Rationale MicroRNA miR145 has been implicated in vascular smooth muscle cell differentiation, but its mechanisms of action and downstream targets have not been fully defined. Objective Here, we sought to explore and define the mechanisms of miR145 function in smooth muscle cells. Methods and Results Using a combination of cell culture assays and in vivo mouse models to modulate miR145, we characterized its downstream actions on smooth muscle phenotypes. Our results show that the miR-143/145 gene cluster is induced in smooth muscle cells by coculture with endothelial cells. Endothelial cell-induced expression of miR-143/145 is augmented by Notch signaling and accordingly expression is reduced in Notch receptor-deficient cells. Screens to identify miR145-regulated genes revealed that the TGFβ pathway has a significantly high number of putative target genes, and we show that TGFβ receptor II (TGFBR2) is a direct target of miR145. Extracellular matrix (ECM) genes that are regulated by TGFBR2 were attenuated by miR145 overexpression, and miR145 mutant mice exhibit an increase in ECM synthesis. Furthermore, activation of TGFβ signaling via angiotensin II infusion revealed a pronounced fibrotic response in the absence of miR145. Conclusions These data demonstrate a specific role for miR145 in the regulation of matrix gene expression in smooth muscle cells, and suggest that miR145 acts to suppress TGFβ-dependent ECM accumulation and fibrosis, while promoting TGFβ-induced smooth muscle cell differentiation. Our findings offer evidence to explain how TGFβ signaling exhibits distinct downstream actions via its regulation by a specific microRNA.

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IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27–induced CCL5 production is IL-27R–dependent. In CD4+ T cells, IL-27–induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27–induced CCL5 appears to contribute to an IL-27–mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5– and IL-27–treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.

BRD4, a chromatin modifier frequently upregulated in a variety of neoplasms including hepatocellular cancer (HCC), promotes cancer cell growth by activating oncogenes through its interaction with acetylated histone tails of nucleosomes. Here, we determined the anti-HCC efficacy of AZD5153, a potent bivalent BRD4 inhibitor, and elucidated its underlying molecular mechanism of action. AZD5153 treatment inhibited HCC cell proliferation, clonogenic survival and induced apoptosis in HCC cells. In vivo, AZD5153-formulated lipid nanoemulsions inhibited both orthotopic and subcutaneous HCCLM3 xenograft growth in NSG mice. Mapping of BRD4- chromosomal targets by ChIP-seq analysis identified the occupancy of BRD4 with the promoters, gene bodies, and super-enhancers of both mRNA and noncoding RNA genes, which were disrupted upon AZD5153 treatment. RNA-seq analysis of polyadenylated RNAs showed several BRD4 target genes involved in DNA replication, cell proliferation, and anti-apoptosis were repressed in AZD5153-treated HCC cells. In addition to known tumor-promoting genes, e.g., c-MYC, YAP1, RAD51B, TRIB3, SLC17A9, JADE1, we found that NAPRT, encoding a key enzyme for NAD+ biosynthesis from nicotinic acid, was also suppressed in HCC cells by the BRD4 inhibitor. Interestingly, AZD5153 treatment upregulated NAMPT, whose product is the rate-limiting enzyme for NAD+ synthesis from nicotinamide. This may explain why AZD5153 acted in concert with FK866, a potent NAMPT inhibitor, in reducing HCC cell proliferation and clonogenic survival. In conclusion, our results identified novel targets of BRD4 in the HCCLM3 cell genome and demonstrated anti-HCC efficacy of AZD5153, which was potentiated in combination with an NAMPT inhibitor.

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CD200 is overexpressed in human solid tumors such as neuroblastoma and melanoma and is considered as an immune checkpoint molecule for dampening cancer immunity. However, how CD200 affects tumor microenvironment (TME) and host immunity is incompletely understood. In the present study, we evaluated the role of the CD200-CD200R pathway in TME using loss-of-function CD200R-/- mice, along with two clinically relevant, CD200-positive murine tumor models: the mouse neuroblastoma NB9464D and melanoma Yummer1.7. We found that comparing with the wild type mice, CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. Transcriptome analysis by single cell RNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD45+ immune cells, including CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effectors including CD8+ T cells and NK cells in combination, are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling differentially regulates the expression of chemokines in tumor-associated macrophages (TAMs). In the absence of CD200R, TAMs up-regulate CCL24 and CCL8 and down-regulate CXCL3, CXCL2, and CCL3 via activation of ERK and/or p38 MAP kinases. Increased expression of CCL24 in CD200R-deficient tumors resulted in increased infiltration of eosinophils, which also contributes to anti-tumor activity. Overall, we conclude that CD200R-mediated signaling, via interaction with CD200 on tumor cells, contributes to unfavorable TME primarily through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has significant implications in developing CD200-targeted immunotherapy of solid tumors. Citation Format: Cho-Hao Lin, Fatemeh Talebian, Yang Li, Jianmin Zhu, Bolin Zhao, Jin-Qing Liu, Sujit Basu, Xueling Pan, Xi Chen, Pearlly Yan, William E. Carson, Gang Xin, Haitao Wen, Ruoning Wang, Zihai Li, Qin Ma, Xue-Feng Bai. CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5831.