Resistance to chemotherapy is a recurrent issue in all cancer types. Because CML has the propensity to evolve from the CP to the AP and BC, with different responses to targeted therapy such as TK inhibitors, the molecular understanding of the mechanism of resistance in this neoplasia is advancing rapidly.
IM was the first molecularly targeted therapy rationally designed to specifically inhibit BCR-ABL TK activity [38
]. However, despite the effectiveness and good tolerability of IM, drug resistance does emerge. Although a hematological response is observed in over 95% of CP patients, primary resistant can occur [39
]. Otherwise, AP patients initially respond to IM but inevitably relapse with treatment-refractory disease because they acquire other mutations in addition to BCR-ABL amplification or kinase domain mutations [40
Since the demonstration that IM could be extruded from CML cells through Pgp action [43
has become an interesting subject in IM resistance studies. Our previous results indicated that ABCB1
is overexpressed in CML patients with intrinsic and acquired resistance to IM therapy compared to its expression in IM-responsive patients and healthy bone marrow donors see reference [30
]. This finding contrasts the idea that only individuals in the BC stage can exhibit ABCB1
overexpression, as suggested in the literature. Even though we have analyzed a small cohort of patients, our results corroborate those of Vasconcelos and colleagues see reference [24
], ratifying the importance of ABCB1
/Pgp in CML.
Interestingly, in CML, Jamieson and colleagues demonstrated that the granulocyte-macrophage progenitor pools from patients in BC and IM-resistant patients exhibited elevated levels of nuclear β-catenin compared with those in granulocyte-macrophage progenitors from healthy donors. Moreover, these progenitors acquired self-renewal ability [45
]. These data indicated the important role of the Wnt/β-catenin pathway in the self-renewal of CML progenitors and the acquisition of resistance. Wnt signaling involvement in TK inhibitor resistance was also demonstrated through its noncanonical pathway by Gregory and colleagues [46
]. Their results indicated that Wnt/Ca2+
/NFAT signaling maintains the survival of Ph+
leukemic cells under BCR-ABL inhibition. Altogether, we can speculate that the deregulation of Wnt signaling leads to key modifications in the biology of cells, allowing them to become intrinsically more resistant to drug therapy. However, a link between TK inhibitor resistance, Wnt signaling and drug efflux mechanisms such as MDR has never been considered. Despite some recent findings demonstrating that TCF consensus sites for β-catenin were functional in the ABCB1
promoter in other types of cancer, in CML, this regulation has not yet been investigated.
In this work, using MDR (overexpressing ABCB1
) and non-MDR cell lines (Lucena and K562, respectively) as models of CML, we demonstrated through EMSA and ChIP analyses that β-catenin binds to the TCF/LEF consensus binding site in the ABCB1
promoter. RT-qPCR analyses indicated that this binding occurred at 2-fold higher levels in Lucena cells than in K562 cells. As it has been demonstrated that the BCR-ABL protein can establish β-catenin expression in CML via
TK-mediated phosphorylation [47
], this finding suggests that in drug resistance in CML, the canonical Wnt pathway could be more strongly activated to positively regulate ABCB1
transcription, as previously evidenced in other types of cancer. Interestingly we could demonstrate by RT-qPCR, western blot and FACS, β-catenin higher expression in the MDR cell line. Furthermore, it is known that BCR is a negative regulator of the Wnt/β-catenin pathway. The fusion gene BCR-ABL
formed in CML decreases BCR transcription and BCR translation, and thus, there is no more BCR available to complex with β-catenin, leading to the translocation of β-catenin to the nucleus and Wnt/β-catenin pathway activation [48
To verify whether Wnt/β-catenin could regulate ABCB1 mRNA levels, we modulated the canonical pathway in CML cell lines. We demonstrated that activation of β-catenin signaling by LiCl treatment resulted in increased ABCB1 mRNA levels in both cell lines, with higher levels observed in K562 cells. As discussed previously, K562 cells exhibited less β-catenin binding to the ABCB1 promoter than Lucena cells. ABCB1 mRNA levels were not altered significantly in Lucena cells, suggesting that these cells exhibit saturation of the Wnt pathway. By silencing the pathway using a siRNA approach with WNT 1 and β-catenin knockdown, we verified the opposite—a significant decrease in ABCB1 mRNA levels in Lucena cells—indicating that when Wnt/β-catenin signaling is downregulated, ABCB1 transcription is also downregulated.
gene promoter presents seven TCF binding sites that have been until now poorly investigated in the regulation of gene transcriptional activity in cancer cells. However the more proximal TCF binding site have already been demonstrated to be functional in some cancer types see references [13
]. In this study we could demonstrate that all the seven TCF binding sites are potentially functional (supplementary material) and moreover we showed that two of them are functional in vivo
. Transfection experiments suggest that TCF binding sites can function in combination to enhance transcriptional activity.
These findings suggest that the canonical pathway of Wnt signaling regulates ABCB1
in CML. Several studies have demonstrated that quiescent CML stem cells do not undergo apoptosis even in the presence of high-dose or more potent TK inhibitors. Moreover, seminal studies demonstrated that a quiescent population of CML stem cells with BCR-ABL kinase domain mutation that is detectable before the initiation of IM therapy gives rise to leukemic cells that persist after treatment see references [1
]. These findings suggest that CML stem cells contribute to CML persistence and disease progression. A question to be further addressed is whether ABCB1
is also regulated by the canonical Wnt pathway in CML stem cells, as this pathway has also been correlated with self-renewal.
CML stem cells and normal hematopoietic stem cells (HSC) share several characteristics despite exhibiting remarkable differences. Self-renewal is an essential stem cell property, but self-renewal pathway activation has also been increasingly recognized as a hallmark of cancer. Interestingly, HSC and CML stem cells also exhibit increased levels of drug efflux-related molecules such as the product of ABCB1
, Pgp and decreased levels of OCT1, a transporter involved in the uptake of IM, rendering them more resistant to drugs [53