Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1.     

Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

CXCR7 mediates SDF1‐induced melanocyte migration

Melanoblasts are derived from the neural crest and migrate to the dermal/epidermal border of skin and hair bulges. Although melanoblast migration during embryogenesis has been well investigated, there are only a few reports regarding the migration of mature melanocytes. Here, we demonstrate that a chemokine, stromal‐derived factor‐1 (SDF1, also known as CXCL12), and one of its receptor CXCR7 regulate normal human epidermal melanocyte (NHEM) migration. We found that SDF1 induces the directional migration of NHEMs. Interestingly, although both CXCR4 and CXCR7 are expressed in NHEMs, blockade of CXCR4 using a CXCR4‐specific neutralizing antibody did not exert any influence on the SDF1‐induced migration of NHEMs, whereas blockade of CXCR7 using a CXCR7‐specific neutralizing antibody did influence migration. Furthermore, SDF1‐induced NHEMs migration exhibited the early hallmark events of CXCR7 signaling associated with MAP kinase activation. It is known that the phosphorylation of ERK through CXCR7 signaling is mediated by β‐arrestins. The treatment of NHEMs with SDF1 resulted in the phosphorylation of ERK in a β‐arrestin 2‐dependent manner. These results suggest that melanocytes may have a unique mechanism of migration via SDF1/CXCR7 signaling that is different from that of other cell types.     

Phosphoinositide 3‐kinases in cell migration

Cell migration is essential for many biological processes in animals and is a complex highly co‐ordinated process that involves cell polarization, actin‐driven protrusion and formation and turnover of cell adhesions. The PI3K (phosphoinositide 3‐kinase) family of lipid kinases regulate cell migration in many different cell types, both through direct binding of proteins to their lipid products and indirectly through crosstalk with other pathways, such as Rho GTPase signalling. Emerging evidence suggests that the involvement of PI3Ks at different stages of migration varies even within one cell type, and is dependent on the combination of external stimuli, as well as on the signalling status of the cell. In addition, it appears that different PI3K isoforms have distinct roles in cell polarization and migration. This review describes how PI3K signalling is regulated by pro‐migratory stimuli, and the diverse ways in which PI3K‐mediated signal transduction contributes to different aspects of cell migration.     

CXCR4‐SF1 bifunctional adipose‐derived stem cells benefit for the treatment of Leydig cell dysfunction‐related diseases

Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29⁺ CD44⁺ CD34^? CD45^? ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.     

Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells

CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and G(i) protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.     

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

Background

HIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown.

microRNA‐145‐3p inhibits non‐small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway

The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA‐145‐3p (miR‐145‐3p) in the development and progression of non‐small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR‐145‐3p, PDK1, and mTOR levels were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR‐145‐3p and siPDK1 to confirm the effect of miR‐145‐3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR‐145‐3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR‐145‐3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR‐145. Finally, levels of PDK1, mTOR, and phosphorylated‐mTOR were lower in cells transfected with miR‐145‐3p as well as those with siPDK1. These findings indicate that miR‐145‐3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.     

Paracrine effect of CXCR4‐overexpressing mesenchymal stem cells on ischemic heart injury

It has been reported that CXCR4‐overexpressing mesenchymal stem cells (MSC^CX4) can repair heart tissue post myocardial infarction. This study aims to investigate the MSCCX4‐derived paracrine cardio‐protective signaling in the presence of myocardial infarction. Mesenchymal stem cells (MSCs) were divided into 3 groups: MSC only, MSC^CX4, and CXCR4 gene‐specific siRNA‐transduced MSC. Mesenchymal stem cells were exposed to hypoxia, and then MSCs‐conditioned culture medium was incubated with neonatal and adult cardiomyocytes, respectively. Cell proliferation–regulating genes were assessed by real‐time polymerase chain reaction (RT‐PCR). In vitro: The number of cardiomyocytes undergoing DNA synthesis, cytokinesis, and mitosis was increased to a greater extent in MSC^CX4 medium‐treated group than control group, while this proproliferative effect was reduced in CXCR4 gene‐specific siRNA‐transduced MSC–treated cells. Accordingly, the maximal enhancement of vascular endothelial growth factor, cyclin 2, and transforming growth factor‐β2 was observed in hypoxia‐exposed MSC^CX4. In vivo: MSCs were labeled with enhanced green fluorescent protein (EGFP) and engrafted into injured myocardium in rats. The number of EGFP and CD31 positive cells in the MSC^CX4 group was significantly increased than other 2 groups, associated with the reduced left ventricular (LV) fibrosis, the increased LV free wall thickness, the enhanced angiogenesis, and the improved contractile function. CXCR4 overexpression can mobilize MSCs into ischemic area, whereby these cells can promoted angiogenesis and alleviate LV remodeling via paracrine signaling mechanism.     

Mitofusin2, a rising star in acute‐on‐chronic liver failure,triggers macroautophagy via the mTOR signalling pathway

Acute‐on‐chronic liver failure (ACLF) is a life‐threatening syndrome with poor prognosis. Several studies have begun to prove that mitochondria play a crucial role in liver failure. Mitofusin2 (Mfn2) plays a key role in maintaining the integrity of mitochondrial morphology and function. However, the role and underlying mechanisms of Mfn2 on cell autophagy of ACLF remain unclear. Our aim was to explore the effect of Mfn2 on several biological functions involving cell autophagy in ACLF. In this study, we constructed an ACLF animal model and a hepatocyte autophagy model, using adenovirus and lentivirus to deliver Mfn2 to liver cells, in order to assess the effect of Mfn2 on autophagy and apoptosis in ACLF. Furthermore, we explored the biological mechanism of Mfn2‐induced autophagy of ACLF using Western blotting, RT‐PCR and electron microscopy. We found that Mfn2 significantly attenuated ACLF, characterized by ameliorated gross appearance and microscopic histopathology of liver, and reduced serum AST, ALT, and TBIL levels. Mfn2 improved the expressions of LC3‐II, Atg5 and Bcl‐2 and down‐regulated the expression of P62 and Bax in ACLF. Like rapamycin, Mfn2 also significantly inhibited the expressions of p‐PI3K, p‐Akt and p‐mTOR in ACLF. In conclusion, our findings suggest that Mfn2 influences multiple biological functions of ACLF via the PI3K/Akt/mTOR signalling pathway. This study will provide a reliable theoretical basis for the application of Mfn2 as an effective target for ACLF treatment, reversing or delaying the process of ACLF.     

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.     

CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell

Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway.     

Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.