable cisplatin-based chemotherapy and clinical outcomes in non-small cell lung cancer by targeting Bcl-2.

MicroRNA-630 (miR-630) plays dual roles in tumor progression in various human cancers. However, the role of miR-630 in chemoresistance and prognosis in non-small cell lung cancer (NSCLC) remains to be elucidated. This retrospective study enrolled 114 surgically resected patients with NSCLC who experienced tumor relapse and underwent cisplatin-based chemotherapy. The aim was to examine the possible association between miR-630 (and its targeting of Bcl-2 expression) and the response to cisplatin-based chemotherapy. Patients with tumors expressing low miR-630, high Bcl-2, and a combination of both were more likely than their counterparts to show unfavorable responses to cisplatin-based chemotherapy. Kaplan-Meier and Cox regression analysis indicated that low miR-630, high Bcl-2, and a combination of both may independently predict poor overall survival and short relapse-free survival in patients with NSCLC. Six types of NSCLC cells were collected to determine the inhibitory concentration of cisplatin yielding 50% viability (IC50) by the MTT assay. The IC50 value for cisplatin was negatively correlated with miR-630 expression levels among these cell types, except for A549 cells. Mechanistically, low miR-630 expression conferred cisplatin resistance and colony formation by de-targeting Bcl-2 in NSCLC cells. We therefore suggest that low miR-630, high Bcl-2, and a combination of both may potentially predict an unfavorable chemotherapeutic response and poor outcome in patients with NSCLC.

PD-L1 confers resistance to EGFR mutation-independent tyrosine kinase inhibitors in non-small cell lung cancer via upregulation of YAP1 expression.

Programmed death ligand (PD-L1) expression was associated with tumor immune escape and subsequent poor prognosis in non-small cell lung cancer (NSCLC). This expression was higher in patients with EGFR-mutated NSCLC tumors than in those with EGFR-wild-type (WT) NSCLC tumors. We therefore hypothesized that poor prognosis mediated by higher PD-L1 may be partially through conferring resistance to tyrosine kinase inhibitor (TKI) in NSCLC regardless of EGFR mutation. The change in PD-L1 expression following gene manipulation corresponded with changes in expression of HIF-1α and YAP1. The expression of HIF-1α and YAP1 was concomitantly decreased by PD-L1 silencing or by ROS scavenger treatment (N-acetylcysteine, NAC); however, a ROS inducer treatment (pyocyanin) completely reversed the decreased expression of both genes in EGFR-mutated and -wild-type (WT) NSCLC cells. The MTT assay indicated that the inhibitory concentration of gefitinib yielding 50% cell viability (IC50) depended on PD-L1-mediated YAP1 expression. Mechanistic studies indicated that upregulation of YAP1 by PD-L1 might be responsible for EGFR mutation-independent TKI resistance via the ROS/HIF-1α axis. An unfavorable TKI response was more common in patient tumors with high PD-L1 or YAP1 mRNA expression than in patient tumors with low mRNA expression of these genes. In conclusion, PD-L1 might confer EGFR mutation-independent TKI resistance in NSCLC cells via upregulation of YAP1 expression.

Hepatitis B virus X protein represses LKB1 expression to promote tumor progression and poor postoperative outcome in hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus X (HBx) protein plays critical roles in hepatitis B virus (HBV)-associated hepatocellular tumorigenesis through different molecular mechanisms, including inactivation of p53, a key transcription factor of liver kinase B1 (LKB1). We hypothesized that p53 inactivation by HBx protein could decrease LKB1 expression, thereby promoting tumor progression and poor outcomes in patients with HBV-associated hepatocellular carcinoma. METHODS: Manipulation strategies for HBx protein and/or p53 were used to verify that loss of LKB1 could promote colony formation and invasiveness in HepG2 and Hep3B cells. The expressions of HBx protein and LKB1 in 93 hepatocellular carcinomas (HCC) were also evaluated by immunohistochemistry. Kaplan-Meier and Cox regression models were used to assess the prognostic value of both HBx protein and LKB1 proteins in patients with hepatocellular carcinoma. RESULTS: Mechanistically, LKB1 expression was decreased at the transcriptional level after inactivation of p53 by HBx protein. Decreases in LKB1 expression were also associated with HBx protein-mediated colony formation and invasive capabilities. HBx protein, LKB1, and a combination of both proteins had prognostic significance for overall survival and relapse-free survival in our study population. CONCLUSION: The results from cell line experiments and evaluation of patient prognosis according to expression of HBx protein and LKB1 in their HCC strongly support the hypothesis that decreases in LKB1 expression by HBx protein-mediated p53 inactivation may play an important role in HBV-associated hepatocellular tumorigenesis.

Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression.

Resistance to tyrosine kinase inhibitors (TKIs) results in tumor relapse and poor prognosis in patients with lung adenocarcinoma. TKI resistance caused by epidermal growth factor receptor (EGFR) mutations at T790M and c-Met amplification occurs through persistent activation of the MEK/ERK and PI3K/AKT signaling pathways. We therefore expected that dual inhibitors of both signaling pathways could overcome TKI resistance in lung adenocarcinoma. Here, dioscin was selected from a product library of Chinese naturally occurring compounds and overcame TKI resistance in EGFR-mutated lung adenocarcinoma cells. Mechanistically, dioscin may down-regulate the expression of SH2 domain-containing phosphatase-2 (SHP2) at the transcription level by increasing p53 binding to the SHP2 promoter due to reactive oxygen species (ROS). Simultaneous inhibition of MEK/ERK and PI3K/AKT activation via decreased SHP2 expression and its interaction with GAB1 may be responsible for dioscin-mediated TKI sensitivity. A higher unfavorable response to TKI therapy occurred more commonly in patients with high SHP2 mRNA expression than in patients with low SHP2 mRNA expression. Therefore, we suggest that dioscin may act as a dual inhibitor of the MEK/ERK and PI3K/AKT signaling pathways to overcome TKI resistance via dysregulation of SHP2 expression in lung adenocarcinoma.

An increase in BAG-1 by PD-L1 confers resistance to tyrosine kinase inhibitor in non-small cell lung cancer via persistent activation of ERK signalling.

High programmed cell death 1 ligand 1 (PD-L1) expression in tumour tissues was associated with poor outcomes in non-small cell lung cancer (NSCLC) due to evasion of tumour immune surveillance. However, the role of PD-L1 in tumour invasion and resistance to tyrosine kinase inhibitor (TKI) treatments is not fully understood. Here, we provide evidence to support the involvement of PD-L1 expression in the invasiveness and TKI resistance in NSCLC cells by increased Bcl-2-associated athanogene-1 (BAG-1) expression. The upregulation of BAG-1 transcription by PD-L1 was verified by constructing the BAG-1 promoters using the polymerase chain reaction (PCR) and deletion mutations for luciferase reporter assays. The results indicated that C/EBPß phosphorylation by extracellular signal-regulated kinase (ERK) signalling was responsible for PD-L1-mediated BAG-1 transcription. Mechanistically, the PD-L1-induced BAG-1 expression reciprocally increased PD-L1 expression due to persistent activation of ERK signalling, and it consequently conferred TKI resistance in NSCLC cells. The mechanistic action of this cell model was further confirmed by an animal model, affirming that PD-L1 conferred tumour invasiveness and TKI resistance via persistent activation of ERK signalling by the PD-L1/BAG-1 axis. We therefore suggest a combination of an ERK inhibitor with a TKI as a potential strategy for conquering PD-L1-mediated tumour invasion and TKI resistance in NSCLC patients whose tumours harbour high PD-L1/high BAG-1 expression.

A combination of anti-PD-L1 mAb plus Lm-LLO-E6 vaccine efficiently suppresses tumor growth and metastasis in HPV-infected cancers.

