ADRB3 induces mobilization and inhibits differentiation of both breast cancer cells and myeloid-derived suppressor cells

Metastatic tumors are mainly composed of neoplastic cells escaping from the primary tumor and inflammatory cells egressing from bone marrow. Cancer cell and inflammatory cell are remained in the state of immaturity during migration to distant organs. Here, we show that ADRB3 is crucial in cell mobilization and differentiation. Immunohistochemistry revealed ADRB3 expression is significantly more frequent in breast cancer tissues than in adjacent noncancerous tissues (92.1% vs. 31.5%). Expression of ADRB3 correlated with malignant degree, TNM stage and poor prognosis. Moreover, ADRB3 expression was markedly high in activated disseminated tumor cells, myeloid-derived suppressor cells (MDSCs), lymphocytes and neutrophil extracellular traps of patients. Importantly, ADRB3 promoted the expansion of MDSC through stimulation of bone marrow mobilization and inhibiting of the differentiation of immature myeloid cells. Furthermore, ADRB3 promoted MCF-7 cells proliferation and inhibited transdifferentiation into adipocyte-like cell by activating mTOR pathway. Ultimately, the MDSC-deficient phenotype of ADRB3 ^-/- PyMT mice was associated with impairment of mammary tumorigenesis and reduction in pulmonary metastasis. Collectively, ADRB3 promotes metastasis by inducing mobilization and inhibiting differentiation of both breast cancer cells and MDSCs.Subject terms: Breast cancer, Breast cancer     

Deregulation of Apoptotic Factors Bcl-xL and Bax Confers Apoptotic Resistance to Myeloid-derived Suppressor Cells and Contributes to Their Persistence in Cancer

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.     

WDR13 promotes the differentiation of bovine skeletal muscle‐derived satellite cells by affecting PI3K/AKT signaling

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet β cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle‐derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.     

miR-143 inhibits proliferation and metastasis of nasopharyngeal carcinoma cells via targeting FMNL1 based on clinical and radiologic findings

Mounting evidence has reported that microRNA-143 (miR-143) is involved in the development of multiple cancers. To investigate the underlying mechanisms of miR-143 regulating proliferation and metastasis in nasopharyngeal carcinoma (NPC) cells, we evaluated the levels of miR-143 and formin-like protein 1 (FMNL1) in NPC tissues. The results of qRT-PCR and Western blot analysis showed that the expression of miR-143 was decreased, while FMNL1 was increased in NPC tissues. The expression of miR-143 was significantly elevated in NPC cells compared with that of human nasopharyngeal epithelial cells. The results of MiRcode prediction, dual-luciferase reporter, and Western blot analysis assays indicated that miR-143 negatively regulated the expression of FMNL1 (r² = 0.4365P = 0.0001). Overexperssion of miR-143 or FMNL1 knockdown inhibited cell proliferation, migration, and invasion in NPC cells (P < 0.05). Ectopic expression of FMNL1 undermined the inhibition effect of miR-143 on proliferation, migration, and invasion in NPC cells. The findings of this study revealed that miR-143 functioned as a tumor suppressor and inhibited the NPC progression by targeting FMNL1.     

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.     

Negative pressure wound therapy promotes muscle‐derived stem cell osteogenic differentiation through MAPK pathway

Negative pressure wound therapy (NPWT) has been revealed to be effective in the treatment of open fractures, although the underlying mechanism is not clear. This article aimed to investigate the effects of NPWT on muscle‐derived stem cell (MDSC) osteoblastic differentiation and the related potential mechanism. The cell proliferation rate was substantially increased in NPWT‐treated MDSCs in comparison with a static group for 3 days. There was no observable effect on the apoptosis of MDSC treated with NPWT compared with the control group for 3 days. The expression levels of HIF‐1α, BMP‐2, COL‐I, OST and OPN were increased on days 3, 7 and 14, but the expression level of Runx2 was increased on days 3 and 7 in the NPWT group. Pre‐treatment, the specific inhibitors were added into the MDSCs treated with NPWT and the control group. ALP activity and mineralization were reduced by inhibiting the ERK1/2, p38 and JNK pathways. The expression levels of Runx2, COL‐I, OST and OPN genes and proteins were also decreased using the specific MAPK pathway inhibitors on days 3, 7 and 14. There were no significant effects on the expression of BMP‐2 except on day 3. However, the expressions of the HIF‐1α gene and protein slightly increased when the JNK pathway was inhibited. Therefore, NPWT promotes the proliferation and osteogenic differentiation of MDSCs through the MAPK pathway.     

