Jia-Wen Xu

Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial–mesenchymal transition of breast cancer cells

The epithelial–mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor κB (NF-κB) activation. On the other hand, we also found that the adriamycin-activated NF-κB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-κB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-κB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-κB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component.

RACK1: A superior independent predictor for poor clinical outcome in breast cancer.

We aimed to investigate the expression of RACK1 in breast cancer, evaluate its role in predicting prognosis and compare with commonly used biomarkers: Ki67, ER, PR and HER‐2 for patients with breast cancer. The RACK1 expression and its clinical significance were examined in 160 breast carcinoma patients using immunohistochemistry. Correlations of RACK1 expression with other commonly used biomarkers and survival analyses were assessed. Immunohistochemistry results showed that the number of RACK1 cases scoring 0, 1, and 2 were 66, 54, and 40, respectively. RACK1 staining was strongly related to clinical stage, histological grade, Ki67, ER, PR and HER‐2 (all p < 0.05). Consistently, all of the cases exhibiting RACK1 staining score 0 were survivors, whereas the majority (55.0%) of those exhibiting RACK1 staining score 2 were deaths. Kaplan‐Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter overall survival times (p < 0.001). Univariate and multivariate analyses revealed that RACK1, tumor size, lymph node metastasis, and HER‐2 were independent prognostic factors (all p < 0.05). Interestingly, receiver operator characteristic (ROC) curves showed that the ROC areas for RACK1, Ki67, ER, PR and HER‐2 were 0.833, 0.766, 0.446, 0.387, and 0.689, respectively, and the superiority of RACK1 in sensitivity and specificity as biomarker was demonstrated. To our knowledge, it is the first time to investigate the expression of RACK1, and identified that RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely used diagnostic index in breast carcinoma.

Involvement of EGFR in the promotion of malignant properties in multidrug resistant breast cancer cells.

Multidrug resistance is the most predominant phenomenon leading to chemotherapy treatment failure in breast cancer patients. Despite many studies having suggested that overexpression of epidermal growth factor receptor (EGFR) is a potent predictor of malignancy in cancers, systematic research of EGFR in multidrug resistant (MDR) breast cancer cells is lacking. In order to clarify the role of EGFR in MDR breast cancer cells, MCF7/Adr expressing relatively higher EGFR, and its parental cell line MCF7 expressing relatively lower EGFR, were chosen for this study. Knockdown of EGFR by siRNA in MCF7/Adr cells showed that EGFR siRNA inhibits cell migration, invasion and proliferation in vitro; converse effects were observed in MCF7 cells transfected with pcDNA3.0-EGFR plasmid. Moreover, we found that EGFR upregulated migration and invasion via EMMPRIN, MMP2 and MMP9 in addition to promoting cell cycle passage via elevation of cyclin D1 and CDK4 in MDR breast cancer cells. Interestingly, MCF7/Adr cells not expressing EGFR showed significant decrease of P-glycoprotein (P-gp) and ABCG2 expression levels, and became more sensitive to treatment of adriamycin (ADR) and paclitaxel (Taxol); the above results indicated that MDR of cancer cells is related to S-phase arrest. In conclusion, EGFR is an important factor enhancing the malignancy of MDR breast cancer cells, partially, inducing MDR. Anti-EGFR therapy may improve outcome in chemorefractory breast cancer patients.

CD44/Cellular prion protein interact in multidrug resistant breast cancer cells and correlate with responses to neoadjuvant chemotherapy in breast cancer patients

