The Mechanism Of Wnt5a Alone Or Cooperates With CCL25 Regulating Adult T-cell Acute Lymphoblastic Leukemia Metastasis

Background and objective:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematopoietic disease, in which patients have been observed to exhibit diffuse infiltration of the bone marrow by immature T cell lymphoblasts, high white blood cell counts, mediastinal masses and frequent infiltration of the central nervous system at diagnosis. The T-ALL juvenile cells could infiltrate into extra marrow tissues, including meninges, thymus, gonad, liver, lung, spleen and lymph nodes, leading to corresponding lesions. The immortalized malignant proliferation and invasion degree of T-ALL cells have been closely related with prognosis in patients. The prognosis of T-ALL has been observed to gradually improve with the introduction of intensified chemotherapy, with cure rates in modern protocols reaching over 70% in children, with prognosis in adults still being poor.CCR9, a chemokine receptor, is highly expressed in acute T lymphocyte leukemia, were it has been reported to promote infiltration of leukemia cells, upon specific binding with its unique ligand CCL25. Wnt family are a type of secretory glycoprotein which are enriched with cysteine, and have been reported to be widely involved in tumor cell proliferation, migration and invasion, and is closely related with chemokines and their receptors. Recent reports have shown that Wnt can promote the expression of chemokines, which in turn promotes the expression of specific Wnt members. Furthermore, others researchers have reported that Wnt proteins are involved in specific chemokine mediated cell metastasis, with few reports focusing on mechanism related to CCL25/CCR9 mediated T-ALL infiltration. However, the exactly relationship between CCL25/CCR9 and Wnt signaling pathways in the acute T lymphocyte leukemia has not been fully elucidated.In this study, we aimed at investigating the relationship between CCL25/CCR9 and Wnt family in adult T-ALL, focusing on the specific Wnt members that might interact and regulate the CCL25/CCR9 pathway, in order to provide theoretical foundation for demonstrating the mechanisms by which CCL25/CCR9 and Wnt promoting infiltration (metastasis) in adult T-ALL, thus providing new potential therapeutic targets.Research contents and methods:Using CCR9 high expressing adult T-ALL cell line MOLT4 as a cell model. RT-PCR was used to detect the effect of CCL25 on the expression of Wnt members in MOLT4 cells. Upon screening the possibly altered Wnt members in MOLT4 cells, Real time PCR and Western Blot were used to verify the effect of CCL25 on the expression of Wnt5a in MOLT4 cells. Western Blot was also used to analyze the effect of PKC on CCL25 induced Wnt5a expression in MOLT4 cells, in order to demonstrate the effect and mechanism of CCL25 on the expression of Wnt5a in MOLT4 cells.Gene Set Enrichment Analysis was utilized to predict the possible biological functions that Wnt5a involves in adult T-ALL. Western Blot, Transwell assay and Matrigel-Transwell assay were used to investigate the effect of Wnt5a, and the effect of inhibition or silencing of PI3K/Akt or RhoA on Wnt5a induced MOLT4 cell migration and invasion. Laser confocal microscopy and scanning electron microscopy were used to analyze the effect of Wnt5a on MOLT4 polarization and pseudopodia formation, respectively. The effects of inhibition or silencing PI3K/Akt or RhoA on Wnt5a induced MOLT4 cell polarization and pseudopodia formation also investigated, which were used to demonstrate the effect and mechanism of Wnt5a on MOLT4 cell migration and invasion.Transwell assay and Matrigel-Transwell assay were used to examine the effect of Wnt5a on CCL25 induced MOLT4 cell migration and invasion in vitro, respectively. MOLT4 cell xenograft experiment was used to analyze the effect of Wnt5a on CCL25 mediated MOLT4 cell infiltration in SCID mice. It was further utilized to assess the effect of Wnt5a on CCL25 induced RhoA activation in MOLT4 cells, which were used to determine the effect and mechanism of Wnt5a on CCL25 induced MOLT4 cell infiltration.Results:Wnt2b, Wnt5a and Wnt10b were found to be expressed in MOLT4 cells, however, only Wnt5a expression levels were significantly increased after CCL25 treatment. Inhibition of PKC significantly reduced CCL25 induced Wnt5a expression in MOLT4 cells, while activation of PKC significantly promoted Wnt5a expression.Gene Set Enrichment Analysis results showed that Wnt5a is associated with several migration and invasion related biological processes in adult T-ALL. Transwell assay and Matrigel-Transwell assay showed that Wnt5a induced MOLT4 cell migration and invasion, which was then significantly reduced by inhibition or silencing of PI3K/Akt or RhoA in vitro. Laser confocal microscopy results showed that Wnt5a induced MOLT4 cells polarization, which could be significantly reduced by silencing or inhibition of PI3K/Akt or RhoA. Scanning electron microscopy results further showed that Wnt5a significant induced MOLT4 cell lamellipodium and filopodia formation, and silence or inhibition of PI3K/Akt or RhoA significantly reduced Wnt5a induced MOLT4 cell lamellipodium and filopodia formation.In vitro results show that Wnt5a Silencing significantly reduced CCL25 induced MOLT4 cell migration and invasion, of which exogenous Wnt5a significantly promoted CCL25 induced MOLT4 cell migration and invasion. Furthermore, MOLT4 cell xenograft results showed that MOLT4 cell ratio in bone marrow of Wnt5a treatment group was significantly lower than in control group. Similary, MOLT4 cells in peripheral blood of CCL25 group, Wnt5a group and CCL25+Wnt5a group were also significantly lower as compared to the control group. The body weight of 4 MOLT4 cell xenograft groups were observed to be lower than normal, however, the organs/body weight ratio were higher than normal. Only CCL25+Wnt5a group was observed to have lesion in the liver. H&E staining results showed that ratio of MOLT4 cell infiltration into the liver of CCL25, Wnt5a and CCL25+Wnt5a groups are significantly higher than normal. Additionally, the ratio of MOLT4 cell infiltration into the liver of CCL25+Wnt5a group are significantly higher than CCL25 and Wnt5a groups, thus demonstrating that Wnt5a could alone or in combination with CCL25 promote adult acute T lymphocyte leukemia infiltrating into liver. Lastly, Western Blot results indicated that Wnt5a enhanced CCL25 induced MOLT4 RhoA activation, indicating that Wnt5a cooperates with CCL25 in promoting MOLT4 infiltration via enhancing CCL25 induced RhoA activation.Conclusion:1. CCL25/CCR9 promotes Wnt5a expression by promoting PKC expression and activation in MOLT4 cells.2. Wnt5a promoting MOLT4 cell migration and invasion via PI3K/AKT-RhoA pathway to enhance cell polarization and pseudopodium formation in vitro, Wnt5a is a potential target for therapy of adult T-ALL.3. Wnt5a cooperates with CCL25 to stimulate adult T-ALL cell metastasis (infiltration) via enhancing CCL25 induced RhoA activation.