Cancer initiation and progression are defined by the behavior of cancer cells per se and the development of tumor tissues, both of which are modulated by crosstalk between cancer cells and the surrounding microenvironment. Advances in cancer research have highlighted the significance of constant evolution of the tumor microenvironment, leading to tumor formation, metastasis and refractoriness to therapy. MicroRNAs (miRNAs) are small non-coding RNAs that function as major players of posttranscriptional gene regulation in diverse biological processes. They function as both tumor suppressors and promoters in many aspects of the autonomous behavior of cancer cells. Theoretically, dysfunction in the gene regulatory networks of cancer cells is one of the major driving forces for alterations of ostensibly normal surrounding cells. In this context, the core targets of miRNAs, termed miRNA regulons, are currently being expanded to include various modulators of the tumor microenvironment. Recent advances have highlighted two important roles played by miRNAs in the evolution of tumor microenvironments: miRNAs in tumor cells transform the microenvironment via non-cell-autonomous mechanisms, and miRNAs in neighboring cells stabilize cancer hallmark traits. These observations epitomize the distal and proximal functions of miRNAs in tumor microenvironments, respectively. Such regulation by miRNAs affects tumor angiogenesis, immune invasion and tumor-stromal interactions. This review summarizes recent findings on the mechanisms of miRNA-mediated regulation of tumor microenvironments, with a perspective on the design of therapeutic interventions.

Lymphatic vessels (LVs) play critical roles in the maintenance of fluid homeostasis and in pathological conditions, including cancer metastasis. Although mutations in ALK1, a member of the transforming growth factor (TGF)-β/bone morphogenetic protein (BMP) receptor family, have been linked to hereditary hemorrhagic telangiectasia, a human vascular disease, the roles of activin receptor-like kinase 1 (ALK-1) signals in LV formation largely remain to be elucidated. We show that ALK-1 signals inhibit LV formation, and LVs were enlarged in multiple organs in Alk1-depleted mice. These inhibitory effects of ALK-1 signaling were mediated by BMP-9, which decreased the number of cultured lymphatic endothelial cells. Bmp9-deficient mouse embryos consistently exhibited enlarged dermal LVs. BMP-9 also inhibited LV formation during inflammation and tumorigenesis. BMP-9 downregulated the expression of the transcription factor prospero-related homeobox 1, which is necessary to maintain lymphatic endothelial cell identity. Furthermore, silencing prospero-related homeobox 1 expression inhibited lymphatic endothelial cell proliferation. Our findings reveal a unique molecular basis for the physiological and pathological roles of BMP-9/ALK-1 signals in LV formation.lymphangiogenesis | angiogenesis | blood vascular endothelial cells

Asymmetric selection of single-stranded guide RNAs from double-stranded RNA precursors is crucial in RNA silencing-mediated gene regulation. However, the precise mechanisms of small-RNA asymmetry are unclear, especially because asymmetric selection can still occur when the putative asymmetry sensors Drosophila R2D2 and mammalian Dicer are depleted. Here we report a direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5'-uridine or 5'-adenosine and thermodynamically unstable 5' ends in parallel through its two sensor regions, which contact the 5' nucleobases and 5'-phosphates of prospective guide strands. Hence, miRNA asymmetry shows superposed patterns reflecting 5'-end nucleotide identity ('digital' pattern) and thermodynamic stability ('analog' pattern). Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle, to aid in comprehensive understanding of miRNA function and development of new RNA-silencing therapies in precision medicine.

Zinc finger E‐box binding protein 1 (ZEB1) and ZEB2 induce epithelial‐mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1‐regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1‐bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL‐6 and IL‐8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid‐derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT.

Transforming growth factor‐β (TGF‐β) plays central roles in endothelial–mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial–mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA‐31 (miR‐31) is a positive regulator of TGF‐β‐induced EndMT. Although the expression is not induced by TGF‐β, miR‐31 is required for induction of mesenchymal genes including α‐SMA, actin reorganization and MRTF‐A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF‐A activity, as a miR‐31 target. Global transcriptome analysis further showed that miR‐31 positively regulates EndMT‐associated unique secretory phenotype (EndMT‐SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL‐6 and Angptl2. As a mechanism for this phenomenon, TGF‐β and miR‐31 suppress Stk40, a negative regulator of NF‐κB pathway. Interestingly, TGF‐β induces alternative polyadenylation (APA)‐coupled miR‐31‐dependent Stk40 suppression without concomitant miR‐31 induction, and APA‐mediated exclusion of internal poly(A) sequence in Stk40 3′UTR enhances target efficiency of Stk40. Finally, miR‐31 functions as a molecular hub to integrate TGF‐β and TNF‐α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT.

