HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.     

LncRNA CRNDE promotes cell proliferation,migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

     

LASP1 promotes proliferation,metastasis, invasion in head and neck squamous cell carcinoma and through direct interaction with HSPA1A

LIM and SH3 protein 1 (LASP1) is a specific focal adhesion protein that promotes metastasis in a variety of tumours. However, its role in head and neck squamous cell carcinoma (HNSCC) has not been fully validated. The purpose of this study was to analyse the interaction of LASP1 and its binding partner in HNSCC. The expression of LASP1 and HSPA1A in HNSCC was analysed by real‐time PCR and Western blot. The effects of LASP1 on the biology behaviour of HNSCC cell lines were observed in vivo and in vitro. Co‐immunoprecipitation analysis was performed to confirm the interaction between LASP1 and HSPA1A. LASP1 was highly expressed in HNSCC and associated with poor prognosis for patients. LASP1 also promoted cell proliferation, colony formation, invasion and cell cycle G2/M phase transition. Heat shock protein family A member 1A (HSPA1A) is identified as a chaperone protein of LASP1 and co‐localized in the cytoplasm. HSPA1A positively regulates the interaction of LASP1 with P‐AKT and enhances the malignant behaviour of HNSCC cells. LASP1 and HSPA1A are both up‐regulated in HNSCC, and directly binds to each other. Double inhibition of LASP1 and HSPA1A expression may be an effective method for the treatment of HNSCC.     

Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43/AIMP1

AIMP1 (previously known as p43) is first found as a factor associated with a macromolecular tRNA synthetase complex. However, it is also secreted and acts on diverse target cells such as endothelial cells, macrophages, and fibroblasts to control angiogenesis, inflammation, and dermal regeneration, respectively. We previously showed that AIMP1 induces the death of endothelial cell but proliferation of fibroblasts and activates macrophages. In this work, we found that elastase 2-cleaved AIMP1 retained its pro-apoptotic activity to endothelial cells but lost the growth-stimulatory activity to fibroblasts. To determine the functional domains responsible for each activity, we generated several deletion fragments of AIMP1 and compared the activities to the target cells. AIMP1 promoted endothelial cell death and caspase-3 activation through its 101-114 amino acid region, fibroblast proliferation through its 6-46 amino acid region, and endothelial migration through its 114-192 amino acid region as revealed by deletion mapping. Thus, this work revealed that AIMP1 uses different regions for its diverse extracellular activities.     

PTBP1 promotes the growth of breast cancer cells through the PTEN/Akt pathway and autophagy

Invasion and migration is the hallmark of malignant tumors as well as the major cause for breast cancer death. The polypyrimidine tract binding, PTB, protein serves as an important model for understanding how RNA binding proteins affect proliferation and invasion and how changes in the expression of these proteins can control complex programs of tumorigenesis. We have investigated some roles of polypyrimidine tract binding protein 1 (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in breast cancer tissues compared with normal tissues and the same result was confirmed in breast cancer cell lines. Knockdown of PTBP1 substantially inhibited tumor cell growth, migration, and invasion. These results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of metastasis. We further analyzed the relationship between PTBP1 and clinicopathological parameters and found that PTBP1 was correlated with her‐2 expression, lymph node metastasis, and pathological stage. This will be a novel target for her‐2(⁺) breast cancer. PTBP1 exerts these effects, in part, by regulating the phosphatase and tensin homolog‐phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/protein kinase B (PTEN‐PI3K/Akt) pathway and autophagy, and consequently alters cell growth and contributes to the invasion and metastasis.     

AIMP1/p43 protein induces the maturation of bone marrow-derived dendritic cells with T helper type 1-polarizing ability

AIMP1 (ARS-interacting multifunctional protein 1), previously known as p43, was initially identified as a factor associated with a macromolecular tRNA synthetase complex. Recently, we demonstrated that AIMP1 is also secreted and acts as a novel pleiotropic cytokine. In this study, we investigated whether AIMP1 induces the activation and maturation of murine bone marrow-derived dendritic cells (DCs). AIMP1-treated DCs exhibited up-regulated expression of cell-surface molecules, including CD40, CD86, and MHC class II. Additionally, microarray analysis and RT-PCR determinations indicated that the expression of known DC maturation genes also increased significantly following treatment with AIMP1. Treatment of DCs with AIMP1 resulted in a significant increase in IL-12 production and Ag-presenting capability, and it also stimulated the proliferation of allogeneic T cells. Importantly, AIMP1-treated DCs induced activation of Ag-specific Th type 1 (Th1) cells in vitro and in vivo. AIMP1-stimulated DCs significantly enhanced the IFN-gamma production of cocultured CD4+ T cells. Immunization of mice with keyhole limpet hemocyanin-pulsed AIMP1 DCs efficiently led to Ag-specific Th1 cell responses, as determined by flow cytometry and ELISA. The addition of a neutralizing anti-IL-12 mAb to the cell cultures that had been treated with AIMP1 resulted in the decreased production of IFN-gamma, thereby indicating that AIMP1-stimulated DCs may enhance the Th1 response through increased production of IL-12 by APCs. Taken together, these results indicate that AIMP1 protein induces the maturation and activation of DCs, which skew the immune response toward a Th1 response.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Long non‐coding RNA H19 promotes the proliferation and invasion of breast cancer through upregulating DNMT1 expression by sponging miR‐152

