Lihua Lai

MicroRNA-494 Is Required for the Accumulation and Functions of Tumor-Expanded Myeloid-Derived Suppressor Cells via Targeting of PTEN

Myeloid-derived suppressor cells (MDSCs) potently suppress the anti-tumor immune responses and also orchestrate the tumor microenvironment that favors tumor angiogenesis and metastasis. The molecular networks regulating the accumulation and functions of tumor-expanded MDSCs are largely unknown. In this study, we identified microRNA-494 (miR-494), whose expression was dramatically induced by tumor-derived factors, as an essential player in regulating the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway. TGF-β1 was found to be the main tumor-derived factor responsible for the upregulation of miR-494 in MDSCs. Expression of miR-494 not only enhanced CXCR4-mediated MDSC chemotaxis but also altered the intrinsic apoptotic/survival signal by targeting of PTEN, thus contributing to the accumulation of MDSCs in tumor tissues. Consequently, downregulation of PTEN resulted in increased activity of the Akt pathway and the subsequent upregulation of MMPs for facilitation of tumor cell invasion and metastasis. Knockdown of miR-494 significantly reversed the activity of MDSCs and inhibited the tumor growth and metastasis of 4T1 murine breast cancer in vivo. Collectively, our findings reveal that TGF-β1–induced miR-494 expression in MDSCs plays a critical role in the molecular events governing the accumulation and functions of tumor-expanded MDSCs and might be identified as a potential target in cancer therapy.

Inducible MicroRNA-223 Down-Regulation Promotes TLR-Triggered IL-6 and IL-1β Production in Macrophages by Targeting STAT3

MicroRNAs are small non-coding RNA molecules that regulate gene expression by either translational inhibition or mRNA degradation. MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses, including TLR-triggered inflammatory response. Here we reported that the expression of microRNA-223 (miR-223) was significantly decreased in murine macrophages during activation by lipopolysaccharide (LPS) or poly (I∶C) stimulation. The inducible miR-223 down-regulation resulted in the activation of signal transducer and activator of transcription 3 (STAT3), which is directly targeted by miR-223, thus promoting the production of pro-inflammatory cytokines IL-6 and IL-1β, but not TNF-α. Interestingly, IL-6 was found to be a main factor in inducing the decrease in miR-223 expression after LPS stimulation, which formed a positive feedback loop to regulate IL-6 and IL-1β. Herein, our findings provide a new explanation characterizing the molecular mechanism responsible for the regulation of IL-6 production after TLR-triggered macrophage activation.

MicroRNA-92a negatively regulates Toll-like receptor (TLR)-triggered inflammatory response in macrophages by targeting MKK4 kinase.

Background: microRNAs (miRNAs) participate in innate immune responses. Results: miR-92a decreases rapidly in macrophages once stimulated with TLR ligands, and miR-92a controls inflammatory response by targeting MKK4/JNK/c-Jun pathway. Conclusion: TLR-mediated miR-92a reduction feedback enhances TLR-triggered inflammatory response. Significance: Our findings reveal a novel positive feedback loop in which TLR-reduced miR-92a expression functions to regulate the innate inflammatory responses. Toll-like receptors (TLRs) play a critical role in the initiation of immune responses against invading pathogens. MicroRNAs have been shown to be important regulators of TLR signaling. In this study, we have found that the stimulation of multiple TLRs rapidly reduced the levels of microRNA-92a (miRNA-92a) and some other members of the miRNA-92a family in macrophages. miR-92a mimics significantly decreased, whereas miR-92a knockdown increased, the activation of the JNK/c-Jun pathway and the production of inflammatory cytokines in macrophages when stimulated with ligands for TLR4. Furthermore, mitogen-activated protein kinase kinase 4 (MKK4), a kinase that activates JNK/stress-activated protein kinase, was found to be directly targeted by miR-92a. Similar to the effects of the miR-92a mimics, knockdown of MKK4 inhibited the activation of JNK/c-Jun signaling and the production of TNF-α and IL-6. In conclusion, we have demonstrated that TLR-mediated miR-92a reduction feedback enhances TLR-triggered production of inflammatory cytokines in macrophages, thus outlining new mechanisms for fine-tuning the TLR-triggered inflammatory response.

Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model

Aim:To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms.Methods:Methods: Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times.Results:Results: Piperine (35–280 μmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC50 values were 105±1.08 and 78.52±1.06 μmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70–280 μmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 μmol/L) significantly increased the percentage of cells in G2/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 μmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis.Conclusion:Conclusion: These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.

Downregulation of microRNA 99a in oral squamous cell carcinomas contributes to the growth and survival of oral cancer cells

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by either translational inhibition or mRNA degradation. miRNAs play pivotal roles in physiological functioning and pathological progression. The function of microRNA-99a (miR-99a) in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, we investigated the roles of miR-99a in OSCC development and the underlying mechanisms in 25 cases of primary OSCC tissues and Tca-8113 cells. The cells were analyzed using FACS analysis and western blotting. Results showed that the expression levels of miR-99a were markedly decreased in OSCC tissues compared with the adjacent non-tumor tissues (n=25). The results of in vitro experiments showed that miR-99a mimics significantly inhibited the proliferation of Tca-8113 cells, a tongue squamous carcinoma cell line, and that miR-99a mimics markedly induced the apoptosis of Tca-8113 cells. Furthermore, we demonstrated that mammalian target of rapamycin (mTOR) was directly targeted by miR-99a, as the overexpression of miR-99a in Tca-8113 cells downregulated the protein expression level of mTOR. Thus, our findings suggest that the downregulation of miR-99a in OSCC tissues is associated with tumor development. miR-99a regulates the growth and survival of OSCC cells and may be exploited as a biomarker and therapeutic target for patients with OSCC.

Cyclosporine A inhibits breast cancer cell growth by downregulating the expression of pyruvate kinase subtype M2.

The high proliferative rate of tumor cells leads to metabolic needs distinct from those of their normal counterparts. An embryonic- and tumor-specific isoform of the enzyme pyruvate kinase M2 (PKM2) is overexpressed in cancer cells to increase the use of glycolytic intermediates for macromolecular biosynthesis and tumor growth. We report that Cyclosporin A (CsA) can regulate the expression and activity of PKM2 in breast cancer cell lines MCF-7, MDA-MB-435 and MDA-MB-231. PKM2 was found to be highly expressed in the three breast cancer cell lines compared to normal primary breast cells. Treatment with CsA inhibited the viability of breast cancer cells in a time- and dose-dependent manner. CsA significantly downregulated the expression of PKM2 in breast cancer cells and decreased adenosine triphosphate (ATP) synthesis, which induced cancer cells to undergo necrosis. Furthermore, the growth suppression effect of CsA was impaired in MCF-7 cells when they were transfected with the PKM2 overexpression plasmid, suggesting that CsA was an effective inhibitor of PKM2-dependent proliferation of breast cancer cells. These results may provide new insights into the mechanism of CsA in cancer therapy.

IFN-γ producing T cells contribute to the increase of myeloid derived suppressor cells in tumor-bearing mice after cyclophosphamide treatment.

It has been reported that treatment with cyclophosphamide (CTX) as a chemotherapeutic drug in cancer patients or tumor-bearing mice can result in an increase in the proportion of myeloid derived suppressor cells (MDSCs) in blood and lymphoid organs. Here we sought to clarify the possible mechanism of this unwanted increase in proportion of MDSCs in tumor-bearing mice after CTX treatment. We found that both CD4(+) T cells and CD8(+) T cells underwent an expansion and activation before the increase of MDSCs in the early period of CTX treatment in 4T1 breast tumor-bearing mice. The proportion of MDSCs in nude mice lacking T cells after CTX therapy was comparable to that in nude mice without CTX treatment. T cell transfer to 4T1-bearing nude mice enhanced the proportion of MDSCs in tumor-bearing mice after CTX therapy. The co-culture of MDSCs and T cells in vitro also showed that CD4(+) T cells and CD8(+) T cells could facilitate the expansion and survival of MDSCs, and this effect was mediated by IFN-γ released by T cells. These results gave an explanation of the unwanted consequence resulted from CTX treatment in tumor-bearing mice. It also provided some insights into the strategies for eliminating the bad side of CTX treatment and to make it more effective in cancer therapy.

