De Wei Wu

Phosphorylation of paxillin confers cisplatin resistance in non-small cell lung cancer via activating ERK-mediated Bcl-2 expression.

Paxillin (PXN) is required for receptor tyrosine kinase-mediated ERK activation, and the activation of the Raf/MEK/ERK cascade has been linked with Bcl-2 expression. We hypothesized that phosphorylation of PXN by the EGFR/Src pathway might contribute to cisplatin resistance via increased Bcl-2 expression. We show that cisplatin resistance was dependent on PXN expression, as evidenced by PXN overexpression in TL-13 and TL-10 cells and PXN knockdown in H23 and CL1-5 cells. Specific inhibitors of signaling pathways indicated that the phosphorylation of PXN at Y118 and Y31 via the Src pathway was responsible for cisplatin resistance. We further demonstrated that ERK activation was also dependent on this PXN phosphorylation. Bcl-2 transcription was upregulated by phosphorylated PXN-mediated ERK activation via increased binding of phosphorylated CREB to the Bcl-2 promoter. A subsequent increase in Bcl-2 levels by a PXN/ERK axis was responsible for the resistance to cisplatin. Animal models further confirmed the findings of in vitro cells indicating that xenograft tumors induced by TL-13-overexpressing cells were successfully suppressed by cisplatin combined with Src or ERK inhibitor compared with treatment of cisplatin, Src inhibitor or ERK inhibitor alone. A positive correlation of phosphorylated PXN with phosphorylated ERK and Bcl-2 was observed in lung tumors from NSCLC patients. Patients with tumors positive for PXN, phosphorylated PXN, phosphorylated ERK and Bcl-2 more commonly showed a poorer response to cisplatin-based chemotherapy than did patients with negative tumors. Collectively, PXN phosphorylation might contribute to cisplatin resistance via activating ERK-mediated Bcl-2 transcription. Therefore, we suggest that Src or ERK inhibitor might be helpful to improve the sensitivity for cisplatin-based chemotherapy in NSCLC patients with PXN-positive tumors.

C-Myc suppresses microRNA-29b to promote tumor aggressiveness and poor outcomes in non-small cell lung cancer by targeting FHIT

The dual role of the microRNA-29 (miR-29) family in tumor progression and metastasis in solid tumors has been reported. Evidence for the role of miR-29 in tumor malignancy and its prognostic value in overall survival (OS) and relapse-free survival (RFS) in non-small cell lung cancer (NSCLC) remains conflicting. Mechanistic studies presented herein demonstrated that c-Myc suppressed the expression of miR-29b, promoting soft agar growth and invasion capability in lung cancer cells. Interestingly, the decrease in the expression of miR-29b by c-Myc is responsible for soft agar growth and invasiveness mediated by FHIT loss due to promoter methylation. Among patients, low expression of miR-29b and FHIT was more common in tumors with high c-Myc expression than in tumors with low c-Myc expression. Kaplan–Meier and Cox regression analysis showed that tumors with high c-Myc, low miR-29b and low FHIT expression had shorter OS and RFS periods than their counterparts. In conclusion, the decrease in the expression of miR-29b by c-Myc may be responsible for FHIT loss-mediated tumor aggressiveness and for poor outcome in NSCLC. Therefore, we suggest that restoration of the miR-29b expression using the c-Myc inhibitor might be helpful in suppressing tumor aggressiveness mediated by FHIT loss and consequently improving outcomes in NSCLC patients with tumors with low expression of FHIT.