PD-1/PD-L1 immunotherapy is viewed as having clinical benefits in advanced cancers but is effective in only a few patients, suggesting that an efficient combination approach is needed to improve efficacy. Immunohistochemistry analysis indicated that PD-L1 expression was correlated with the E6 expression in tumors from 122 lung cancer patients. The poorest survival occurred in PD-L1-positive/E6-positive tumor. PD-L1 expression was increased by the expression of E6, but not the E7, oncoprotein in lung and cervical cancer cells. PD-L1 expression was responsible for E6-mediated colony formation and soft agar growth. Therefore, PD-L1 secreted from tumor cells may directly promote tumor progression, particularly in E6-positive tumors. Immune deficiency nude mice were used to test the possibility that combining anti-PD-L1 mAb with Lm-LLO-E6 vaccine could have a higher antitumor activity compared with anti-PD-L1 mAb or Lm-LLO-E6 vaccine alone. A greater antitumor activity was obtained with anti-PD-L1 mAb + Lm-LLO-E6 vaccine than with anti-PD-L1 mAb or Lm-LLO-E6 alone in subcutaneous and metastatic tumors induced by TL-1 and SiHa cells. The longest survival time for nude mice was observed in the anti-PD-L1 mAb + Lm-LLO-E6 vaccine group. In conclusion, an anti-PD-L1 mAb + Lm-LLO-E6 vaccine may be an efficient treatment for suppression of tumor growth and metastasis induced by HPV-infected cells.

The YAP1/SIX2 axis is required for DDX3-mediated tumor aggressiveness and cetuximab resistance in KRAS-wild-type colorectal cancer.

The mechanism underlying tumor aggressiveness and cetuximab (CTX) resistance in KRAS-wild-type (KRAS -WT) colorectal cancer remains obscure. We here provide evidence that DDX3 promoted soft agar growth and invasiveness of KRAS-WT cells, as already confirmed in KRAS-mutated cells. Mechanistically, increased KRAS expression induced ROS production, which elevated HIF-1α and YAP1 expression. Increased HIF-1α persistently promoted DDX3 expression via a KRAS/ROS/HIF-1α feedback loop. DDX3-mediated aggressiveness and CTX resistance were regulated by the YAP1/SIX2 axis in KRAS-WT cells and further confirmed in animal models. Kaplan-Meier and Cox regression analysis indicated that DDX3, KRAS, and YAP1 expression had prognostic value for OS and RFS in KRAS-WT and KRAS-mutated tumors, but SIX2 and YAP1/SIX2 were prognostic value only in KRAS-WT patients. The observation from patients seemed to support the mechanistic action of cell and animal models. We therefore suggest that combining YAP1 inhibitors with CTX may therefore suppress DDX3-mediated tumor aggressiveness and enhance CTX sensitivity in KRAS-WT colorectal cancer.

Targeting insulin-like growth factor-binding protein-3 by microRNA-125b promotes tumor invasion and poor outcomes in non-small-cell lung cancer.

Insulin-like growth factor-binding protein-3 acts as a tumor suppressor that inhibits the PI3K/AKT signaling pathway due to blocking insulin growth factor-1 binding to its receptor. We hypothesized that insulin-like growth factor-binding protein-3 might be targeted by microRNA-125b and promote tumor invasion and poor outcome in non-small-cell lung cancer via activation of the PI3K/AKT signaling pathway. Real-time polymerase chain reaction and immunohistochemistry were performed to determine the level of microRNA-125b, insulin-like growth factor-binding protein-3 messenger RNA, and phosphorylated-AKT expression in 105 tumors from non-small-cell lung cancer patients. Low insulin-like growth factor-binding protein-3 messenger RNA levels and positive phosphorylated-AKT expression were more commonly found in patients with high microRNA-125b tumors than low microRNA-125b tumors. A poorer overall survival and relapse-free survival were observed in patients with high microRNA-125b tumors than low-microRNA-125b tumors in p53-mutated patients, but not in p53-wild-type patients. Mechanistically, microRNA-125b promotes invasion ability in p53-mutated cells via the PI3K/AKT activation by targeting of insulin-like growth factor-binding protein-3, but this effect was not observed in p53-wild-type cells. An increase in phosphorylated-AKT expression due to targeting of insulin-like growth factor-binding protein-3 by microRNA-125b was responsible for cell invasion in p53-mutated cells. In conclusion, the microRNA-125b level promotes invasive ability in p53-mutated cells via PI3K/AKT activation by targeting of insulin-like growth factor-binding protein-3, thereby resulting in p53-mutated non-small-cell lung cancer patients with poor outcomes.