miR-34a-mediated regulations in the micro-environment of colorectal cancer

自Virchow以来，炎症过程和肿瘤形成已被联系起来，但其潜在机制尚未完全了解。我们已经证明，由原型肿瘤抑制因子p53诱导的microRNA miR-34a是上皮细胞对细胞因子IL-6介导的炎症反应的一个组成部分（Rokavec et al. (2014) JCI）： miR-34a在由IL-6受体（IL-6R）、转录因子STAT3和miR-34a组成的正反馈回路中发挥作用。该循环在人类crc中被激活，并与远处转移和低生存率相关。我们最近确定，miR-34a和p53在散发性CRC小鼠模型中协同抑制侵袭和转移，并且它们的联合失活与人类CRC中单独失活的生存率较低相关（Öner等人，2018,Gastroenterology）。在本提案中，我们的目标是利用遗传方法和CRC和结肠炎相关结肠癌（CAC）的小鼠模型进一步表征IL-6R/STAT3/miR-34a反馈回路的组成部分和下游效应物。由于我们获得了肿瘤相关巨噬细胞（tam）中miR-34a表达在侵袭性cac表型中的作用的证据，我们将详细描述miR-34a及其在tam和骨髓源性巨噬细胞（bmdm）中直接受miR-34a调节的两个靶点的作用。其中一个是集落刺激因子1 （CSF1）的受体CSF1R，另一个是白细胞介素4的受体IL4R。这两种细胞因子/受体对都是已知的，是TAM M1/M2极化的中枢调节因子，这是TAM抗或促肿瘤潜能的核心。因此，我们将分析巨噬细胞特异性缺失CSF1R或IL4R合并miR-34a缺失的小鼠。我们将评估miR-34a对来自这些小鼠的bmdm和tam的炎症信号、mRNA表达和极化的影响，以及它们对共培养CRC细胞侵袭性的影响。此外，我们将在小鼠CAC模型中确定巨噬细胞miR-34a与抑制侵袭性的相关性。此外，我们计划在CAC和散发性CRC小鼠模型中，通过基因和治疗抑制mir -34a缺陷小鼠中的这些受体，来探究CSF1R和IL4R激活的治疗相关性。此外，我们将在APCmin小鼠模型中确定miR-34a与髓样p53抑制腺瘤形成能力的相关性。总之，计划中的分析对理解miR-34a作为肿瘤抑制介质的作用做出了重要贡献。在未来，这些结果的临床转化可能与散发性结肠癌和结肠炎相关癌症的治疗和预后有关。

Inflammatory processes and tumor formation have been linked since Virchow, however the underlying mechanisms have not been fully understood. We have shown that the microRNA miR-34a, which is induced by the prototypic tumor suppressor p53, is an integral component of the response of epithelial cells to inflammation mediated by the cytokine IL-6 (Rokavec et al. (2014) JCI): miR-34a functions in the context of a positive feedback-loop consisting of the IL-6 receptor (IL-6R), the transcription factor STAT3 and miR-34a. This loop is activated in human CRCs and associated with distant metastases and poor survival. We recently determined that miR-34a and p53 cooperate in the suppression of invasion and metastasis in a sporadic CRC mouse model and that their combined inactivation is associated with poorer survival than their singular inactivation in human CRCs (Öner et al., 2018, Gastroenterology). In this proposal we aim to further characterize components of the IL-6R/STAT3/miR-34a feed-back loop and downstream effectors using genetic approaches and mouse models of CRC and colitis-associated colon cancer (CAC). Since we obtained evidence for a role of miR-34a expression in tumor associated macrophages (TAMs) for the invasive phenotype of CACs we will characterize the role of miR-34a and two of its targets, that we found to be directly regulated by miR-34a, in TAMs and bone marrow derived macrophages (BMDMs) in detail. One of them is CSF1R, the receptor for colony-stimulating factor 1 (CSF1) and the other IL4R, the receptor for interleukin 4. Both cytokine/receptor pairs are known, central regulators of TAM M1/M2 polarization, which is central for the anti- or pro-tumorigenic potential of TAMs. Therefore, we will analyze mice with macrophage-specific deletion of CSF1R or IL4R combined with miR-34a deletion. We will evaluate the influence of miR-34a on inflammatory signalling, mRNA expression and polarization in BMDMs and TAMs derived from these mice, as well as their effects on the invasiveness of co-cultured CRC cells. Furthermore, we will determine the relevance of macrophage miR-34a for suppression of invasiveness in mouse models of CAC. In addition, we plan to interrogate the therapeutic relevance of CSF1R and IL4R activation by genetically and therapeutically inhibiting these receptors in miR-34a-deficient mice in mouse models of CAC and sporadic CRC. In addition, we will determine the relevance of miR-34a for the ability of myeloid p53 to suppress the formation of adenomas in the APCmin mouse model. In summary, the planned analyses represent an important contribution to understanding the role of miR-34a as a mediator of tumor suppression. In the future, the clinical translation of these results may be relevant for the treatment and prognostication of sporadic colon and colitis-associated cancer.