Mu Hao

Bone marrow stromal cells protect myeloma cells from bortezomib induced apoptosis by suppressing microRNA-15a expression.

Despite unsurpassed anti-tumor activity of bortezomib for multiple myeloma (MM), drug resistance has emerged as a challenge, especially when MM cells adhere to the stroma. This study aimed to determine whether bone marrow stromal cells (BMSCs) have a role in the development of chemoresistance in MM. Our data demonstrate that the secretion of interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and cell-to-cell contact with microenvironment-derived stromal cells from patients with multiple myeloma (MM-BMSCs) significantly decreased the sensitivity of myeloma cells to bortezomib treatment. Mechanistically, we found that microRNA (miRNA)- 15a expression was up-regulated in U266 and NCI-H929 cells treated by bortezomib, which was inhibited by MM-BMSCs. miRNA-15a transfected myeloma cells were arrested in G1/S checkpoint and secreted less VEGF compared to control transfected cells, although no significant difference was found in VEGF mRNA levels. In conclusion, our data suggest that via suppressing miRNA-15a expression, BMSCs provide survival support and protect myeloma cells from bortezomib induced apoptosis.

Umbilical Cord-Derived Mesenchymal Stem Cells Isolated by a Novel Explantation Technique Can Differentiate into Functional Endothelial Cells and Promote Revascularization

Stem cells transplantation holds great promise for the treatment of ischemic diseases through functional revascularization. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are also an ideal candidate for cell-based bioengineering. Herein, we report on the development of a simple and effective protocol to isolate UC-MSCs, and confirm their endothelial potential both in vitro and in vivo. UC-MSCs were isolated by a novel explantation technique and induced to differentiate into endothelial-like cells. Then UC-MSCs were transplanted into ischemic mouse model and cultured on 3D gel/MMT-CS composite scaffolds. Morphological and proliferation assessments show that sufficient UC-MSCs can be generated during a relatively short culture period with explantation technique. Increased expression of endothelial-specific markers (KDR and vWF), and functional markers (ac-LDL uptake and UEA-1 binding), indicate that functional endothelial progenitor cells are induced after 9 days of in vitro culture. In an ischemic hindlimb mouse model, the ratio of ischemic/nonischemic limb perfusion 4 weeks after MSCs transplantation reached 0.84 +/- 0.09. The capillary density of this group was 2.57-fold greater than that of sham-injected mice (P < 0.05). Immunofluorescence and immunohistological analyses indicate that MSCs may act to salvage the ischemic tissue by incorporating into the local vasculature. In vitro, UC-MSCs were observed to incorporate into 3D gel/MMT-CS composite scaffolds, to secrete extracellular matrix, to remain viable, and to retain their proliferation capacity. In conclusion, UC-MSCs isolated by novel yet simple explantation technique are well suited for application in the development of novel stem cell-based revascularization therapies.

The Impact of Clone Size on the Prognostic Value of Chromosome Aberrations by Fluorescence In Situ Hybridization in Multiple Myeloma

Purpose: Accumulating evidence indicates that intratumor heterogeneity is prevalent in multiple myeloma and that a collection of multiple, genetically distinct subclones are present within the myeloma cell population. It is not clear whether the size of clonal myeloma populations harboring unique cytogenetic abnormalities carry any additional prognostic value. Experimental Design: We analyzed the prognostic impact of cytogenetic aberrations by fluorescence in situ hybridization at different cutoff values in a cohort of 333 patients with newly diagnosed myeloma and 92 patients with relapsed myeloma. Results: We found that nearly all IgH-related arrangements were observed in a large majority of the purified plasma cells; however, 13q deletion, 17p deletion, and 1q21 amplification appeared in different percentages within the malignant plasma cell population. Based on the size of subclones carrying these cytogenetic aberrations, the patients were divided into four groups: 0%–10%, 10.5%–20%, 20.5%–50%, and >50%. Receiver-operating characteristics analysis was applied to determine the optimal cutoff value with the greatest differential survival and showed that the most powerful clone sizes were 10% for 13q deletion, 50% for 17p deletion, and 20% for 1q21 gains, which provided the best possible cutoffs for predicting poor outcomes. Conclusions: Our study indicated that the impact of clone size on prognostic value varies between specific genetic abnormalities. Prognostic value was observed for even a subgroup of plasma cells harboring the cytogenetic aberration of 13q deletion and 1q21 gains; however, 17p deletion displayed the most powerful cutoff for predicting survival only if the predominant clones harbored the abnormality. Clin Cancer Res; 21(9); 2148–56. ©2015 AACR.

