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

肿瘤相关性缺氧，即氧气供应减少，是肿瘤微环境的一个标志，由于它选择耐药细胞并促进转移，导致临床预后较差。此前，P53状态被认为是低氧对肿瘤细胞影响的关键决定因素。然而，对P53功能缺失的缺氧细胞提供选择性优势的下游通路还没有被表征。缺氧也是结直肠癌(CRC)转移的核心，因为它介导了上皮-间充质转化/EMT，这为原代肿瘤细胞提供了侵袭、迁移和抗凋亡的特性，以及增加的干性和化疗耐药性。最近，我们报道了在低氧条件下，P53缺陷的细胞表现为EMT，而P53熟练的细胞则经历了MET。此外，我们还发现在p53基因缺失的细胞中，miR-34a在缺氧过程中受到HIF-1A的抑制。这种miR-34a的下调是低氧诱导的大肠癌细胞系EMT、侵袭和迁移以及STAT3激活所必需的。为了充分了解p53/miR-34a通路在肿瘤抑制中的功能，有必要鉴定和鉴定在低氧条件下上调并介导miR-34a下调效应的miR-34a靶点。我们最近确定XBP-1是一个可能的miR-34a靶标。重要的是，XBP-1是已知的缺氧诱导内质网/内质网应激的效应因子，它的失控与结肠炎相关性和散发性结直肠癌的进展有关。此外，我们还发现，作为XBP1miRNA剪接调控因子的IRE1miRNAs也可能受到P53诱导的的负调控。在这项提案中，我们计划确认XBP-1作为miR-34a的直接靶标，并确定这些法规的功能影响。此外，我们将通过建立结直肠癌转移的异种小鼠模型，确定P53/HIF1a/miRNA/IRE1A/XBP-1连接在低氧诱导的EMT中的作用。为了评估所建议的调控环对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基因敲除小鼠中分析它。此外，我们将使用CRC衍生的类肿瘤来分析缺氧诱导的EMT和化疗耐药中的这一信号通路。最后，我们将分析环组件在病理研究所提供的人类结直肠癌样本队列中的表达，以评估这些调控在人类中的保守性及其放松调控的预后价值。综上所述，这项研究可能为肿瘤中P53和/或miR-34a的频繁失活提供一个机制解释，并揭示这些变化是如何促进转移的。此外，这一新的信号通路的组成部分代表了有吸引力的预后标志物和潜在的肿瘤治疗方法的干预点。

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.