Metadherin Involved In The Pathogenesis Of Chronic Lymphocytic Leukemia Via Wnt/β-catenin And BCR Signaling Pathways

Chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL) is most popular leukemia in the elderly population, accounting for about 1/3 of the total number of all adult leukemia. The American Cancer Society estimates new patients with CLL will reach to 15,680 people in 2013. The prognosis between different individual differences obviously in CLL and it has no obvious correlation with the clinical classification of this disease. In recent years, with the application of fludarabine, cyclophosphamide combined with rituximab (Fludarabin + Cyclophosphamide + Rituximab, FCR) chemimmunology therapy regimens as the first-line treatment of CLL, complete remission rate and long-term survival of patients improved significantly. Even so, there are still 50% untreated CLL patients can not achieve complete remission treated with FCR. The chioce of regimens for patients who relapse or carry with poor prognostic factor is still need to further investigation. In recent years, the application of gene expression profiles enhanced the understanding of the pathogenesis in CLL, and a large number of genes were found involving in cancer development and signaling pathways regulation of CLL. Their dysregulation results in aberration regulation in signaling pathways which realize their pro-tumor effects in CLL, such as BCR, NF-κB, NOTCH and Wnt/β-catenin signaling pathway. Some of them can be treated as theraputic targets in CLL. CLL is a highly heterogeneous disease, with regard to the pathogenesis of CLL, there are still many unknowns field waiting to be discovered.Metadherin (MTDH) was first found in astrocyte cells which treated with TNF-a or infected with HIV. The research shows that MTDH is broad participating in a variety of physiological and pathological processes of the body, including the development, neurodegeneration and inflammation. The tumor related evidence about this multifunctional protein was first verified in a variety of tumor cell lines, and further experiments proven its role in metastasis of breast cancer lung in mice. MTDH almost expressed in all tumor cells and promoted tumors via a variety of mechanisms involved in tumor cell proliferation, survival and metastasis activity. As a key factor to promote the development of tumors, MTDH have been treated as oncogenes which can also used to evaluate prognostic of some tumours. This function of MTDH is mediated by multiple signaling pathways such as Wnt, PI3K/AKT, NF-κB, MAPK signaling pathways.Wnt signaling pathway plays important roles in embryonic development, nerve regeneration, and the body homeostasis and tumorigenesis process. There are three downstream transduction ways of Wnt signaling, and the canonical Wnt/β-catenin pathway have the closely relationship with tumors. The dysregulation of Wnt signaling was associated with the pathogenesis of gastric cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, thyroid cancer, ovarian cancer, skin cancer, endometrial cancer, breast cancer and so on. Wnt/β-catenin signaling pathway involved in development of a variety of non-Hodgkin’s lymphoma (NHL), such as mantle cell lymphoma, extranodal marginal zone lymphoma, cutaneous lymphoma, and diffuse large B-cell lymphoma. In CLL, Wnt family proteins are proven to be overexpression, such as Wnt3, Wnt5b, Wnt6 and LEF-1 and so on. Inhibiting the activity of the Wnt signaling pathway can reduce the survival of CLL cells and perform a therapeutic role.Microenvironment plays an important role in the development of CLL. In CLL, moleculars in microenvironment reacted with CLL cells and mediated its proliferation and resistant to apoptosis, as well as drug resistance. Inhibition of these signaling pathways is providing an alternative therapeutic way beyond traditional chemical immunotherapy regimen. BCR signaling pathway in CLL is one of the most important pathway. Clinical application of inhibiors such as fostamatinib, ibrutinib and idelalisib targeting SYK, BTK and PI3K8 in CLL highlights the importance role of BCR signaling pathway. Further investigate the function of it will helpful for us to understand the pathogenesis of CLL. Here, we also further explored the relationship between MTDH expression and BCR signaling pathway in CLL.Part I Metadherin expression and clinical significance in patients with chronic lymphocytic leukemiaObjective:MTDH is a new gene which cloned in recent years. Studies have shown that MTDH expression is associated with the biological behavior of tumor growth, invasion, and metastasis and so on. MTDH is a key factor to promote tumor development, which promote tumor progression by regulating multiple signaling pathways such as NF-KB, PI3K/AKT and Wnt/β-catenin signaling pathway. There was no