Youshan Zhao

Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production

Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.

Down-regulation of Dicer1 promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells in patients with myelodysplastic syndrome.

Although it has been reported that mesenchymal stromal cells are unable to provide sufficient hematopoietic support in myelodysplastic syndrome, the underlying mechanisms remain elusive. In this study, we found that mesenchymal stromal cells from patients with myelodysplastic syndrome displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21. Senescent mesenchymal stromal cells from patients had decreased differentiation potential and decreased stem cell support capacity. Gene knockdown of Dicer1, which was down-regulated in mesenchymal stromal cells from patients, induced senescence. The differentiation and stem cell-supporting capacities were significantly inhibited by Dicer1 knockdown. Overexpression of Dicer1 in mesenchymal stromal cells from patients reversed cellular senescence and enhanced stem cell properties. Furthermore, we identified reduced expression in the microRNA-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression, a key senescence mediator, in Dicer1 knockdown cells. Moreover, we found that miR-93 and miR-20a expression levels were significantly reduced in mesenchymal stromal cells from patients and miR-93/miR-20a gain of function resulted in a decrease of cellular senescence. Collectively, the results of our study show that mesenchymal stromal cells from patients with myelodysplastic syndrome are prone to senescence and that Dicer1 down-regulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells. Dicer1 down-regulation seems to contribute to the insufficient hematopoietic support capacities of mesenchymal stromal cells from patients with myelodysplastic syndrome.

Senescence of bone marrow mesenchymal stromal cells is accompanied by activation of p53/p21 pathway in myelodysplastic syndromes

The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony‐forming potential, alteration of cytoskeleton, and increased senescence‐associated β‐galactosidase (SA‐β‐Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower risk but not higher risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addition, BMMSCs from MDS patients had impaired hematopoietic supporting function. Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not changed in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.

Impaired osteogenic differentiation of mesenchymal stem cells derived from bone marrow of patients with lower-risk myelodysplastic syndromes

The pathogenesis of myelodysplastic syndromes (MDS) has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) and osteoblasts are key components of the hematopoietic microenvironment. Here, we measured the expression of multiple osteogenic genes in 58 MSCs from MDS patients with different disease stages and subtypes by real-time PCR and compared the osteogenic differentiation of MSCs from 20 MDS patients with those of MSCs from eight normal controls quantitatively and dynamically. The mRNA level of Osterix and RUNX2, two key factors involved in the early differentiation process toward osteoblasts, was significantly reduced in undifferentiated MSCs from lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers; however, higher-risk MDS was normal. Finally, in bone marrow biopsy, the number of osteoblasts was significantly decreased in lower-risk MDS. These results indicate that MSCs from lower-risk MDS have impaired osteogenic differentiation functions, suggesting their insufficient stromal support in MDS.

Interleukin-17 enhances the production of interferon-γ and tumour necrosis factor-α by bone marrow T lymphocytes from patients with lower risk myelodysplastic syndromes

Lower risk myelodysplastic syndromes (MDSs) are characterised by increased apoptosis of haematopoietic cells in the bone marrow (BM). The mechanism driving this excessive apoptosis involves multiple immune molecules, including inflammatory cytokines such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukins (ILs). Interleukin‐17 (IL‐17) is the hallmark cytokine produced by CD4+ Th17 cells, and IL‐17 mediates activation of the adaptive T‐cell response inducing an inflammatory cytokine environment. However, little is known about the role of IL‐17 in MDS‐associated immune dysfunction.

Whole-exome and targeted sequencing identify ROBO1 and ROBO2 mutations as progression-related drivers in myelodysplastic syndromes

The progressive mechanism underlying myelodysplastic syndrome remains unknown. Here we identify ROBO1 and ROBO2 as novel progression-related somatic mutations using whole-exome and targeted sequencing in 6 of 16 (37.5%) paired MDS patients with disease progression. Further deep sequencing detects 20 (10.4%) patients with ROBO mutations in a cohort of 193 MDS patients. In addition, copy number loss and loss of heterogeneity (LOH) of ROBO1 and ROBO2 are frequently observed in patients with progression or carrying ROBO mutations. In in vitro experiments, overexpression of ROBO1 or ROBO2 produces anti-proliferative and pro-apoptotic effects in leukaemia cells. However, this effect was lost in ROBO mutants and ROBO-SLIT2 signalling is impaired. Multivariate analysis shows that ROBO mutations are independent factors for predicting poor survival. These findings demonstrate a novel contribution of ROBO mutations to the pathogenesis of MDS and highlight a key role for ROBO-SLIT2 signalling in MDS disease progression.