Loss of TIMP-3 Promotes Tumor Invasion via Elevated IL-6 Production and Predicts Poor Survival and Relapse in HPV-Infected Non–Small Cell Lung Cancer

Human papillomavirus (HPV) 16/18 E6 oncoprotein is expressed in lung tumors and is associated with p53 inactivation. The tissue inhibitor of metalloproteinase 3 (TIMP-3) is essential for limiting inflammation; therefore, we expected that TIMP-3 loss might induce chronic inflammation, thereby promoting tumor malignancy as well as poor survival and relapse in patients with HPV-infected non-small cell lung cancer. In this study, the loss of TIMP-3 by loss of heterozygosity and/or promoter hypermethylation was more frequent in HPV16/18 E6-positive tumors than in E6-negative tumors. To explore the possible underlying mechanism, E6-negative TL4 and CL1-0 cells were transfected with an E6 cDNA plasmid. A marked decrease in TIMP-3 expression was caused by promoter hypermethylation via increased DNA (cytosine-5-)-methyltransferase 1 (DNMT1) expression. Mechanistic studies indicated that TIMP-3 loss promoted interleukin-6 (IL-6) production, which led to cell invasion and anchorage-independent growth on soft agar plates. Kaplan-Meier and Cox regression models showed that patients with low-TIMP-3/high-IL-6 tumors had shorter overall survival and relapse-free survival periods when compared with patients with high-TIMP-3/low-IL-6 tumors. In summary, loss of TIMP-3 may increase IL-6 production via the tumor necrosis factor α/nuclear factor κB axis, thereby promoting tumor malignancy and subsequent relapse and poor survival in patients with HPV-infected non-small cell lung cancer.

MicroRNA-21 promotes tumour malignancy via increased nuclear translocation of β-catenin and predicts poor outcome in APC-mutated but not in APC-wild-type colorectal cancer

MiR-21 has been associated with poor prognosis in colon adenocarcinomas. However, in our preliminary data, the prognostic value of miR-21 levels was observed only in adenomatous polyposis coli (APC)-mutated tumours, not in APC-wild-type tumours. We explored whether β-catenin nuclear translocation was synergistically promoted by miR-21 in APC-mutated cells but not in APC-wild-type cells. We enrolled 165 colorectal tumour to determine APC mutation, miR-21 levels and nuclear β-catenin expression by direct sequencing, real-time PCR and immunohistochemistry. Overall survival and relapse-free survival were analysed by Kaplan-Meier and Cox regression models. The mechanistic action of β-catenin nuclear translocation modulated by miR-21 and its effect on cell invasion were evaluated in a cell model. Positive nuclear β-catenin expression was more commonly occurred in APC-mutated tumours than in APC-wild-type tumours. High miR-21 levels were relatively more common in tumours with positive nuclear β-catenin expression than in those with negative nuclear β-catenin expression. APC-mutated tumours with high miR-21 levels had shorter overall survival and relapse-free survival periods compared with others. However, the prognostic value of miR-21 levels was not observed in APC-wild-type tumours. Phosphorylation of β-catenin at Ser552 via the miR-21-mediated PTEN/AKT axis plays a critical role in β-catenin nuclear translocation in APC-mutated cells but not in APC-wild-type cells. Moreover, nuclear β-catenin expression increased by miR-21 is responsible for the capability of invasiveness. In summary, nuclear translocation of β-catenin increased by miR-21 promotes tumour malignancy and a poor outcome in APC-mutated patients but not in APC-wild-type colorectal cancer.

Paxillin promotes tumor progression and predicts survival and relapse in oral cavity squamous cell carcinoma by microRNA-218 targeting

High-risk human papillomavirus (HPV) 16-infected oral cavity squamous cell carcinoma (OCSCC) differs significantly from non-HPV-infected OCSCC. However, the molecular pathogenesis of HPV-infected OCSCC remains unclear. Paxillin (PXN) has been reported to promote lung tumor progression by miR-218 targeting. In addition, expression of miR-218 has been shown to be reduced by HPV16 E6 in cervical cancer. We thus asked whether PXN can promote tumor progression by E6-reduced miR-218 in OCSCC, especially in HPV-infected OCSCC. Mechanistic studies demonstrated that PXN expression increased markedly upon E6-mediated reductions in miR-218, resulting in increased colony formation and invasion capabilities in HPV-infected OCSCC cells. Among tumor specimens, HPV16/18 infection was negatively associated with miR-218 expression and positively associated with PXN expression. Kaplan-Meier and Cox regression models demonstrated that patients with low-miR-218 tumors or high-PXN tumors exhibited shorter overall survival (OS) and relapse-free survival (RFS) than those with high-miR-218 tumors or low-PXN tumors. Interestingly, HPV-infected patients with low-miR-218, high-PXN tumors and both combinations exhibited the worst OS and RFS compared with patients in their counterparts. These observations in patients were consistent with the findings from the cell model. Therefore, we suggest that PXN might be targeted to suppress tumor progression and consequently to improve outcomes in OCSCC, especially in HPV-infected OCSCC.

FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slug-mediated PUMA reduction

Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor—perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome.

Reduction of microRNA-184 by E6 oncoprotein confers cisplatin resistance in lung cancer via increasing Bcl-2

MicroRNA-184 suppresses cell growth and survival via targeting c-Myc and Bcl- 2. We recently reported that miR-184 promotes tumor progression in non-small cell lung cancer via targeting CDC25A and c-Myc. We here hypothesized that miR-184 could be down-regulated by E6 oncoprotein to confer cisplatin resistance in NSCLC. Human papillomavirus (HPV) 16-positive lung cancer TL-1 and cervical cancer SiHa cells compared with HPV16-negative TL-10 and C33A cells were enrolled for E6 manipulation. MiR-184 expression levels were increased by E6-knockdown in TL-1 and SiHa cells, but decreased by E6-overexpression in TL-10 and C33A cells. The MTT assay showed that the inhibition concentration of cisplatin yielding for 50% cell viability was dependent on miR-184 levels. Bcl-2 de-targeted by E6-mediated miR- 184 reduction was responsible for cisplatin resistance. Luciferase reporter assay and real- time PCR analysis indicated that the miR-184 promoter activity and its expression were modulated by E6 and/or p53 manipulation. Chromatin immunoprecipitation (ChIP) assay confirmed that p53 was bound onto the miR-184 promoter and its binding activity was modulated by E6 and/or p53 manipulation. Among patients, high miR184 and high Bcl-2 mRNA expression was more commonly occurred in E6- positive tumors than in E6-negative tumors. Fifty-nine out of 136 patients receiving cisplatin-based chemotherapy were available for the retrospective study. Patients with low-mR-184, E6-positive, high-Bcl-2 tumors, and both combinations were more prevalently occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. In conclusion, a decrease in miR-184 level by E6 oncoprotein may predict unfavorable response to cisplatin-based chemotherapy in HPV-infected NSCLC patients via increasing Bcl-2 expression.

Bcl-2 stabilization by paxillin confers 5-fluorouracil resistance in colorectal cancer

5-Fluorouracil (5-FU) is chemotherapeutic agent widely used for the treatment of colorectal cancer. Unfortunately, advanced colorectal cancer is often resistance to such chemotherapy and poor outcome. An adaptor protein paxillin (PXN) is phosphorylated at Y31/Y118 (pPXN-Y31/Y118) by Src contributes to cell mobility and Ser (S)272 of PXN in LD4 domain is important to the interaction between PXN and Bcl-2. We thus hypothesized that pPXN-Y31/Y118 may be required for Bcl-2 protein stability via PXN interacting with Bcl-2 to confer 5-FU resistance in colorectal cancer. Mechanistically, pPXN-S272 is phosphorylated through pPXN-Y31/Y118-mediated p21 protein-activated kinase 1 (PAK1) activation and pPXN-S272 is required for PXN to interact with Bcl-2. The interaction between PXN and Bcl-2 is essential for Bcl-2 protein stability through phosphorylation of Bcl-2 at S87 (pBcl-2-S87) by pPXN-Y31/Y118-mediated ERK activation. An increase in Bcl-2 expression by PXN is responsible for resistance to 5-FU. The resistance to 5-FU can be abolished by inhibitor of Src and PAK1 or Bcl-2 antagonist in cell and animal models. Among patients, Bcl-2 expression is positively correlated with expression of PXN and pPXN-S272, respectively. Patients with high PXN/high Bcl-2 or high pPXN-S272/high Bcl-2 tumors are commonly to have an unfavorable response to 5-FU-based chemotherapy, compared with patients who have high PXN, high pPXN-S272 or high Bcl-2 tumors alone. Therefore, we suggest that Src, PAK1 or Bcl-2 inhibitor may potentially overcome the resistance of 5-FU-based chemotherapy and consequently to improve outcomes in patients with PXN/Bcl-2 and pPXN-S272/Bcl-2-positive tumors.

