Analysis of the p53/HIF-1A/miR-34a pathway in colorectal cancer

肿瘤相关的缺氧，即氧气可用性降低，是肿瘤微环境的一个标志，它会导致不良的临床结果，因为它会选择治疗耐药细胞并促进转移。p53的状态先前已被确定为缺氧对肿瘤细胞影响的关键决定因素。然而，为缺氧细胞提供选择优势的下游通路，p53功能丧失尚未被描述。缺氧也是结直肠癌（CRC）转移的核心，因为它介导上皮-间充质转化/EMT，使原发肿瘤细胞具有侵袭性、迁移性和抗凋亡特性，并增加干性和化疗耐药性。最近，我们报道了缺氧条件下p53缺乏的细胞表现出EMT，而p53精通的细胞则表现出MET。此外，我们可以证明miR-34a在p53缺陷细胞缺氧时被HIF-1A抑制。miR-34a的下调对于低氧诱导的EMT、侵袭和迁移以及CRC细胞系的STAT3激活是必要的。为了充分了解p53/miR-34a通路在肿瘤抑制中的作用，有必要对miR-34a靶点进行鉴定和表征，这些靶点在缺氧条件下上调并介导miR-34a下调的作用。我们最近发现XBP-1可能是miR-34a的靶点。重要的是，XBP-1是缺氧引起的内质网/内质网应激的已知效应者，其失调与结肠炎相关和散发性结直肠癌的进展有关。此外，我们发现XBP1剪接的调节因子IRE1也可能受到p53诱导的miRNAs的负调控。在本提案中，我们计划验证XBP-1作为miR-34a的直接靶点，并确定这些调控的功能含义。此外，我们将通过使用CRC转移的异种移植小鼠模型来确定p53/HIF1A/miRNA/IRE1A/XBP-1连接在缺氧诱导的EMT中的作用。为了评估所提出的调控环与肿瘤进展的体内相关性，我们将在遗传性和散发性结直肠癌模型的背景下，在miR-34a和p53敲除小鼠中分析它。此外，我们将分析这种信号通路在缺氧诱导的EMT和化疗耐药中使用crc衍生的类肿瘤。最后，我们将分析在病理研究所提供的人类结直肠癌样本队列中环路组件的表达，以评估这些调控在人类中的保存及其解除管制的预后价值。综上所述，该研究可能为肿瘤中p53和/或miR-34a的频繁失活提供了机制解释，并揭示了这些改变如何促进转移。此外，这一新信号通路的组成部分代表了治疗crc的肿瘤治疗方法的有吸引力的预后标志物和潜在干预点。

Tumor-associated hypoxia, i.e. decreased availability of oxygen, is a hallmark of the tumor microenvironment which results in a poor clinical outcome since it selects for therapy-resistant cells and promotes metastasis. The p53 status has previously been identified as a critical determinant of the effect of hypoxia on tumor cells. However, the down-stream pathways providing a selective advantage to hypoxic cells with loss of p53 function have not been characterized yet. Hypoxia is also central for metastasis of colorectal cancer (CRC) since it mediates epithelial-mesenchymal transition/EMT, which provides primary tumor cells with invasive, migratory and anti-apoptotic properties, as well as increased stemness and chemo-resistance. Recently, we reported that under hypoxia p53-deficient cells display EMT, whereas p53-proficient cells undergo MET. In addition, we could show that miR-34a is repressed by HIF-1A during hypoxia in p53-deficient cells. This down-regulation of miR-34a was necessary for hypoxia-induced EMT, invasion and migration as well as STAT3 activation of CRC cell lines. To fully understand the function of the p53/miR-34a pathway in tumor suppression, it is necessary to identify and characterize the miR-34a targets, which are up-regulated under hypoxic conditions and mediate the effects of miR-34a down-regulation. We recently identified XBP-1 as a putative miR-34a target. Importantly, XBP-1 is a known effector of endoplasmatic reticulum/ER-stress elicited by hypoxia and its deregulation has been implicated in the progression of colitis-associated and sporadic colorectal cancer. In addition, we found that IRE1, a regulator of XBP1 splicing, may also be under negative control of p53-induced miRNAs. In this proposal we plan to validate XBP-1 as a direct target of miR-34a and determine the functional implications of these regulations. Furthermore, we will determine the role of the p53/HIF1A/miRNA/IRE1A/XBP-1 connection in hypoxia-induced EMT by using xenograft mouse models of CRC metastasis. To evaluate the in vivo relevance of the proposed regulatory loop for tumor progression we will analyse it in miR-34a and p53 knock-out mice in the context of models of hereditary and sporadic colorectal cancer. In addition, we will analyse this signalling pathway in hypoxia-induced EMT and chemo-resistance using CRC-derived tumoroids. Finally, we will analyze the expression of the loop components in cohorts of human colorectal cancer samples available at the Institute of Pathology to evaluate the conservation of these regulations in humans and the prognostic value of its deregulation. Taken together, this study may offer a mechanistic explanation for the frequent inactivation of p53 and/or miR-34a in tumors and reveal how these alterations contribute to metastasis. In addition, the components of this new signaling pathway represent attractive prognostic markers and potential intervention points for tumor therapeutic approaches to treat CRCs.