The High Expression of CFTR in Ph+ Acute Leukemia Regulates the BCR-ABL and Wnt/ -Catenin Signaling Pathway Via Interaction with PP2A Protein

Purpose The cystic fibrosis transembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily and mediates the transportion of Cl- and HCO3-. Recent studies have found that CFTR is not only an anion channel protein, but also is used as a regulator to regulate the function of other proteins through interactions of its PDZ domain with other proteins. Here, the expression of CFTR in Ph+ acute leukemia, and the role of CFTR in the regulation of BCR-ABL and its mediated classical Wnt/β-catenin signaling were investigated.

Methods The expression of CFTR protein in the normal human airway epithelial cell line HBE, Ph+ chronic granulocytic leukemia (CML) cell line K562 and Ph+ acute lymphoblastic leukemia (ALL) cell line SUP-B15, and Ph+ primary leukemic cells were examined by Western-blot assay; the effect of CFTRinh-172, a CFTR inhibitor, on BCR-ABL and Wnt/β-catenin signaling was investigated by Western-blot assay in Ph+ acute leukemia cell lines; the interaction between the CFTR, BCR-ABL and PP2A proteins in K562 cell line was analyzed by co-immunoprecipitation (co-IP)/Western-blot assay.

Results 1.Compared with the normal human airway epithelial cell line HBE (a positive control, expression value of CFTR =1), CFTR protein expression by Western-blot assay in K562 (value=2.921) and SUP-B15 (value=2.042) were higher; the CFTR expression in normal mononuclear cells(MNCs) showed very low level (mean value =0.068 ± 0.005); however, in 40 cases of B-ALL primary leukemia cells, the expression of CFTR ( values = 1.613) in 20 cases of Ph+ B-ALL was higher than that in 20 cases of Ph- B-ALL (CFTR values = 0.432); similarly, in 12 cases of CML primary leukemic cells, the expression of CFTR ( values = 1.360) in 6 cases of CML-blast crisis was higher than that in 6 cases of CML-chronic phase (CFTR values = 0.060). These results demonstrated that CFTR were over-expression in Ph+ acute leukemia. 2.CFTRinh-172 down-regulated the expression of CFTR, p-BCR-ABL and Wnt/β-catenin signaling (Dvl-2 and β-catenin were significantly reduced and p-GSK3β was significantly upregulated) in K562 and SUP-B15 cell lines. Imatinib also down-regulated expression of p-BCR-ABL and Wnt/β-catenin signaling, however, had no effect on CFTR expression. 3. After CFTR was down-regulated by CFTRinh-172 and CFTR shRNA, the expression of PP2A_A subunit increased and p-PP2A_C(Y307)/PP2A_C rate (the phosphorylation on Y307 site was responsible for PP2A inactivation) significantly reduced, meaning that the down-regulation of CFTR increased PP2A function. 4.The results of Co-IP confirmed that CFTR could combine with PP2A_A subunit (rather than PP2A_C subunit), and PP2A_A subunit binding with CFTR were reduced after the treatment of CFTRinh-172. 5.The combination of CFTRinh-172 and PP2A inhibitor okadaic acid not only inhibited CFTRinh-172-mediated up-regulation of PP2A_A subunit and significantly up-regulated p-PP2A_C/PP2A_C rate, and more importantly, restored CFTRinh-172-mediated down-regulation of p-BCR-ABL and β-catenin partially. The results demonstrated that CFTR directly regulate the expression and activity of BCR-ABL, and further regulate the classical Wnt/β-catenin signaling via the interaction with PP2A.

Conclusion The high expression of CFTR in Ph+ acute leukemia regulates the BCR-ABL and Wnt/-catenin signaling pathway via interaction with PP2A protein.