Knockdown of a Wnt Receptor FZD1 reduces MDR1/P-Glycoprotein Expression in Human Leukemia Cells through the Wnt/β-Catenin Signaling Pathway

Background: Multidrug resistance (MDR) expressed in leukemia cells is a major obstacle in the use of chemotherapy for leukemia treatment. The Wnt signaling pathway is essential for maintenance, activation, regulated self-renewal and proliferation in both normal hematopoietic and leukemia cells. Frizzled-1 (FZD1), part of the Wnt receptor family, has been reported to be associated with drug resistance in a number of solid cancers, but the involvement of FZD1 in leukemia is yet to be studied. Here, we investigated whether FZD1 is associated with MDR1/P-glycoprotein (P-gp) in multidrug resistance of leukemic cell lines and examined FZD1 and MDR1/P-gp expression levels in AML cells from patients.

Methods and Patients: Drug resistant cell lines K562/ADM, K562/Vin and NOMO-1/ADM as well as their parental cell lines were transduced with a GFP-containing FZD1 lentiviral vector (shFZD1) or a scrambled control (shC). The effect of FZD1 knockdown and change in MDR1/P-gp were confirmed by quantitative RT-PCR, western blot analysis, and flow cytometry. Cell growth was evaluated by cell counts over time and colony formation assays. The P-gp-mediated MDR phenotype was verified by assessing the rhodamine123 (Rh123) efflux. Drug sensitivity was examined in both the presence and absence of adriamycin (ADM), daunomycin (DNR) or vincrine (VCR). We also analyzed target genes downstream of the Wnt/β-catenin signaling pathway including β-catenin. Clinical samples were obtained from 112 AML patients with a normal karyotype at initial diagnosis after acquiring written informed consent.

Results: The expression level of FZD1 in drug resistant cells was higher than that of their parent cells. Effective knockdown of FZD1 was confirmed and FZD1 expression was reduced by 82% (K562/Vin) and 50%-61% (NOMO-1/ADM and K562/ADM) when compared to shC. Interestingly, knockdown of FZD1 also resulted in a significant reduction of MDR1 by 40%-50% in resistant cells. Western blotting showed a reduction in the P-gp level as well as the FZD1 protein in shFZD1 cells. Mean fluorescence intensities of P-gp were also reduced significantly in K562/Vin (50.6%±16.4%), K562/ADM (68.4%±16.3%) and NOMO-1/ADM (56.9%±0.6%) cells. Rh123 intracellular content in these shFZD1 cells significantly increased 2.5-3.4 folds, indicating a reduction in P-gp function. The cytotoxicity assay revealed restoration of drug sensitivity to a varying extent. In addition, cell growth and colony formation were significantly suppressed. To investigate whether these results were caused by the activation of the Wnt/β-catenin pathway, immunofluorescence of β-catenin was performed, and the results showed that accumulation of nucleic β-catenin was reduced after inhibition of FZD1. We then assessed β-catenin, TCF7L2, LEF1, CyclinD1, p21 and p27 target genes in shFZD1 cells. The expression levels of proteins revealed that β-catenin and TCF7L2 were significantly decreased in shFZD1 cells. Elevated p27 and decreased cyclin D1 protein levels were also observed.

A total of 112 untreated AML patients with normal karyotypes who received anthracycline-based chemotherapy were analyzed. Eighty-five patients achieved complete remission (CR), and the remaining 27 patients had no response (NR). Thirty-four of these 85 patients relapsed (CR-Re) after remission, and the remaining 51 patients maintained CR. No significant difference in FZD1 expression was observed between the CR and NR groups. However, a statistical difference was observed between the NR group and the CR group when the CR-Re group was excluded from the analysis (median 5.989 vs. 1.856; p = 0.003). Expression of FZD1 was also significantly higher in the CR-Re group than in the CR group (10.738; p = 0.002). The NR group had significantly higher MDR1/P-gp levels than the CR group. Furthermore, a statistically significant relationship was observed in the NR group between FZD1 and MDR1 (p = 0.005), MDR1 and P-gp expressions (p <0.05).

Conclusions: These data indicate that FZD1 promotes the activation of β-catenin/TCF7L2 and increases the proliferative capacity of cells through the regulation of cyclin D1 expression. FZD1 seems to modulate multidrug resistance through the Wnt/β-catenin signaling pathway, suggesting that AML patients who have higher FZD1 expression at diagnosis tend to relapse and show chemotherapy resistance. Therefore, FZD1 likely participates in the development of MDR in leukemic cells.