| Multiple myeloma(MM) is one of the most common hematological malignancy, with plasma cells monoclonal hyperplasia and secretion of a large number of monoclonal immunoglobulin, characterized by easy relapse, metastasis and poor prognosis of patients. Twist1 is a transcription factor belongs to basic helix-loop-helix(b HLH) protein family. Twist1 plays an important role in embryonic development, especially in the formation of mesoderm and differentiation. In addition, Twist1 plays an important role in tumor cell proliferation, senescence, apoptosis, epithelial- mesenchymal transition, tumor microenvironment, metastasis, characteristic of cancer stem cell, angiogenesis, chemoresistance, etc. High expression of Twist1 was found in breast cancer, prostate cancer, liver cancer, lung cancer, ovarian cancer, bladder cancer, head and neck squamous cell carcinom and some non-epithelial tumors, such as glioma, osteosarcoma, malignant melanoma, acute myeloid leukemia, chronic lymphocytic leukemia and some types of lymphomas, and the expressions were related to tumor progression, metastasis and poor prognosis. In cancer, Twist1 decreased epithelial markers expression, increased mesenchymal markers expression, led to epithelial to mesenchymal transition. Epithelial cells acquire easy motion, migration of mesenchymal characteristics to promote the tumor invasion and metastasis.Myeloma patients prone to marrow infiltration, invasion of soft tissue. Extensive metastasis are seen in advanced stage, the spine is more often involved. High levels of IL- 6 and TNF-α were detected in the blood and bone marrow of patients with multiple myeloma, which associated with tumor progression and poor prognosis. IL-6 induces STAT3 phosphorylation in WM-266-4 human melanoma cells, resulting in transient upregulation of Twist, which is a key regulator of metastasis. The expression of N-cadherin, a protein downstream of Twist1, was also increased on the cell surface after treatment with IL-6. These cells showed enhanced invasiveness. In breast cancer, epithelial-mesenchymal transition induced by TNF-α requires NF-κB-mediated transcriptional upregulation of Twist1. But as to multiple myeloma, the relationship of IL- 6,TNF-α and Twist1 expression was not well understood.Currently, Twist1 expression and its significance in myeloma has not yet been reported. It is of great significance for the evaluation of prognosis and treatment to explore whether Twist1 involved in the occurrence and development of myeloma and its mechanism. Therefore, this study was to investigate expression and significance of Twist1 in multiple myeloma. Research paper includes three parts, Part one: High nuclear expression of Twist1 in the skeletal extramedullary disease of myeloma patients predicts inferior survival. Part two: The influences of biological behavior in myeloma cells after overexpression of Twist1. Part three: The effects on Twist1 expression by IL- 6,TNF-α in tumor microenvironment.In a word, this paper discusses the role of Twist1 in myeloma progress and the probable mechanism, which provides theoretical basis for the targeted treatment of multiple myeloma. Part one High nuclear expression of Twist1 in the skeletal extramedullary disease of myeloma patients predicts inferior survivalObjective: The aim of the study was to investigate the expression of epithelial to mesenchymal transition(EMT)-inducing transcription factors, including Twist1 and ZEB1, in skeletal extramedullary disease(EMD) of multiple myeloma(MM) patients and to clarify the effects on clinical outcomes.Methods:1 The expression of Twist1 and ZEB1 in the bone marrow(BM) and the masses of skeletal EMD from 70 MM cases with skeletal EMD and 30 MM patients without skeletal EMD were determined by immunohistochemistry.2 MVD in the BM and masses from skeletal EMD was highlighted by immunostaining endothelial cells with a rabbit monoclonal antibody to CD34.Results:1 the percentage of high nuclear staining for Twist1 was 24.3%(17/70) in skeletal EMD, which was significantly higher than in the BM of these patients as well as those without skeletal EMD(P=0.030 and P=0.011).2 The high nuclear expression rate of ZEB1 in the BM without skeletal EMD, BM with EMD, and mass of skeletal EMD was 30.0%(9/30), 30.3%(10/33), and 45.7%(32/70), respectively. The nuclear expression rate of ZEB1 in the masses of skeletal EMD was not significantly increased compared to the BM sections without and those with skeletal EMD(P = 0.185, P=0.197).3 Spearman correlation analysis demonstrated a significant positive association between Twist1 and ZEB1 expression in the nuclei of myeloma cells of skeletal EMD(r=0.350, P=0.003).The microvessel density(MVD, P=0.004) was significantly higher in patients with high nuclear expression of Twist1(Twist1-high) than in those with low expression. However, there was no significant difference in the MVD between the patients with strong nuclear ZEB1 expression and those with light nuclear expression( P=0.623).4 Patients with Twist1-high experienced a lower rate of progression-free survival(PFS, 11.8% vs. 35.0%, P= 