Role Of MicroRNA-494 In Regulating The Activity Of Tumor-expanded Myeloid-derived Suppressor Cells

In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation, in part based on the phenomena that inflammatory cells were present in biopsied samples from tumors. But the causal relationship between inflammation, innate immunity and cancer is generally accepted only in recent years. Mounting evidence indicates tumor-infiltrating inflammatory cells contribute significantly to tumor progression through suppressing the host immunity, facilitating tumor cells invasion and participating in the formation of the new blood vessels.The growth and metastasis of solid tumors are often associated with aberrant myelopoiesis, including the significant accumulation of myeloid-derived suppressor cells (MDSCs) in patients and various mouse tumor models that have the potential to promote tumor growth. MDSCs represent a heterogeneous population of myeloid cells comprising immature dendritic cells (DCs), macrophages, granulocytes, and other myeloid cells in early differential stages that can be identified in mice by expression of CD11b and Gr-1. Recent studies classified Gr1+CD11b+ cells as either Granulocytic MDSCs or Monocytic MDSCs based on nuclear morphology and the expression of Ly6G or Ly6C, respectively, and indicated both populations suppress antigen-specific T-cell responses, but through distinct effector molecules and signaling pathways. In addition to their depressive effect on host immune surveillance, MDSCs also facilitate tumor cell invasion and metastasis. We and others have previously reported that Tumor-derived factors mobilize MDSCs from bone marrow into the site of tumor where they produce multiple MMPs that contribute to tumor invasion.Previous studies have revealed that, in addition to the complex transcriptional programmes, the existence of potentially widespread post-transcriptional regulatory mechanisms that have crucial roles in regulating the development and function of immune cells. The mediators of these processes are known as microRNAs (miRNAs)—an abundant class of endogenous small non-coding RNAs of approximately 22 nucleotides in length that can form imperfect Watson-Crick base pairs at multiple sites within the 3'-untranslated region (UTR) of their cognate mRNA targets to repress their expression. miRNAs affect all aspects of immune system development from hematopoiesis to activation. At the same time, miRNA dysregulation in immune cells can lead to various immune-related pathological disorders, such as inflammation and cancer. Our previous studies shown microRNAs participate in the regulation of the function of macrophage. Mounting evidence indicates that MDSCs elicited by tumor-derived factors contribute significantly to tumor progression, however, it remains unclear about whether noncoding RNA is involved. In this study, we have reported the increased expression of miR-494 in MDSCs induced by tumor derived factors is critically involved in tumor growth and metastasis. First, we found the expression of miR-494 in MDSCs derived from all 6 tumor models on two different mouse strains was higher than MDSCs from naive mice, indicating the high level of miR-494 expression is the common characteristics of tumor-associated MDSCs. Second, our in vitro assays showed that tumor cells culture medium (TCCM) can induce the overtly level of miR-494 in MDSCs in a dose-dependent manner. Finally, we further found TGF-?1 in TDF was a main cytokine responsible for up-regulation of miR-494 in tumor-expanded MDSCs.We also observed that tumor-infiltrating MDSCs had a significant high level of miR-494 expression compared with splenic MDSCs, more intriguing, the expression level seemed be correlated between miR-494 and ARG1, iNOS2 and MMPs which known as closely related to the activity of MDSCs. Using overexpression or active inhibition of miR-494 approaches, we further determined miR-494 participated in regulating these genes expression and played a pivotal role in regulating both suppressive activity and the ability of facilitating tumor cell invasion and metastasis of MDSCs. The growth of the primary tumors was retarded and colonies of metastatic cells in the lungs were rarely detected when endogenous miR-494 activity in vivo was inhibited. Depletion of CD8+ T cells restored primary tumor growth, suggesting LV-sponge suppressed primary tumor growth mainly by blocking MDSCs-mediated tumor-specific T-cell tolerance. Whereas the phenomenon, that inhibition of miR-494 significantly reduced metastatic cancer cells in lung even CD8+T cells were depleted, in combination with the data from in vitro invasion assays suggested miR-494 regulated the ability of facilitating tumor cell invasion and metastasis of MDSCs via regulating MMPs expression. Interestingly, the number of tumor-infiltrating MDSCs significantly decreases in LV-sponge infected mice even combination of 2.43 mAb treatment that resulted in the primary tumor size comparable with LV-ctrl infected mice. We have further proven that the decrease number of tumor-infiltrating MDSCs due to the increased apoptosis and the decreased migration into tumor site of MDSCs induced by LV-sponge.Recent studies have determined that JAK/STAT signalling pathways play crucial roles in the suppressive activity of MDSCs. However, it remains unclear whether another signalling pathways are involved, especially the intracellular signal mechanisms responsible for Non-immunological functions of MDSCs. As a multifunctional tumor suppressor, PTEN is mutated or otherwise inactivated is frequently observed in a wide variety of human cancers. In addition to its tumor suppressive function, PTEN also play a critical role in the maintenance of the normal physiological functions of many organ systems. As far as immune system is concerned, published work has shown that depending on the cell type, PTEN is important for proper development, cell fate and cell function.Our study has now shown that PTEN is a functional target of miR-494 in MDSCs and PI3K/Akt pathway plays an "omnipotent" role of the regulation of MDSCs promoting tumor progression. As a functional miR-494 target, PTEN expression in MDSCs was suppressed by up-regulation of miR-494 induced by TDF that resulted in the enhanced ability of infiltrating into tumor site via SDF-1/CXCR4 axe. Moreover, down-regulation of PTEN activates PI3K/Akt pathway that alters the intrinsic apoptotic/survival signal to prolong the lifespan of MDSCs, which further contributed to the accumulation of MDSCs into tumor site. Furthermore, the activated PI3K/Akt pathway promotes the expression of ARG1, iNOS2 and MMPs via mTOR dependent pathway that results in the enhanced inhibitory activity and the ability of facilitating tumor cell invasion and metastasis that is attributed to tumor progress.In summary, our results indicate that upregulation of miR-494 promotes tumor growth and metastasis by enhancing the accumulation and activity of MDSCs in tumor tissue via suppressing PTEN expression, thereby resulting in increased Akt activity and subsequent activation of mTOR. By targeting miR-494, not only generated a strong antitumor immunity but also inhibited tumor metastasis, having the effect of "killing two birds with one stone".