report about whether MTDH expression or not in CLL. Here we detected MTDH expression in CLL comparing with B cells in normal controls which were collected from different tissue sources. And analyzed the correlation between MTDH expression and clinical characteristics of patients to preliminary assessment of whether MTDH involved in the pathogenesis of CLL.Methods:1. Specimen collection;2. Density gradient centrifugation of peripheral blood mononuclear cells. B cells were isolated by immunomagnetic beads and the purity was identified using flow cytometry;3. RNA extraction by Trizol method, and then performed with real-time quantitative RT-PCR analysis;4. Protein extraction for Western blot analysis;5. MTDH expression by flow cytometry;6. Statistical analysis.Results:1. MTDH is overexpression in CLL cell line MEC-1 in mRNA and Protein levels;2. Upregulation of MTDH in mRNA level was correlated with clinical staging of CLL The purity of B cells we used in these studies was over 85%. MTDH expression in 31 CLL B cells was significantly higher than 15 cases of age-related normal human peripheral blood B cells (P<0.0001). According to Rai staging classification, MTDH expression gradually increased from stage Ⅰ to stage Ⅳ CLL patients, but there is no statistically significant difference between patients with stage Ⅲ and Ⅳ. MTDH is overexpression in patients with higher expression of serum β2-MG (P=0.002) and lactate dehydrogenase (Lactate dehydrogenase, LDH, P= 0.004) than patients with its expression in the normal range. It’s irrelevant between MTDH expression and age (P= 0.378) or gender (P=0.223) of CLL.3. Overexpression of MTDH in protein level in CLL B cells: MTDH expression were detected in 27 out of 31 CLL cases, while there were no MTDH detected in 15 normal age-related peripheral blood mononuclear expression was not detected. (P<0.0001) Taking into account the differences in purity of B cells between the two groups, we further detected MTDH expression in normal B cells by flow cytometry. MEC-1 was used as a positive control for the experiment. In CLL patients the percentage of MTDH+ B cells is about 95%. The percentage of MTDH+ B cells coming from peripheral blood, cord blood Bone marrow and tonsils were 12.49%,2.13%,2.70% and 6.66%.Conclusion:1. MTDH expression increased in primary B cells of patients and CLL cell lines MEC-1. In CLL patients, MTDH overexpression in nearly 87% CLL patients which indicated MTDH as a tumor promoting oncogenes in CLL;2. mRNA expression of MTDH associated with Rai clinical stage classification of CLL and prognostic indicator β2-MG and LDH of serum, but no correlation with gender and age;3. For the less CLL patients with mutated IGVH or TP53, here we did not analysis its correlation with MTDH expression. The prognosis of CLL is intensively correlated with abnormal expression of these genes, which is one of our future research areas to developing the role of MTDH in CLL.Part Ⅱ The effect of Metadherin on the biological behavior of chronic lymphocytic leukemiaObjective:MTDH is highly expressed in many tumors and participates in the proliferation, metastasis, and chemo-resistance of tumor. Inhibiting MTDH expression in tumor cells can inhibite its proliferation and promote apoptosis. In our previous research, we confirmed MTDH is over-expressed in CLL, and in this part, we try to investigate the function of MTDH in CLL cell line MEC-1. And provide theoretical basis for a new biological therapy targeted for CLL.Methods:1. cell culture;2. Lentivirus -mediated small interference RNA silencing targeting MTDH gene in chronic lymphocytic leukemia;3. Immunofluorescence microscopy and flow cytometry to detect transfection efficiency of MEC-1;4. RNA extraction by Trizol method, and then performed with real-time quantitative RT-PCR analysis;5. Protein extraction for Western blot analysis;6. CCK-8 assay of cell proliferation;7. Annexin V-PE/7-AAD double staining to measure apoptosis;8. Statistical analysis.Results:1. To detect GFP fluorescence intensity by fluorescence microscopy, and flow cytometry to identify the percentage of GFP-positive cells, the results indicate that the transfection efficiency of MEC-1 is approximately 60-70%;2. MTDH gene interference successful in MEC-1 cells in mRNA and protein level;3. MTDH gene interference reduce proliferation 45% of MEC-1 cell (106±14.08 vs 60.55±8.321);4. MTDH gene interference prompted 17% of apoptosis in MEC-1 cell (6.550 ± 1.518 vs 23.79±3.753).Conclusion:1. MTDH participate in CLL proliferation and anti-apoptotic effects, and its a potential therapeutic target for CLL;2. Further investigate the relationship between MTDH and other signaling pathways in CLL will help us further understanding the pathogenesis of CLL.Part III Metadherin is involved in the regulation of Wnt/β-catenin pathway in chronic