The tumor microenvironment contains various components, including cancer cells, tumor vessels, and cancer‐associated fibroblasts, the latter of which are comprised of tumor‐promoting myofibroblasts and tumor‐suppressing fibroblasts. Multiple lines of evidence indicate that transforming growth factor‐β ( TGF ‐β) induces the formation of myofibroblasts and other types of mesenchymal (non‐myofibroblastic) cells from endothelial cells. Recent reports show that fibroblast growth factor 2 ( FGF 2) modulates TGF ‐β‐induced mesenchymal transition of endothelial cells, but the molecular mechanisms behind the signals that control transcriptional networks during the formation of different groups of fibroblasts remain largely unclear. Here, we studied the roles of FGF 2 during the regulation of TGF ‐β‐induced mesenchymal transition of tumor endothelial cells ( TEC s). We demonstrated that auto/paracrine FGF signals in TEC s inhibit TGF ‐β‐induced endothelial‐to‐myofibroblast transition (End‐MyoT), leading to suppressed formation of contractile myofibroblast cells, but on the other hand can also collaborate with TGF ‐β in promoting the formation of active fibroblastic cells which have migratory and proliferative properties. FGF 2 modulated TGF ‐β‐induced formation of myofibroblastic and non‐myofibroblastic cells from TEC s via transcriptional regulation of various mesenchymal markers and growth factors. Furthermore, we observed that TEC s treated with TGF ‐β were more competent in promoting in vivo tumor growth than TEC s treated with TGF ‐β and FGF 2. Mechanistically, we showed that Elk1 mediated FGF 2‐induced inhibition of End‐MyoT via inhibition of TGF ‐β‐induced transcriptional activation of α‐smooth muscle actin promoter by myocardin‐related transcription factor‐A. Our data suggest that TGF ‐β and FGF 2 oppose and cooperate with each other during the formation of myofibroblastic and non‐myofibroblastic cells from TEC s, which in turn determines the characteristics of mesenchymal cells in the tumor microenvironment.

Multiple microRNAs (miRNAs) regulate epithelial-mesenchymal transition and endothelial-mesenchymal transition (EndMT). Here we report that microRNA-27b (miR-27b) positively regulates transforming growth factor-β (TGF-β)-induced EndMT of MS-1 mouse pancreatic microvascular endothelial cells. TGF-β induced miR-23b/24-1/27b expression, and inhibition of miR-27 suppressed TGF-β-mediated induction of mesenchymal genes. Genome-wide miRNA target analysis revealed that miR-27 targets Elk1, which acts as a competitive inhibitor of myocardin-related transcription factor-serum response factor signalling and as a myogenic repressor. miR-27b was also found to regulate several semaphorin receptors including Neuropilin 2, Plexin A2 and Plexin D1. These results suggest important roles of miR-27 in TGF-β-driven EndMT.

The precise control of microRNA (miRNA) biosynthesis is crucial for gene regulation. Lin28A and Lin28B are selective inhibitors of biogenesis of let-7 miRNAs involved in development and tumorigenesis. Lin28A selectively inhibits let-7 biogenesis through cytoplasmic uridylation of precursor let-7 by TUT4 terminal uridyl transferase and subsequent degradation by Dis3l2 exonuclease. However, a role of this uridylation pathway remains unclear in let-7 blockade by Lin28B, a paralog of Lin28A, while Lin28B is reported to engage a distinct mechanism in the nucleus to suppress let-7. Here we revisit a functional link between Lin28B and the uridylation pathway with a focus on let-7 metabolism in cancer cells. Both Lin28A and Lin28B interacted with Dis3l2 in the cytoplasm, and silencing of Dis3l2 upregulated uridylated pre-let-7 in both Lin28A- and Lin28B-expressing cancer cell lines. In addition, we found that amounts of let-7 precursors influenced intracellular localization of Lin28B. Furthermore, we found that MCPIP1 (Zc3h12a) ribonuclease was also involved in degradation of both non-uridylated and uridylated pre-let-7. Cancer transcriptome analysis showed association of expression levels of Lin28B and uridylation pathway components, TUT4 and Dis3l2, in various human cancer cells and hepatocellular carcinoma. Collectively, these results suggest that cytoplasmic uridylation pathway actively participates in blockade of let-7 biogenesis by Lin28B.