High expression of APAF-1 elevates erythroid apoptosis in iron overload myelodysplastic syndrome

Apoptotic protease-activating factor 1 (APAF-1) is a central component of the intrinsic pathway of apoptosis. Our study aims at searching the role of APAF-1 in iron overload myelodysplastic syndrome (MDS). Erythroid apoptosis rate, mRNA expression levels of APAF-1, and caspase-9 activity were determined by flow cytometry, quantitative real-time PCR, and colorimetric assay in MDS patients, respectively. In addition, K562 and MDS-L cell lines were incubated with different concentrations of ferric ammonium citrate (FAC) or ferric ammonium citrate + desferrioxamine (FAC + DFO) in vitro to observe the alteration in erythrocyte apoptosis rate, APAF-1 mRNA, and protein expression levels. Moreover, as control, erythroid apoptosis rate and APAF-1 mRNA expression were detected after silencing APAF-1 expression by endoribonuclease-prepared small interfering RNAs (esiRNAs) in K562 and MDS-L cell lines. Both erythroid apoptosis rate and APAF-1 mRNA expression of the iron overload (IO) group were significantly higher than those of the non-IO group (P < 0.001 and P < 0.001). There is a significant difference of caspase-9 activity between the IO group and the non-IO group (P < 0.001). Erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines significantly elevated after FAC incubation in different concentrations (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L), while erythroid apoptosis rate and APAF-1 mRNA expression in the FAC + DFO group declined (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). After silencing of APAF-1 expression with specific esiRNAs, erythroid apoptosis rate and APAF-1 mRNA expression of K562 and MDS-L cell lines markedly decreased (P < 0.001 and P < 0.001 for K562; P < 0.001 and P < 0.001 for MDS-L). APAF-1 plays an important role in iron-induced erythroid apoptosis increase in MDS.

Genomic loss of EZH2 leads to epigenetic modifications and overexpression of the HOX gene clusters in myelodysplastic syndrome

The role of EZH2 in cancer is complex and may vary depending on cancer type or stage. We examined the effect of altered EZH2 levels on H3K27 methylation, HOX gene expression, and malignant phenotype in myelodysplastic syndrome (MDS) cell lines and an in vivo xenograft model. We also studied links between EZH2 expression and prognosis in MDS patients. Patients with high-grade MDS exhibited lower levels of EZH2 expression than those with low-grade MDS. Low EZH2 expression was associated with high percentages of blasts, shorter survival, and increased transformation of MDS into acute myeloid leukemia (AML). MDS patients frequently had reductions in EZH2 copy number. EZH2 knockdown increased tumor growth capacity and reduced H3K27me3 levels in both MDS-derived leukemia cells and in a xenograft model. H3K27me3 levels were reduced and HOX gene cluster expression was increased in MDS patients. EZH2 knockdown also increased HOX gene cluster expression by reducing H3K27me3, and H3K27 demethylating agents increased HOX gene cluster expression in MDS-derived cell lines. These findings suggest genomic loss of EZH2 contributes to overexpression of the HOX gene clusters in MDS through epigenetic modifications.

Rigosertib as a selective anti-tumor agent can ameliorate multiple dysregulated signaling transduction pathways in high-grade myelodysplastic syndrome

Rigosertib has demonstrated therapeutic activity for patients with high-risk myelodysplastic syndrome (MDS) in clinical trials. However, the role of rigosertib in MDS has not been thoroughly characterized. In this study, we found out that rigosertib induced apoptosis, blocked the cell cycle at the G2/M phase and subsequently inhibited the proliferation of CD34+ cells from MDS, while it minimally affected the normal CD34+ cells. Further studies showed that rigosertib acted via the activation of the P53 signaling pathway. Bioinformatics analysis based on gene expression profile and flow cytometry analysis revealed the abnormal activation of the Akt-PI3K, Jak-STAT and Wnt pathways in high-grade MDS, while the p38 MAPK, SAPK/JNK and P53 pathways were abnormally activated in low-grade MDS. Rigosertib could markedly inhibit the activation of the Akt-PI3K and Wnt pathways, whereas it activated the SAPK/JNK and P53 pathways in high-grade MDS. A receptor tyrosine kinase phosphorylation array demonstrated that rigosertib could increase the activation of RET and PDGFR-β while reducing the activation of Tie2 and VEGFR2 in MDS cells. Taken together, these data indicate that rigosertib is a selective and promising anti-tumor agent that could ameliorate multiple dysregulated signaling transduction pathways in high-grade MDS.

Iron overload promotes erythroid apoptosis through regulating HIF-1a/ROS signaling pathway in patients with myelodysplastic syndrome

Erythroid apoptosis increases significantly in myelodysplastic syndrome (MDS) patients with iron overload, but the underlying mechanism is not fully clear. In this study, we aim to explore the effect of HIF-1a/ROS on erythroid apoptosis in MDS patients with iron overload. We found that iron overload injured cellular functions through up-regulating ROS levels in MDS/AML cells, including inhibited cell viability, increased cell apoptosis and blocked cell cycle at G0/G1 phase. Interestingly, overexpression of hypoxia inducible factor-1a (HIF-1a), which was under-expressed in iron overload models, reduced ROS levels and attenuated cell damage caused by iron overload in MDS/AML cells. And gene knockdown of HIF-1a got the similar results as iron overload in MDS/AML cells. Furthermore, iron overload caused high erythroid apoptosis was closely related with ROS in MDS patients. Importantly, the HIF-1a protein levels of erythrocytes elevated obviously after incubation with desferrioxamine (DFO) from MDS patients with iron overload, accompanied by ROS levels inhibited and erythroid apoptosis reduced. Taken together, our findings determine that the HIF-1a/ROS signaling pathway plays a key role in promoting erythroid apoptosis in MDS patients with iron overload.