Long non‐coding RNA (lncRNA) H19 in tumors played important roles in various biological processes. However, the biological role and molecular mechanism of H19 in breast cancer are unclear. Here, we found that H19 was aberrantly upregulated in human breast tumor tissues and cells. A negative correlation between H19 and miR‐152 and positive correlation between H19 and DNMT1 mRNA were observed. Downregulation of H19 and DNMT1 significantly retarded breast cancer cell proliferation and invasion. H19 act as an endogenous sponge by directly binding to miR‐152. miR‐152 directly targeted DNMT1 and was regulated by H19. Besides, H19 overexpression dramatically relieved the inhibition of miR‐152 on DNMT1 expression. miR‐152 inhibition and DNMT1 overexpression obviously reversed the inhibitory effects of H19 downregulation on cell proliferation and invasion. In conclusion, H19 promoted proliferation and invasion of breast cancer through the miR‐152/DNMT1 axis, providing a novel mechanism about the occurrence and development of breast cancer.     

Identification and characterisation of a Theileria annulata proline‐rich microtubule and SH3 domain‐interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria‐induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co‐immunoprecipitation, we identified a CLASP1/CD2AP/EB1‐containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14‐3‐3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta‐p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain‐interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1‐binding SxIP motif, as well as functional SH3 domain‐binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non‐infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.     

Long noncoding RNA LINC00339 promotes laryngeal squamous cell carcinoma cell proliferation and invasion via sponging miR-145

Laryngeal squamous cell carcinoma (LSCC) is a very common neoplasm of the head and neck in the world. Long noncoding RNAs play key roles in cell infiltration, fate, apoptosis, and invasion. However, the functional role and expression of LINC00339 remains unclear in LSCC. In this study, we showed that the expression level of LINC00339 was upregulated in LSCC tissues and cell lines. LINC00339 silencing suppressed the proliferation, invasion, and epithelial-mesenchymal transition (EMT) progression of LSCC cells. In addition, we showed that LINC00339 acted as a sponge of miR-145, and LINC00339 silencing promoted the expression of miR-145 in Hep2 cell. Furthermore, the expression of miR-145 was lower in LSCC tissues than in their paired normal samples and the miR-145 expression level was negatively correlated with LINC00339 expression in LSCC tissues. The knockdown of miR-145 promoted the proliferation, invasion, and EMT progression of LSCC cells. Finally, we indicated that LINC00339 silencing inhibited the proliferation, invasion, and EMT progression of LSCC cells by suppressing the miR-145 expression. These data suggested that LINC00339 acted as an oncogene in the development of LSCC, partly by regulating the miR-145 expression.     

The activity of aminoacyl-tRNA synthetase-interacting multi-functional protein 1 (AIMP1) on endothelial cells is mediated by the assembly of a cytoskeletal protein complex

AIMP1 was first found as a factor associated with the aminoacyl-tRNA synthetase (ARS) complex. However, it is also secreted and acts on different target cells such as endothelial cells, macrophages, and fibroblasts as an extracellular regulator, respectively, of angiogenesis, inflammatory responses and dermal regeneration. AIMP1 has also been reported to suppress in vivo tumor growth. In this study, we investigated the signaling pathways activated by exogenous AIMP1 in an in vitro endothelial model. AIMP1 decreases EC viability through an α5β1 integrin-dependent mechanism and inhibits cell adhesion, is internalized and shows an asymmetric pattern of distribution and accumulation in cell protrusions. Experiments of affinity purification, pull down, and co-immunoprecipitation showed that AIMP1 interacts with four cytoskeletal proteins (filamin-A, α-tubulin, vinculin, and cingulin). α-Tubulin also gets phosphorylated upon cell treatment with AIMP1 and colocalization between AIMP1 and filamin-A as well as between AIMP1 and cingulin was observed through immunofluorescence assays. In this work, we propose that AIMP1 effect on EC adhesion is mediated by the assembly of a cytoskeletal protein complex on the cytosolic face of the cell membrane which could regulate cellular architecture maintenance and remodeling. Moreover, this activity is able to indirectly influence cell viability.     

Reverse Correlation between MicroRNA-145 and FSCN1 Affecting Gastric Cancer Migration and Invasion

MicroRNAs (miRs) play important roles in modulating gene expression during the processes of tumorigenesis and tumor development. Previous studies have found that miR-145 is down-regulated in the stomach neoplasm and is related to tumor migration and invasion. However, both the molecular mechanism and function of miR-145 in gastric cancer remain unclear. The present study is the first demonstration of the significant down-regulation of miR-145 expression in infiltrative gastric cancer compared to expanding gastric cancer. Additionally, correlation analyses revealed strong inverse correlations between miR-145 and FSCN1 expression levels in infiltrative gastric cancer. Furthermore, we demonstrated that miR-145 directly targets FSCN1 and suppresses cell migration and invasion in gastric cancer. Knocking down the expression of FSCN1 led to the suppression of migration and invasion in gastric cancer cells, and re-expressing FSCN1 in miR-145-overexpressing cells reversed their migration and invasion defects. Thus, we concluded that miR-145 regulates cell migration and invasion in gastric cancer primarily by directly targeting FSCN1.     

Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P(1-5). S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P(1), S1P(2) and S1P(3) all contribute positively to S1P-stimulated glioma cell proliferation, with S1P(1) being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P(5) blocks glioma cell proliferation, and inhibits ERK activation. S1P(1) and S1P(3) enhance glioma cell migration and invasion. S1P(2) inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P(2) also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P(2)-stimulated glioma invasion. Thus, while S1P(2) decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.