Multidrug resistance (MDR) is one of the most important factors leading to chemotherapeutic failure in patients with breast cancer. The invasive/metastatic ability of MDR cells is strengthened compared with their parental cells. However, the mechanisms underlying MDR have not been fully elucidated. We found that CD44 and the cellular prion protein (PrPc) were both overexpressed in MDR cells (MCF7/Adr and H69AR). Subsequently, we chose the human breast cancer cell line MCF7/Adr, which is resistant to adriamycin, for further research. We discovered that PrPc physically and functionally interacted with CD44. The knockdown of CD44 or PrPc by siRNA in MCF7/Adr cells inhibited cell migration, invasion and proliferation in vitro. However, when the MCF7/Adr cells transfected with CD44 siRNA were incubated with 10 times the peak plasma concentration (PPC) of taxol, their invasive ability was again enhanced. In the breast‐carcinoma tissue samples, a significant correlation between the CD44 expression and the PrPc expression was observed in the postneoadjuvant‐chemotherapy (NAC) cases. Moreover, in Group 2, which was unresponsive to NAC, the CD44 and PrPc expression levels were significantly increased in the post‐NAC cases compared with the pre‐NAC cases using the paired‐samples t‐test. These data indicate that the CD44/PrPc interaction enhances the malignancy of breast cancer cells and affects the responses to neoadjuvant chemotherapy in breast cancer patients. Therefore, blocking the CD44/PrPc interaction may improve outcomes in chemorefractory breast cancer patients.

BCL2L10 protein regulates apoptosis/proliferation through differential pathways in gastric cancer cells†

The reason for and consequences of BCL2L10 down‐regulation in gastric carcinoma are poorly understood. Our aim was to investigate the function of the protein BCL2L10 in gastric carcinoma. We investigated BCL2L10 expression using quantitative real‐time PCR and immunoblotting. The methylation status of the BCL2L10 gene promoter was examined by bisulphite sequencing in fresh gastric normal and carcinoma tissues. We studied apoptosis and proliferation regulation in gastric cancer cell lines using flow cytometry, fluorescence staining, murine xenografting and immunoblotting. Pathway inhibitors were applied to confirm the major pathways involved in apoptosis or proliferation regulation. We observed significant correlations between lower BCL2L10 expression and CpG island hypermethylation of the BCL2L10 gene promoter in gastric carcinoma, apoptosis induced by over‐expressed BCL2L10 through mitochondrial pathways, and proliferation accelerated by BCL2L10 siRNA via the PI3K–Akt signalling pathway in gastric cancer cell lines. The pro‐apoptotic effect of BCL2L10 and growth promotion by BCL2L10 siRNA in gastric cancer cells suggest that it may be a tumour suppressor. Copyright

C/EBP-α ameliorates CCl(4)-induced liver fibrosis in mice through promoting apoptosis of hepatic stellate cells with little apoptotic effect on hepatocytes in vitro and in vivo.

CCAAT enhancer binding protein-α (C/EBP-α) is a transcript factor that regulates adipocyte differentiation and induces apoptosis in hepatic stellate cells (HSCs) in vivo and in vitro. However, the effect of C/EBP-α on hepatocytes in vivo remains unknown. This study investigated whether C/EBP-α exerts different apoptotic effects on hepatocytes and HSCs in vitro and in vivo. An adenovirus vector-expressing C/EBP-α gene was constructed, and a rat hepatic stellate cell lines (HSC-T6) and hepatocytes were transfected. A CCl4-induced liver fibrosis model in mice was also utilized. C/EBP-α induced apoptosis in hepatocytes and HSCs, but a significant difference between these cell types was observed in vitro. The mitochondrial pathway was involved in the apoptotic process and was predominant in HSC-T6 apoptosis. In the CCl4-induced mice liver fibrosis model, the administration of Ad-C/EBP-α decreased extracellular matrix deposition, including collagen and hydroxyproline content, and γ-GT levels, a marker of liver damage, were reduced significantly. Immunohistochemistry and TUNEL assay results showed an increase of apoptosis in HSCs, but hepatocytes were less affected. C/EBP-α induced differential apoptotic effects in hepatocytes and HSCs in vitro and in vivo. This differential effect could be a potential target for the treatment of hepatic fibrosis with little hepatic toxicity.

Loss of BCL2L10 protein expression as prognostic predictor for poor clinical outcome in gastric carcinoma.

Xu J D, Furuya T, Cao X X, Liu X L, Li Q Q, Wang W J, Xu J W, Xu Z D, Sasaki K & Liu X P
(2010) Histopathology57, 814–824