lymphocytic leukemiaObjective:MTDH involved in the regulation of multiple signaling pathways, its role in Wnt signaling has been elucidated in liver cancer, gastric cancer, and DLBCL. In CLL, there exists abnormal activation of the Wnt signaling pathway, inhibition of Wnt signaling may play a therapeutic role in CLL. In this section, we try to investigated the correlation between MTDH expression and Wnt/β-catenin signaling pathway.Methods:1. cell culture;2. Lentivirus-mediated small interference RNA silencing targeting MTDH gene in chronic lymphocytic leukemia;3. Immunofluorescence microscopy and flow cytometry to detect transfection efficiency of MEC-1;4. RNA extraction by Trizol method, and then performed with real-time quantitative RT-PCR analysis;5. Protein extraction for Western blot analysis;6. Statistical analysis.Results:1. MTDH interference result to the downregulation of Wnt signaling in MEC-1 lentiviral vector-mediated MTDH gene silencing reduce the LEF-1 expression in MEC-1 which consequently reduced mRNA levels of cyclinDl and c-myc gene, while no significant effect on P-catenin level;2. Statistical data indicate that there is a positive correlation between MTDH and LEF-1 expression in 14 CLL cases, r=0.879, P< 0.01. In normal human PBMC, they were both negative expression.Conclusion:1. MTDH involved in regulation of the Wnt signaling pathway;2. MTDH increased with the degree of LEF-1 expression, suggesting that there exist some regulation way which we may directly regulate the expression of LEF-1 will be the impact on the Wnt pathway in CLL MTDH in;3. This part is a preliminary discussion about the role of MTDH to regulate Wnt pathway, and the exact mechanism needs further study.Part IV Metadherin is involved in the regulating BCR signaling pathways in chronic lymphocytic leukemiaObjective:There exist some co-regulation signaling pathways between MTDH and BCR signaling pathway. Here, we try to investigate the relationship between BCR activation and MTDH expression.Methods:1. specimen collection;2. cell cultures3. BCR activation assay4. Lentivirus-mediated small interference RNA silencing targeting MTDH gene in chronic lymphocytic leukemia;5. Immunofluorescence microscopy and flow cytometry to detect transfection efficiency of MEC-1;6. RNA extraction by Trizol method, and then performed with real-time quantitative RT-PCR analysis;7. Protein extraction for Western blot analysis;8. Statistical analysis.Results:1. BCR activation signal promote proliferation of primary cells of CLLStudies have shown that CLL patients who are carrying um-IgVH, CD38 and ZAP-70 expression tend to proliferate under the stimulation of anti-IgM antibody, wherein the state of IgVH can predict the response more clearly. Taking into account these differences, we selected six CLL cells samples which carried um-IgVH and proliferation after stimulation by anti-IgM antibody in vitro stimulation for further research.2. The process of BCR activation is accompanied with MTDH upregulation;3. MTDH involved in the activation of BCR signaling in MEC-1 cell linesMEC-1 cell lines characterize with the um-IgVH, and anti-IgM antibody can stimulate cells to proote proliferate increase about 1.3 fold. Interference MTDH expression in MEC-1 can block the proliferative response which mediated by the activation of BCR signaling in MEC-1.Conclusion:1. BCR-mediated signal activation can increase the expression of MTDH in CLL;2. Interference MTDH expression in MEC-1 can block the proliferative response mediated by BCR activation;3. MTDH is a potential therapeutic target in CLL, and the therapeutic effect of MTDH partially mediated by blocking BCR signaling transduction.4. To investigate the function differences of MTDH in CLL patients with um-IgVH or m-IgVH would help us further understanding the relationship between MTDH and BCR signaling pathway.SignificanceOur findings first confirmed MTDH abnormal expression in CLL B cells in CLL patients and correlated with Rai clinical classification and serum prognostic indicator P2-MG and LDH. In CLL,87% of the patients with high expression of MTDH, and its expression cannot be detected in normal B cells derived from different tissues. The results suggest that MTDH could be a proto-oncogene involved in the development of CLL. Interference MTDH expression can inhibite cell proliferation and promote apoptosis of CLL cells. This is partially realized by interferring activity of Wnt signaling and BCR signaling. This suggests that MTDH is a potential therapeutic target in CLL. Because the small number of cases, we do not research its relationship with other clinical prognostic indicators such as TP53, IGVH rearrangement. The mechanisms by which MTDH regulate Wnt signaling pathway and BCR regulation in CLL are still not clear. This is the direction of our future research.