Myeloid-Derived Suppressor Cells in Murine Retrovirus-Induced AIDS Inhibit T- and B-Cell Responses In Vitro That Are Used To Define the Immunodeficiency

Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b⁺ Gr-1⁺ Ly6C⁺) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1–PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G^low/± Ly6C⁺ CD11b⁺-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.     

Small G protein signalling modulator 2 (SGSM2) is involved in oestrogen receptor-positive breast cancer metastasis through enhancement of migratory cell adhesion via interaction with E-cadherin

The function of small G protein signalling modulators (SGSM1/2/3) in cancer remains unknown. Our findings demonstrated that SGSM2 is a plasma membrane protein that strongly interacted with E-cadherin/β-catenin. SGSM2 downregulation enhanced the phosphorylation of focal adhesion kinase (FAK; Y576/577), decreased the expression of epithelial markers such as E-cadherin, β-catenin, and Paxillin, and increased the expression of Snail and Twist-1, which reduced cell adhesion and promoted cancer cell migration. Oestrogen and fibronectin treatment was found to promote the colocalization of SGSM2 at the leading edge with phospho-FAK (Y397). The BioGRID database showed that SGSM2 potentially interacts with cytoskeleton remodelling and cell-cell junction proteins. These evidences suggest that SGSM2 plays a role in modulating cell adhesion and cytoskeleton dynamics during cancer migration.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

RhoA-mediated FMNL1 regulates GM130 for actin assembly and phosphorylates MAPK for spindle formation in mouse oocyte meiosis

Formin-like 1 (FMNL1) is a member of Formin family proteins which are the actin nucleators. Although FMNL1 activities have been shown to be essential for cell adhesion, cytokinesis, cell polarization and migration in mitosis, the functional roles of mammalian FMNL1 during oocyte meiosis remain uncertain. In this study, we investigated the functions of FMNL1 in mouse oocytes using specific morpholino (MO) microinjection and live cell imaging. Immunofluorescent staining showed that in addition to its cytoplasmic distribution, FMNL1 was primarily localized at the spindle poles after germinal vesicle breakdown (GVBD). FMNL1 knockdown caused the low rate of polar body extrusion and resulted in large polar bodies. Time-lapse microscopic and immunofluorescence intensity analysis indicated that this might be due to the aberrant actin expression levels. Cortical polarity was disrupted as shown by a loss of actin cap and cortical granule free domain (CGFD) formation, which was confirmed by a failure of meiotic spindle positioning. And this might be the reason for the large polar body formation. Spindle formation was also disrupted, which might be due to the abnormal localization of p-MAPK. These results indicated that FMNL1 affected both actin dynamics and spindle formation for the oocyte polar body extrusion. Moreover, FMNL1 depletion resulted in aberrant localization and expression patterns of a cis-Golgi marker protein, GM130. Finally, we found that the small GTPase RhoA might be the upstream regulator of FMNL1. Taken together, our data indicate that FMNL1 is required for spindle organization and actin assembly through a RhoA-FMNL1-GM130 pathway during mouse oocyte meiosis.Key words: actin, FMNL1, golgi, polar body extrusion, spindle organization     

H2O2和水杨酸对陈化马铃薯切片交替呼吸途径影响的比较

外源5.0 mmol/L H2O2和0.1 mmol/L 水杨酸(salicylic acid, SA)处理均可明显提高陈化24 h的马铃薯切片的交替呼吸途径容量(Valt)及其与总呼吸的比值(Valt/Vt).应用交替氧化酶的单克隆抗体进行Western杂交的结果表明,H2O2和SA处理均可明显提高陈化马铃薯切片中交替氧化酶的表达水平.用氧同位素分辨法研究,结果表明:H2O2处理对陈化马铃薯切片中交替呼吸途径的实际运行没有影响,而SA处理对交替呼吸途径的实际运行具有明显的促进作用.上述结果表明,H2O2和SA对植物组织交替呼吸途径的影响存在差异,二者均可促进交替氧化酶的表达从而诱导交替呼吸途径容量的发生,但H2O2不影响其实际运行,而SA还可同时诱导其实际运行.     

The carboxy-terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration

The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1ɣ, but not FMNL1β or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1ɣ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1ɣ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1ɣ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.     