Suppressing miRNA-15a/-16 expression by interleukin-6 enhances drug-resistance in myeloma cells

The bone marrow microenvironment facilitates the survival, differentiation, and proliferation of myeloma (MM) cells. This study identified that microRNA-15a and -16 expressions tightly correlated with proliferation and drug sensitivity of MM cells. miRNA-15a/-16 expression in MM cells was significantly increased after treatment with cytotoxic agents. The interaction of bone marrow stromal cells (BMSC) with MM cells resulted in decreased miRNA-15a/-16 expression and promoted the survival of the MM cells. Interleukin-6 (IL-6) produced by BMSCs suppressed the expression of miRNA-15a and 16 in a time- and dose- dependent pattern, with the suppression on miRNA-15a being more significant than on miRNA-16. miRNA-15a-transfected MM cells were found to be arrested in G1/S checkpoint, and the transfected MM cells had decreased growth and survival. In conclusion, our data suggest that via suppressing miRNA-15a and -16 expressions, IL-6 secreted by BMSCs promotes drug-resistance in myeloma cells.

Low serum miR-19a expression as a novel poor prognostic indicator in multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the clonal expansion of plasma cells. Despite continuing advances, novel biomarkers are needed for diagnosis and prognosis of MM. In our study, we characterized the diagnostic and prognostic potential of circulating microRNAs (miRNAs) in MM. Serum miRNA levels were analyzed in 108 newly diagnosed symptomatic MM patients and 56 healthy donors (HDs). Our analysis identified 95 dysregulated miRNAs in newly diagnosed MM patients. Of the 95 dysregulated miRNAs, dysregulation of miR‐19a, miR‐92a, miR‐214‐3p, miR‐135b‐5p, miR‐4254, miR‐3658 and miR‐33b was confirmed by quantitative reverse transcription PCR (RT‐qPCR). Receiver operating characteristic analysis revealed that a combination of miR‐19a and miR‐4254 can distinguish MM from HD with a sensitivity of 91.7% and specificity of 90.5%. Decreased expression of miR‐19a was positively correlated with international staging system advancement, del(13q14) and 1q21 amplification. Furthermore, downregulation of miR‐19a resulted in significantly decreased progression‐free survival (PFS) and overall survival (OS). Our analysis indicated that the poor prognostic correlation of miR‐19a expression was independent of genetic abnormalities in MM. Multivariate analysis revealed that miR‐19a was a significant predictor of shortened PFS and OS. Interestingly, although miR‐19a levels portend a poor prognosis, patients with low miR‐19a levels had an improved response to bortezomib compared to those with high miR‐19a profile. Patients with downregulated miR‐19a experienced a significantly extended survival upon bortezomib‐based therapy. These data demonstrate that the expression patterns of serum microRNAs are altered in MM, and miR‐19a levels are a valuable prognostic marker to identify high‐risk MM.

Elevated neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio and decreased platelet-to-lymphocyte ratio are associated with poor prognosis in multiple myeloma

Elevated inflammatory markers are associated with poor outcomes in various types of cancers; however, their clinical significance in multiple myeloma (MM) have seldom been explored. This study investigated the prognostic relevance of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio (MLR) in MM. Totally 559 MM patients were included in this study. NLR, PLR and MLR were calculated from whole blood counts prior to therapy. Kaplan-Meier curves and multivariate Cox proportional models were used for the evaluation of the survival. It has shown that newly diagnosed MM patients were characterized by high NLR and MLR. Elevated NLR and MLR and decreased PLR were associated with unfavorable clinicobiological features. Applying cut-offs of 4 (NLR), 100 (PLR) and 0.3 (MLR), elevated NLR, MLR and decreased PLR showed a negative impact on outcome. Importantly, elevated NLR and decreased PLR were independent prognostic factors for progression-free survival. Thus, elevated NLR and MLR, and decreased PLR predict poor clinical outcome in MM patients and may serve as the cost-effective and readily available prognostic biomarkers.

Features of Extramedullary Disease of Multiple Myeloma: High Frequency of P53 Deletion and Poor Survival: A Retrospective Single-Center Study of 834 Cases

BACKGROUND Multiple myeloma (MM) is a heterogeneous disease in which most patients have myeloma restricted to the bone marrow, and some patients develop extramedullary disease (EMD) at the time of diagnosis or during follow-up, and show different clinical characteristics and a dismal prognosis. PATIENTS AND METHODS We studied 834 consecutive MM patients in a single center in China and compared clinical features of patients with and without EMD. RESULTS In general, the prevalence of EMD was 4.8% at the time of diagnosis and 3.4% during follow-up, with a significant increase in recent years. MM patients with EMD at the time of diagnosis had remarkably greater prevalence of P53 deletion determined using fluorescence in situ hybridization (FISH) analysis (34.5% vs. 11.9%; P = .037) and higher level of lactate dehydrogenase (LDH) (P = .003) compared with patients without EMD. EMD relapse/progression during follow-up was correlated with EMD presentation at diagnosis, immunoglobulin (Ig)D subtype and P53 deletion in FISH analysis, but not previous treatment (thalidomide, bortizomib, or transplantation). With respect to prognosis, multivariate analysis showed that EMD was an independent adverse prognostic factor. The overall survival of patients with and without EMD at diagnosis were 16.5 and 40 months, respectively (P < .001), and the time to disease progression of the 2 groups was 11.5 and 25 months, respectively (P < .001). CONCLUSION MM patients with EMD at the time of diagnosis showed remarkably greater prevalence of P53 deletion in FISH analysis and higher LDH levels. EMD relapse/progression was correlated with EMD presentation at diagnosis, IgD subtype, and P53 deletion in FISH analysis, but not previous exposure to new drugs or transplantation. The presence of EMD involvement negatively affected survival.