DDX3 promotes tumor invasion in colorectal cancer via the CK1ε/Dvl2 axis

DDX3, a subunit of CK1ε, phosphorylates Dvl2 to promote β-catenin activation. Overexpression of the Dvl2 protein results in potent activation of β-catenin/TCF signaling in colorectal cancer. Therefore, we hypothesized that DDX3 might promote tumor invasion via the CK1ε/Dvl2 axis due to β-catenin/TCF activation. Western blotting showed that β-catenin expression was decreased by DDX3 knockdown and increased by DDX3 overexpression in colorectal cancer cells. The TCF promoter activity and invasion capability were concomitantly increased and decreased by DDX3 manipulation in these cells. The invasion capability in colon cancer cells and xenograft lung tumor nodules induced by a DDX3-overexpressing T84 stable clone in tail-vein injection model were nearly suppressed by inhibitors of CK1ε (PF4800567) and β-catenin/TCF signaling (XAV939). Among colorectal cancer patients, DDX3 expression was positively correlated with the expression of pDvl2 and nuclear β-catenin in tumor tissues. The expression of pDvl2 occurred more frequently in high-nuclear than in low-nuclear β-catenin tumors. A prognostic significance of DDX3, pDvl2, and nuclear β-catenin on overall survival and relapse free survival was observed in this study population. We therefore suggest CK1ε or β-catenin/TCF signaling as potential targets for improving tumor regression and outcomes in colorectal cancer, particularly tumors with high-DDX3/high-nuclear β-catenin or high-DDX3/high-pDvl2/high-nuclear β-catenin expression.

Cytoplasmic localization of Nrf2 promotes colorectal cancer with more aggressive tumors via upregulation of PSMD4.

Differences in subcellular localization of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) have been associated with poor outcomes in human cancers. However, the prognostic value of subcellular localization of Nrf2 in colorectal cancer and the underlying mechanism in tumor invasion remain unknown. We enrolled tumors from colorectal patients to evaluate Nrf2, NQO1, and HO-1 expression by immunohistochemistry. NQO1 and HO-1 positive tumors showed nearly complete expression of Nrf2 in the nucleus and/or showed partial expression in the nucleus/cytoplasm (nNrf2); however, tumors negative for NQO1 and HO-1 showed almost complete expression of Nrf2 in the cytoplasm and/or partial expression in the nucleus/cytoplasm (cNrf2). Kaplan-Meier and Cox regression analysis indicated poorer overall survival in patients with cNrf2 tumors than with nNrf2 tumors. Cell models provided evidence that cNrf2, rather than nNrf2, was responsible for cell invasion and soft agar growth triggered by activation of the NF-κB/AKT/β-catenin cascade. Mechanistically, cNrf2 persistently increased PSMD4 expression by the HIF1α/β-catenin axis, whereas PSMD4 reciprocally enhanced Nrf2 nuclear export by increasing CRM1 expression through p53 degradation. The mechanistic action of the cell model was further confirmed with a nude mouse animal model in which xenograft tumors induced by cNrf2 were nearly completely suppressed by the proteasomal inhibitor carfilzomib or the β-catenin inhibitor XAV939. We therefore suggest that PSMD4 or β-catenin might be potential targets for suppressing tumor aggressiveness, and consequently, improving outcomes in patients whose tumors express cNrf2.