Interference Of Frizzled1(FZD1) Reverses Multidrug Resistance In Breast Cancer Cells Through The Wnt/β-catenin Pathway

[Background]Worldwide, breast cancer is the most common invasive cancer in women and the incidence has been on the rise. Chemotherapy is one of the most important tools in breast cancer treatment. However, the development of multidrug resistance (MDR), in which tumor cells become resistant to a wide spectrum of anti-cancer agents with different structures or different target sites, becomes the major obstacle in the treatment of breast cancer. The mechanisms of MDR are numerous and complex. The human multidrug resistance1gene (MDR1) encodes the membrane-located efflux pump P-glycoprotein (P-gp). Overexpression of MDR1/P-gp results in an active efflux of anticancer agents from cells, thus lowering intracellular drug concentrations and inducing cancer cells to resist chemotherapeutic drugs.MDRl/P-gp covers about55%of MDR in breast cancer thus reversing MDR mediated by P-gp will play an important role in the chemotherapy. Although several inhibitors/modulators of MDR1/P-gp have been developed, cytotoxic effects and adverse pharmacokinetics have prohibited their use. Immunology treatment, natural drug therapy and gene therapy also lack the potency to reverse the MDR phenotype completely. Recently, molecular targeted therapy has shown promise in preclinical studies, based on numerous signaling pathways that play roles in the transcriptional regulation of MDR1/P-gp.MDR1has been demonstrated to be a direct target gene of the β-catenin/Tcf4transcriptional complex, thus, MDR1expression may be down-regulated by inhibiting the Wnt/β-catenin pathway. The Wnt/β-catenin signaling pathway plays a key role in conlling multiple aspects of tumor development, including breast cancer. In this pathway, interactions of Wnt proteins with the cell surface Frizzled receptors lead to free β-catenin translocating to the nucleus, where it binds to the transcription factor TCF/LEF to regulate target genes expression.The Wnt receptors Frizzled1and2(FZD1/2), which are known to be essential components of Wnt/β-catenin pathway, have been found to be overexpressed in advanced infiltrating ductal breast cancer. Recent report confirmed FZD1mediates chemoresistance in neuroblastoma through activation of the Wnt/beta-catenin pathway.We detected the expression of FZD1in the breast cancer cell lines MCF-7and MDA-MB-231, as well as in the multidrug resistant subline MCF-7/ADM. The results showed a stronger expression of FZD1and MDR1/P-gp at both mRNA and protein levels in ADM cells, compared with the sensitive cell lines MCF-7and MDA-MB-231. In addition, we treated MCF-7and MDA-MB-231cells with different concentration of anticancer drug doxorubicin and detected FZD1and MDR1mRNA expression using realtime PCR. As a result, we found both of them increased significantly inresponse to0.2μM and1μM doxorubicin, in a dose dependent manner. These results suggest potential roles of FZD1and the Wnt/p-catenin pathway in the development of acquired multidrug resistance in breast cancer cells. We propose that the silencing of FZD1by plasmid-mediated expression of small interference RNA (siRNA) might result in the down-regulation of MDR1/P-gp, and thus reverse multidrug resistance in MDR cells through the Wnt/β-catenin pathway.[Methods]1. We examined the expression of FZD1in the breast cancer cell lines MCF-7and MDA-MB-231, as well as in the multidrug resistant subline MCF-7/ADM by RT-PCR and Western blot. To obtain more MDR cell sublines, we transduced MDA-MB-231and MCF-7cell lines with the retroviral vector SF91m3PRE, containing the human MDR1cDNA.2. We treated MCF-7and MDA-MB-231cells with0,0.2μM,1μM and5μM doxorubicin respectively for72h and detected FZD1and MDR1mRNA expression using realtime PCR.3. Small hairpin siRNA sequences targeted at FZD1and non-targeting siRNAs (as negative controls) sequences were designed were selected and synthesized as64 oligonucleotides, annealed, and then cloned into the pSUPER. neo+GFP expression vector respectively. After comfirmed by enzyme digestion and DNA sequencing, the recombinant plasmids were extracted.4. The MDR cells were all transfected with pSUPER-siFZDl and pSUPER-siNotarget respectively. After another48h culture, RT-PCR was adopted to detected the expression of FZD1and MDR1mRNA, Western blot was used to analysis the FZD1and P-gp protein expression. Rh123efflux assays were performed to detecte the function of P-gp. MTT assay was used to assess the effect of FZD1silencing on the chemosensitivity of MDR cells to4anticancer drugs, doxorubicin, paclitaxel, cisplatin, and5-Fu. In addition, nuclear and cytoplasmic β-catenin proteins were detected by Western blot to reveal the activity of Wnt/β-catenin.5. Stably transfected ADM siFZDl cells were transduced with SF91m3PRE (ADM siFZD1/MDR1), to determine if the treatment could reverse increased cell sensitivity to anti-cancer drugs, which was caused by MDR1/P-gp.[Results]1. FZD1was found to have higher mRNA and protein levels in relation to MDR1/P-gp expression in ADM cells, compared to the sensitive cell lines MCF-7and MDA-MB-231.2. Both FZD1and MDR1increased significantly inresponse to0.2μM and1μM doxorubicin in MCF-7and MDA-MB-231cells, in a dose dependent manner.5μ M doxorubicn almost killed all the cells, however, FZD1and MDR1continued to increase in MDA-MB-231. But in MCF-7, there was a significant decrease in FZD1mRNA expression with an increase of MDR1.3. Semi-quantitative RT-PCR was performed to verify the effects of FZD1interference. The expression of MDR1and FZD1mRNA was down-regulated remarkably in comparison to the control. Western blot analyses showed that FZD1and MDR1/P-gp, in addition to cytoplasmic and nuclear β-catenin proteins, were down-regulated in response to FZD1interference. The P-gp efflux function was measured by flow cytometry. The retained fluorescence intensity in MCF-7cells was set as100%. After30min of incubation in Rh123-free culture medium, a rapid reduction in intracellular Rh123was observed in all MDR cells. However, the fluorescent retention in FZD1-silenced MDR cells obviously exceeded that of the control, indicating that the efflux function of biologically active MDR1/P-gp decreased after FZD1knock-down. MTT assay showed that IC50values of the four anti-cancer drugs all decreased remarkably after FZD1interference. In addition, when we transfected ADM siFZDl cells with SF91m3PRE, again allowing the overexpression of MDR1/P-gp, sensitivity to doxorubicin, paclitaxel, cisplatin, and5-Fu was significantly reversed.[Conclusions]1. FZD1was overexpressed in the multidrug resistant breast cancer cells, consistent with MDR1/P-gp expression. The silencing of FZD1decreased MDR1expression and reversed the multidrug resistance in MDR cells.2. The silencing of FZD1in breast cancer multidrug resistant cells blocked β-catenin translocation to the nucleus, revealed the inhibition of Wnt/β-catenin pathway, suggesting that FZD1interference dereased MDR1expression via Wnt/β-catenin pathway.3. FZD1appeared to mediate multidrug resistance by regulating the Wnt/β-catenin pathway, and is possible to be identified as a new molecular marker to predict multidrug resistance and target for MDR reversing in breast cancer cells.