Serum high expression of miR-214 and miR-135b as novel predictor for myeloma bone disease development and prognosis.

Multiple myeloma (MM) originates from malignant plasma cells, leading to multiple destructive lytic bone lesions that occur in more than 80% of MM patients. MicroRNAs have been reported to be involved in development of bone lesions in MM. However, the circulating microRNA as diagnostic and prognostic biomarkers for bone lesions has not been elucidated yet. In this study, we identified differentially expressed miRNAs that are potentially involved in myeloma-related bone disease in serum of MM patients. MiR-214 and miR-135b was shown to be increased in serum of MM patients with bone lesions. Serum level of miR-214 and miR-135b was highly correlated with the severity of lytic bone lesions and demonstrated as a diagnostic tool for identifying bone diseases based on results of a receiver operating characteristic analysis (ROC). In addition, patients with high levels of serum miR-214 had a dismal survival with significantly shortened progression free survival (PFS) and overall survival (OS). Interestingly, bisphosphonates treatment significantly extended PFS and OS in patients with higher level of miR-214 comparing to patients without bisphosphonates treatment. Taken together, our findings revealed the significance of circulating miR-214 and miR-135b levels in detection of bone disease and in prediction of prognosis of patients with multiple myeloma, suggesting its potential clinical applications. The result of this study also set the foundation for searching more circulating miRNA as biomarker for tumor bone lesions.

Epigenetic silencing of miR-137 induces drug resistance and chromosomal instability by targeting AURKA in multiple myeloma

Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Aberrant microRNAs (miRNAs) expression has been shown to be involved in the pathogenesis of MM. In this study, we further demonstrated that miR-137 was significantly downregulated in MM and negatively correlated with clinical prognosis. Moreover, we described the epigenetic regulation of miR-137 and its association with progression-free survival in MM patients. Furthermore, overexpression of miR-137 in MM cell line (miR-137 OE) increased its sensitivity to bortezomib and eprirubicin in vitro. Also, some high-risk genetic abnormalities in MM, including deletion of chromosome 1p22.2, 14q or 17p13, and gain of chromosome 1p22.2 were detected in NCI-H929 empty vector (NCI-H929 EV) treated cells but not in the NCI-H929 miR-137 overexpression (NCI-H929 miR-137 OE) cells. Luciferase reporter assays demonstrated that miR-137 targeted AURKA. Ectopic expression of miR-137 strongly reduced the expression of AURKA and p-ATM/Chk2 in MM cells, and increased the expression of p53, and p21. Importantly, miR-137 overexpression together with bortezomib treatment significantly inhibited tumor growth in MM xenograft model. Taken together, this study demonstrates that miR-137 is epigenetically silenced in MM, and overexpression of miR-137 could reduce drug resistance and overcome chromosomal instability of the MM cells via affecting the apoptosis and DNA damage pathways.

MicroRNA-15a/16-1 cluster located at chromosome 13q14 is down-regulated but displays different expression pattern and prognostic significance in multiple myeloma

MiRNA-15a/16-1 cluster located at chromosome 13q14 has been confirmed to regulate critical genes associated with cell proliferation, apoptosis and drug resistance in multiple myeloma (MM). However, little is known about their expression pattern and prognostic value in MM patients. In this study, we have analyzed the expression levels of miR-15a/16-1 in 117 MM patients (90 newly diagnosed, 11 relapsed and 16 remission patients) and 19 health donors (HDs) by quantitative real-time PCR. Our results indicated that the expression levels of miR-15a and 16-1 were down-regulated in newly diagnosed MM patients as compared to HDs (P = 0.025; P < 0.001) and independent of del(13q14). Downregulation of miR-15a was significantly associated with disease progression and poor prognosis while miR-16-1 seemed to be a good diagnostic marker to distinguish MM from HDs with area under the curve (AUC) of 0.864, sensitivity of 100% and specificity of 73%. Furthermore, patients with miR-15a < 2.35 (low expression group) had significantly shorter PFS (P < 0.001) and OS (P < 0.001). After adjustment of the established prognostic variables including del(13q), del(17p), amp(1q21) and high risk genetic abnormality, low miR-15a expression (<2.35) was still a powerful independent predictor for PFS (P = 0.008) and OS (P = 0.038). In addition, miR-15a combined with high β2-MG and high risk genetic abnormality can further identify the high-risk subpopulations. Therefore, our data suggest that the expression patterns of miR-15a/16-1 are different in MM patients, and miR-15a seems to be linked with disease progression and prognosis while miR-16-1 acts as a valuable diagnostic marker.