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Tumor-associated macrophages (TAM) have important roles in cancer progression, but the signaling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM and significantly associated with mortality in patients with non-small cell lung cancer. Cancer cells markedly induced Mincle expression in bone marrow-derived macrophages (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro. Mincle was predominately expressed in the M2-like TAM in non-small cell lung carcinoma and LLC tumors, and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro. Mechanistically, we discovered a novel Mincle/Syk/NF-kB signaling pathway in TAM needed for executing their TLR4independent protumoral activities. Adoptive transfer of Minclesilenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble-mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-kB signaling, therefore inhibiting the TAM-driven cancer progression in the syngeneic mouse cancer models. Thus, our findings highlight the function of Mincle as a novel immunotherapeutic target for cancer via blocking the Mincle/Syk/NF-kB circuit in TAM.

Natural killer (NK) cells, early effectors in anticancer immunity, are paralyzed by TGFβ1, an immunosuppressive cytokine produced by cancer cells. Development and activity of NK cells are largely inhibited in the Smad3-dependent tumor microenvironment. Here, we used genetic engineering to generate a stable SMAD3-silencing human NK cell line, NK-92-S3KD, whose cancer-killing activity and cytokine production were significantly enhanced under TGFβ1-rich condition compared with the parental cell line. Interestingly, we identified that the IFNG gene is a direct E4BP4 target gene. Thus, silencing of SMAD3 allows upregulation of E4BP4 that subsequently promoting interferon-γ (IFNγ) production in the NK-92-S3KD cells. More importantly, NK-92-S3KD immunotherapy increases the production of not only IFNγ, but also granzyme B and perforin in tumors; therefore, inhibiting cancer progression in two xenograft mouse models with human hepatoma (HepG2) and melanoma (A375). Thus, the NK-92-S3KD cell line may be useful for the clinical immunotherapy of cancer. .

Transforming growth factor β1 (TGF-β1) plays a promoting role in tumor growth via a mechanism associated with hyperactive Smad3 and suppressed Smad7 signaling in the tumor microenvironment. We report that retrieving the balance between Smad3 and Smad7 signaling with asiatic acid (AA, a Smad7 inducer) and naringenin (NG, a Smad3 inhibitor) effectively inhibited tumor progression in mouse models of invasive melanoma (B16F10) and lung carcinoma (LLC) by promoting natural killer (NK) cell development and cytotoxicity against cancer. Mechanistically, we found that Smad3 physically bound Id2 and IRF2 to suppress NK cell production and NK cell-mediated cytotoxicity against cancer. Treatment with AA and NG greatly inhibited Smad3 translation and phosphorylation while it restored Smad7 expression, and, therefore, it largely promoted NK cell differentiation, maturation, and cytotoxicity against cancer via Id2/IRF2-associated mechanisms. In contrast, silencing Id2 or IRF2 blunted the protective effects of AA and NG on NK cell-dependent anti-cancer activities. Thus, treatment with AA and NG produced an additive effect on inactivating TGF-β1/Smad3 signaling, and, therefore, it suppressed melanoma and lung carcinoma growth by promoting NK cell immunity against cancer via a mechanism associated with Id2 and IRF2.

Aims: To investigate roles of miR-29a-DNMT1-SOCS1 axis in cervical cancer invasion and migration. Materials & methods: The methylation level of SOCS1 was determined by methylation specific PCR. The cell apoptosis, proliferation, migration and invasion were examined by Annexin-V/PI staining, MTT and colony formation assays, plus scratch and transwell assays respectively. The expressions of epithelial–mesenchymal transition and NF-κB related proteins were determined by western blotting. Results: MiR-29a was downregulated, accompanied with DNMT1 upregulation and SOCS1 downregulation in cervical cancer tissues. MiR-29a suppressed DNMT1, inhibited SOCS1 promoter methylation and upregulated its expression. Moreover, miR-29a promoted cell apoptosis, suppressed proliferation, inhibited migration and invasion via inactivation of NF-κB signaling pathway in cervical cancer cells. Conclusion: MiR-29a-DNMT1-SOCS1 axis plays an important role on invasion and metastasis in cervical cancer via NF-κB signaling pathway.

Transforming growth factor b (TGF-b) has been shown to promote tumor invasion and metastasis by activating the matrix metalloproteinases (MMPs); however, signaling mechanisms remain controversial and therapies targeting MMPs are still suboptimal. In the present study, we found that combined therapy with Asiatic acid (AA), a Smad7 agonist, and Naringenin (NG), a Smad3 inhibitor, effectively retrieved the balance between Smad3 and Smad7 signaling in the TGF-b-rich tumor microenvironment and thus significantly suppressed tumor invasion and metastasis in mouse models of melanoma and lung carcinoma. Mechanistically, we unraveled that Smad3 acted as a transcriptional activator of MMP2 and as a transcriptional suppressor of tissue inhibitors of metalloproteinase-2 (TIMP2) via binding to 5 0 UTR of MMP2 and 3 0 UTR of TIMP2, respectively. Treatment with NG inhibited Smad3mediated MMP2 transcription while increasing TIMP, whereas treatment with AA enhanced Smad7 to suppress TGF-b/Smad3 signaling, as well as the activation of MMP2 by targeting the nuclear factor-kB (NF-kB)-membrane-type-1 MMP (MT1-MMP) axis. Therefore, the combination of AA and NG additively suppressed invasion and metastasis of melanoma and lung carcinoma by targeting TGF-b/Smad-dependent MMP2 transcription, post-translational activation, and function.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.