Loss of Gadkin Affects Dendritic Cell Migration In Vitro

Migration is crucial for the function of dendritic cells (DCs), which act as outposts of the immune system. Upon detection of pathogens, skin- and mucosa-resident DCs migrate to secondary lymphoid organs where they activate T cells. DC motility relies critically on the actin cytoskeleton, which is regulated by the actin-related protein 2/3 (ARP2/3) complex, a nucleator of branched actin networks. Consequently, loss of ARP2/3 stimulators and upstream Rho family GTPases dramatically impairs DC migration. However, nothing is known yet about the relevance of ARP2/3 inhibitors for DC migration. We previously demonstrated that the AP-1-associated adaptor protein Gadkin inhibits ARP2/3 by sequestering it on intracellular vesicles. Consistent with a role of Gadkin in DC physiology, we here report Gadkin expression in bone marrow-derived DCs and show that its protein level and posttranslational modification are regulated upon LPS-induced DC maturation. DCs derived from Gadkin-deficient mice were normal with regards to differentiation and maturation, but displayed increased actin polymerization. While the actin-dependent processes of macropinocytosis and cell spreading were not affected, loss of Gadkin significantly impaired DC migration in vitro, however, in vivo DC migration was unperturbed suggesting the presence of compensatory mechanisms.     

CHBP induces stronger immunosuppressive CD127+ M-MDSC via erythropoietin receptor

Erythropoietin (EPO) is not only an erythropoiesis hormone but also an immune-regulatory cytokine. The receptors of EPO (EPOR)₂ and tissue-protective receptor (TPR), mediate EPO’s immune regulation. Our group firstly reported a non-erythropoietic peptide derivant of EPO, cyclic helix B peptide (CHBP), which could inhibit macrophages inflammation and dendritic cells (DCs) maturation. As a kind of innate immune regulatory cell, myeloid-derived suppressor cells (MDSCs) share a common myeloid progenitor with macrophages and DCs. In this study, we investigated the effects on MDSCs differentiation and immunosuppressive function via CHBP induction. CHBP promoted MDSCs differentiate toward M-MDSCs with enhanced immunosuppressive capability. Infusion of CHBP-induced M-MDSCs significantly prolonged murine skin allograft survival compared to its counterpart without CHBP stimulation. In addition, we found CHBP increased the proportion of CD11b⁺Ly6G⁻Ly6C^high CD127⁺ M-MDSCs, which exerted a stronger immunosuppressive function compared to CD11b⁺Ly6G⁻Ly6C^high CD127⁻ M-MDSCs. In CHBP induced M-MDSCs, we found that EPOR downstream signal proteins Jak2 and STAT3 were upregulated, which had a strong relationship with MDSC function. In addition, CHBP upregulated GATA-binding protein 3 (GATA-3) protein translation level, which was an upstream signal of CD127 and regulator of STAT3. These effects of CHBP could be reversed if Epor was deficient. Our novel findings identified a new subset of M-MDSCs with better immunosuppressive capability, which was induced by the EPOR-mediated Jak2/GATA3/STAT3 pathway. These results are beneficial for CHBP clinical translation and MDSC cell therapy in the future.Subject terms: Allotransplantation, Inflammatory diseases     

FHL1 regulates myoblast differentiation and autophagy through its interaction with LC3

Four and a half LIM domain protein 1 (FHL1) belongs to the FHL protein family and is predominantly expressed in skeletal and cardiac muscle. FHL1 acts as a scaffold during sarcomere assembly and plays a vital role in muscle growth and development. Autophagy is key to skeletal muscle development and regeneration, with its dysfunction associated with a range of muscular pathologies and disorders. In this study, we constructed FHL1-silenced or FHL1-overexpressed myoblasts to investigate its role in autophagy during the differentiation of chicken myoblasts into myotubules. Our data showed that FHL1 contributes to myoblast differentiation as measured through MyoG, MyoD, Myh3, and Mb mRNA expression, MyoG and MyHC protein expression and the morphological characteristics of myoblasts. The results showed that FHL1 silencing inhibited the expression of ATG5 and ATG7, meanwhile, immunofluorescence and immunoprecipitation showed that FHL1 and LC3 interacted to regulate the correct formation of autophagosomes. FHL1 inhibition increased cleaved caspase-3 and PARP abundance and promoted myoblast apoptosis. Furthermore, FHL1 rescued skeletal muscle atrophy through regulating the expression of Atrogin-1 and MuRF1. Taken together, these data suggested that FHL1 regulates chicken myoblast differentiation through its interaction with LC3.