0.000) and overall survival(OS, 52.5% vs. 83.7%, P=0.001) compared to those with low expression. Multivariate analysis showed that Twist1-high was independently associated with inferior PFS(HR=2.161; 95%CI: 1.116-4.183; P=0.022) and OS(HR=3.111; 95%CI: 1.114-8.685; P=0.030). In the univariate analysis, ZEB1 expression were only associated with OS.We concluded that Twist1-high is associated with a poor prognosis and may be correlated with angiogenesis in the skeletal EMD of MM patients. Part two The influences of biological behavior in myeloma cells after overexpression of Twist1Objective: In part one, we found high expression of Twist1 is associated with poor prognosis. But the specific mechanism of Twist1 in the occurrence and progress of multiple myeloma is not clear. we observed the effect of Twist1 expression through constructing lentivirus overexpression of Twist1 infected stable cell lines using human MM cell lines RPMI 8226 and LP1, and further reveal the role of Twist1 in the progress of MM.Methods:1 To build stable overexpression of Twist1 cell lines using human MM cell lines RPMI 8226 and LP1 with lentivirus.2 Western Blot and Real Time PCR were used to test transfection effect of Twist1.3 Cell proliferation was measured by CCK-8 assay after the cells were transfected with Twist1 gene.4 Apoptosis rate was assessed by flow cytometry(FCM) after the cells transfected with Twist1 gene.5 Transwell chamber test was used to meacure MM cell migration.6 The protein expression levels of VEGF were detected by western blot and ELISA after MM cells transfected with Twist1 gene.7 The protein expression levels of EMT related genes, E-cadherin、Vimentin、N-cadherin were detected by western blot.8 The growth inhibition rate and IC50 were measured by CCK-8 assay after the cells treated with bortezomib.Results:1 Multiple myeloma cells transdused with lentivirusRPMI8226、LP1 cells were transdused with lentiviral particles for Twist1 or negative control, then drug screened by puromycin. Red fluorescence was found in the more than 90% of the MM cells.2 Transfection effect evaluationMeasured by q-PCR and western blot, the expression level of Twist1 m RNA and protein both increased in RPMI 8226- Twist1 and LP1- Twist1, as compared to groups of blank and negative controls.3 The effect of Twist1 overexpression on the cell proliferationCCK-8 results showed that, after 24 h, the cell proliferation of the 8226-Twist1, LP1-Twist1 groups was significantly higher than that of the 8226-NC, LP1-NC groups, respectivly(both, P <0.05).4 Effect of Twist1 overexpression on apoptosis of MM cellsthe apoptosis rate of RPMI8226 cells was(7.14 ± 0.47)% in RPMI8226-NC group,(2.98 ± 0.41)% in RPMI8226-Twist1 group, and the difference of apoptosis rate between RPMI8226-Twist1 group and RPMI8226-NC group was significantly different(P<0.01. Samely, the apoptosis rate of the LP1-NC cells was(4.27 ± 0.34) %, and(1.75 ± 0.32)% in LP1-Twist1 group, and the difference of apoptosis rate between LP1-NC group and LP1-Twist1 group was significantly different(P<0.01). FCM results showed Twist1 inhibited apoptosis of MM cells.5 Effect of Twist1 overexpression on migration ability of multiple myeloma cells。For the transwell chamber test, the average number of cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the 8226-NC group,8226-Twist1 group were 58.00±6.56, 102.33±12.58 respectively. The difference between the 8226-NC group and the 8226-Twist1 group was statistically significant(P<0.05). Samely, The average number of cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the LP1-NC group, LP1-Twist1 group was 67.00±5.00, 125.33±9.61 respectively. And the difference between the LP1-NC group and the LP1-Twist1 group was statistically significant(P<0.05). The result showed that Twist1 overexpression improved migration ability of multiple myeloma cells。6 Expression of VEGF in MM cells was improved after Twist1 overexpression.VEGF expression, Measured by western blot, were found increased in cells of 8226-Twist1, LP1-Twist1 groups, as compared with groups of negative controls. At the same time, we detected the concentration of VEGF in tumor cell supernatant of Twist1 overexpressed and NC groups by Elisa, finding that 8226-Twist1, LP1-Twist1 groups had an elevated level, Compared with NC groups(both, P <0.05).7 Effect on protein expression of EMT related genes after Twist1 overexpression.Mesenchymal markers, Vimentin and N-cadherin expressions increased in RPMI 8226- Twist1 and LP1- Twist1, as compared to groups of blank and negative controls, while epithelial marker E-cadherin decreased.8 Changes on sensitivity to bortezomib of MM cells after Twist1 over expressionThe IC50 of 8226-NC and 8226-Twist1 treated with bortezomib for 48 h were 21.63±2.30 nmol/L vs 28.63±8.52 nmol/L by CCK 8 method. The resistance ratio of 8226-NC cells was higher than that of 8226-Twist1 cells treated with different concentration of bortezomib(P<0.05). Samely, The IC50 of LP1-NC and LP1-Twist1 treated with bortezomib for 48 h were 25.59±4.07nmol/L vs 29.55±11.32nmol/L by CCK 8 method. The resistance ratio of LP1-NC