We have previously demonstrated that the CCR9/CCL25 signaling pathway plays an important role in drug resistance in human acute T-lymphocytic leukemia (T-ALL) by inducing activation of ERM protein with polarized distribution in T-ALL cell line MOLT4. However, the mechanism of action of the activated ERM protein in the drug resistance of MOLT4 cells induced by CCL25 remains uncharacterized. Here we investigated the mechanism of CCR9/CCL25-initiated drug resistance in CCR9-high-expressing T-ALL cells. Our results showed that 1) the function of P-gp was increased after treatment with CCL25; 2) P-gp colocalized and co-immunoprecipitated with p-ERM and F-actin in CCL25 treated cells; and 3) ERM-shRNA conferred drug sensitivity coincident with release of ERM interactions with P-gp and F-actin after treatment with CCL25. These data suggest it is pivotal that P-gp associate with the F-actin cytoskeleton through p-ERM in CCR9/CCL25 induced multidrug resistance of T-ALL cells. Strategies aimed at inhibiting P-gp-F-actin cytoskeleton association may be helpful in increasing the efficiency of therapies in T-ALL.

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Adult T-cell acute lymphoblastic leukemia (T-ALL) is a refractory leukemia. We previously showed that CCL25/CCR9 promotes T-ALL metastasis. In the present study, we assessed the effects of CCL25 on Wnt expression and the effects of Wnt5a and CCL25 on PI3K/Akt and RhoA activation. Transwell assays and mouse xenograft experiments were utilized to assess the effects of Wnt5a and CCL25 on MOLT4 cell invasion, migration and metastasis. The effects of Wnt5a on MOLT4 cell actin polarization and pseudopodium formation were examined using laser scanning confocal microscopy and scanning electron microscopy. CCL25 induced Wnt5a expression in MOLT4 cells by promoting protein kinase C (PKC) expression and activation. Wnt5a promoted MOLT4 cell migration, invasion, actin polarization, and lamellipodium and filopodia formation via PI3K/Akt-RhoA pathway activation. These effects were rescued by PI3K/Akt or RhoA knockdown or inhibition. Additionally, Wnt5a in cooperation with CCL25 promoted MOLT4 cell mouse liver metastasis and stimulated RhoA activation. These results show that CCL25/CCR9 upregulates Wnt5a by promoting PKC expression and activation in MOLT4 cells. This in turn promotes cell migration and invasion via PI3K/Akt-RhoA signaling, enhancing cell polarization and pseudopodium formation. These findings indicate that the PI3K/Akt-RhoA pathway is likely responsible for Wnt5a-induced adult T-ALL cell migration and invasion.

Metastatic disease remains the primary cause of death for individuals with T cell acute lymphoblastic leukemia (T‐ALL). microRNAs (miRNAs) play important roles in the pathogenesis of T‐ALL by inhibiting gene expression at posttranscriptional levels. The goal of the current project is to identify any significant miRNAs in T‐ALL metastasis. We observed miR‐146b‐5p to be downregulated in T‐ALL patients and cell lines, and bioinformatics analysis implicated miR‐146b‐5p in the hematopoietic system. miR‐146b‐5p inhibited the migration and invasion in T‐ALL cells. Interleukin‐17A (IL‐17A) was predicted to be a target of miR‐146b‐5p; this was confirmed by luciferase assays. Interestingly, T‐ALL patients and cell lines secreted IL‐17A and expressed the IL‐17A receptor (IL‐17RA). IL‐17A/IL‐17RA interactions promoted strong T‐ALL cell migration and invasion responses. Gene set enrichment analysis (GSEA) and quantitative polymerase chain reaction (qPCR) analysis indicated that matrix metallopeptidase‐9 (MMP9), was a potential downstream effector of IL‐17A activation, and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling was also implicated in this process. Moreover, IL‐17A activation promoted T‐ALL cell metastasis to the liver in IL17A −/− mouse models. These results indicate that reduced miR‐146b‐5p expression in T‐ALL may lead to the upregulation of IL‐17A, which then promotes T‐ALL cell migration and invasion by upregulating MMP9 via NF‐κB signaling.