E3 ligase FBXW7 is critical for RIG-I stabilization during antiviral responses

Viruses can escape from host recognition by degradation of RIG-I or interference with the RIG-I signalling to establish persistent infections. However, the mechanisms by which host cells stabilize RIG-I protein for avoiding its degradation are largely unknown. We report here that, upon virus infection, the E3 ubiquitin ligase FBXW7 translocates from the nucleus into the cytoplasm and stabilizes RIG-I. FBXW7 interacts with SHP2 and mediates the degradation and ubiquitination of SHP2, thus disrupting the SHP2/c-Cbl complex, which mediates RIG-I degradation. When infected with VSV or influenza A virus, FBXW7 conditional knockout mice (Lysm+FBXW7f/f) show impaired antiviral immunity. FBXW7-deficient macrophages have decreased RIG-I protein levels and type-I interferon signalling. Furthermore, PBMCs from RSV-infected children have reduced FBXW7 mRNA levels. Our results identify FBXW7 as an important interacting partner for RIG-I. These findings provide insights into the function of FBXW7 in antiviral immunity and its related clinical significance.

Melatonin inhibits osteosarcoma stem cells by suppressing SOX9-mediated signaling

Aims: Melatonin (N‐acetyl‐5‐methoxytryptamine) has been reported to suppress epithelial‐mesenchymal transition and cancer stem cells in some types of cancer. However, the effects of melatonin on the osteosarcoma stem cells, epithelial‐mesenchymal transition and metastasis of osteosarcoma are still not clear. The present study was conducted to dissect the activity of melatonin on the osteosarcoma stem cells and the underlying mechanisms. Main methods: MTT, wound healing, transwell assay and western blotting were conducted to determine the effect of melatonin on osteosarcoma cell invasion and migration and downregulation of SOX9‐mediated signaling. Tumor spheroid assay and FACS analysis were performed to analyze the inhibition of the osteosarcoma stem cells. In vivo model for tumor formation and metastasis from single cell clone was used to evaluate the suppression of osteosarcoma stem cells by melatonin. Key findings: We demonstrated that melatonin potently suppresses the migration and invasion of osteosarcoma cells. Furthermore, melatonin significantly inhibits the sarcosphere formation of osteosarcoma stem cells and regulates EMT markers of osteosarcoma cells. In vivo mice model showed that melatonin significantly inhibits the initiation and metastasis of osteosarcoma. SOX9 is the key transcription factor mediating the effect of melatonin. Melatonin inhibited of cancer stem cell by down‐regulation of SOX9‐mediated signaling pathway in osteosarcoma. Significance: Collectively, these results deepen the understanding of the biological functions of melatonin and provide new insights for the intervention of osteosarcoma stem cells.

Protective function of interleukin 27 in colitis-associated cancer via suppression of inflammatory cytokines in intestinal epithelial cells

ABSTRACT Numerous studies have demonstrated that inflammation contributes to a variety of cancer formation, among them, colitis-associated cancer (CAC) represents a typical inflammation-related cancer. Interleukin 27 (IL-27) has been demonstrated to play an important role in inflammation-related disease. The effect of IL-27 in intestinal inflammation is controversial and its role in CAC is not elucidated yet. In our present study, we found that IL-27 has protective function in murine model of CAC through suppression of inflammatory cytokines in intestinal epithelial cells (IECs). IL-27Rα (WSX-1) deficiency promotes the CAC development in mice, which is driven by enhanced tumor cell proliferation, more intensive myeloid-derived suppressor cells (MDSC) accumulation in colon lamina propria and higher level of inflammatory cytokines and chemokines in IECs. The levels of IL-6, TNF-α, GM-CSF and CXCL1 triggered in vitro by toll-like receptor ligands are significantly upregulated in IECs from WSX-1 KO mice. Removal of commensal microorganism through antibiotic treatment in mice to eliminate TLR ligands deprives the protective function of IL-27 on CAC tumor growth. Thus, IL-27 suppresses CAC formation through an anti-inflammation mechanism targeting IECs and in turn resists the tumorigenesis. Hence, our study explained how IL-27 exerts its anti-inflammatory function on epithelial cells to fight against chronic-inflammation-associated cancer, which might provide new insights on the potential therapeutic strategies for cancer.