cells was higher than that of LP1-Twist1 cells treated with different concentration of bortezomib(P<0.05).The results implied that Twist1 mediated bortezomib resistance.These results indicated that overexpression of Twist1 in MM cells resulted in higher cell proliferation, decreased apoptosis, increased migration ability, improved VEGF expression and bortezomib resistance. In addition, Twist1 induced EMT, facilitated multiple myeloma progression. Part three The expression of Twist1 in myeloma cells treated with IL-6 or TNFαObjective: In part one, we found that patients with high expression of Twist1 experienced a poor prognosis. Study myeloma in vitro in the second part showed that Twist1 overexpression can promote the progress of MM through improving cell proliferation, migration, angiogenesis, EMT, and inhibting apoptosis and sensitivity to chemotherapy drugs. But where high expression of Twist1 in patients came from?And what factors influenced the expression of Twist1?it is not clear. Previous studies identified that IL-6 and TNFα were significantly increased in serum and bone marrow samples of patients with active MM compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFα than those with MM in plateau phase. In order to iluminate the expression of Twist1 and possible mechanism under different tumor microenvironment, Twist1, EMT related genes, NF-κB p65, p-NF-κBp65, STAT3 and p-STAT3 expressions at protein level were detected in RPMI8226 cells after treated with TNFα, IL-6 by Western Blot assay.Methods:1 Twist1 expressions at protein level were detected in RPMI8226 cells after treated with TNFα, IL-6 using Western Blot assay.2 EMT related genes, NF-κBp65, p-NF-κBp65, STAT3 and p-STAT3 expressions at protein level were detected in RPMI8226 cells after treated with TNFα, IL-6 by Western Blot assay.3 Pretreatment in RPMI8226 cells with NF-κB inhibitorRPMI8226 cells in logarithmic growth phase were preincubated for 2 h with 100μM PTDC(NF-κB inhibitor). Then the cells were treated with 40ng/ml TNFα for 24 h. At last, collecting cells and the expression of Twist1 was measured by western blot.4 Pretreatment in RPMI8226 cells with STAT3 inhibitorRPMI8226 cells in logarithmic growth phase were up-front treated with 1μM Cpd188(STAT3 inhibitor) for 1h. Then the cells were treated with 40ng/ml IL-6 for 24 h. At last, collecting cells and the expression of Twist1 was measured by western blot.Results:1 The effect on Twist1 expression with TNFα or IL-6 treatment.The results showed that Twist1 level was significantly increased in RPMI8226 cells treated with different concentration of TNFα or IL-6, which indicated that TNFα and IL-6 can both induce Twist1 expression. In addition, this effect was dose-dependent.2 Twist1 involved in the EMT- like effect induced by TNFα and IL-6.Mesenchymal markers Vimentin, Twist1, N-cadherin level increased in RPMI8226 cells when treated with TNFα or IL-6, while epithelial marker E-cadherin decreased. It suggested that TNFα and IL-6 can induce EMT. Twist1 involved in the process.3 TNFα induces Twist1 expression through the NF-κB Signaling Pathway.Treated with TNFα, nuclear NF-κBp65, p-NF-κBp65 and Twist1 expression increased in RPMI 8226 cells. When preincubated with PTDC(NF-κB inhibitor), Twist1 expression decreased. Therefore, It prompts that TNFα activates NF-κB pathway, TNFα induces Twist1 expression through the NF-κB Signaling Pathway.4 IL-6 induces Twist1 Expression through the STAT3 Signaling Pathway.Treated with IL-6, Twist1 and p-Stat3 expression increased in RPMI 8226 cells, when preincubated with Cpd188(STAT3 inhibitor), Twist1 expression decreased. Thus, we speculate IL-6 activates Twist1 Expression through the STAT3 Signaling Pathway.These results indicated that epithelial-mesenchyme transition induced by TNFα requires NF-κB-mediated transcriptional upregulation of Twist1. Epithelial-mesenchyme transition induced by IL-6 requires STAT3-mediated transcriptional upregulation of Twist1.Conclusions:1 the nuclear expression rate of Twist1 on myeloma cells in the masses of skeletal EMD from MM patients was significantly higher than in the BM of these patients as well as those without skeletal EMD. Twist1-high is associated with a poor prognosis and may be correlated with angiogenesis in the skeletal EMD of MM patients.2 Overexpression of Twist1 with lentivirus in MM cells resulted in high cell proliferation, decreasing apoptosis, increased migration ability, improved VEGF expression and bortezomib resistance. In addition, Twist1 induces EMT, facilitates multiple myeloma progression. Twist1 is a central regulator of epithelial-mesenchymal transition in myeloma cells. Twist1 might be a potential new therapeutic agent for patients with multiple myeloma.3 TNFα and IL-6 in the tumor microenviroment can both induce Twist1 expression.4 Epithelial-mesenchyme transition induced by TNFα requires NF-κB-mediated transcriptional upregulation of Twist1. Epithelial-mesenchyme transition induced by IL-6 requires STAT3-mediated transcriptional upregulation of Twist1. |
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