缺氧诱导因子调控钙网蛋白在维持乳腺癌干细胞表型中的机制研究

Hypoxia-inducible factors (HIFs) play important roles in promoting specification and/or maintenance of the breast cancer stem cell (BCSC) phenotype in response to hypoxia, but the underlining mechanism needs further investigation. It has been reported that calreticulin (CALR) expression was elevated on pancreatic cancer stem-like cells (P-CSLCs), and the reactive oxygen species (ROS) scavenger, which could destabilize HIFα, decreased CALR expression on P-CSLCs, suggesting a role of CALR in CSC phenotype and HIFs involvement. Data from TCGA database shows that CALR is highly expressed in breast cancer tissue compared with normal tissue, and its mRNA level is positively correlated with HIF target genes. In vitro study also found CALR mRNA was significantly induced by hypoxia in HCC1954 and MDA-MB-435 breast cancer cell lines. The human genome sequence was searched for matches to the consensus HIF binding site 5’-(A/G)CGTG-3’ that were located within DNase I-hypersensitive domains at the CALR locus, and several suspicious HIF binding sites were found. So we hypothesize that HIFs could regulate CALR expression to induce BCSC phenotype. In the present study, we’re going to test the CALR expression in breast cancer tissues and analyze its relationship with patients’ clinicopathologic characteristics and survival. Enrich BCSCs through mammosphere formation assay and Aldefluor assay, and test the CALR expression in BCSCs and bulk breast cancer cells, to investigate the relationship between CALR and BCSCs. Establish CALR knockdown subclones and study the changes of percentage of BCSCs, the tumor initiation potential and metastatic capacity of breast cancer cells, to verify the role of CALR in BCSCs. Upregulate HIFs activity by hypoxia exposure and downregulate HIFs by RNAi technique, test the change of CALR expression. Further determine the direct regulation of HIFs on CALR expression through ChIP and Luciferase reporter assay. Our study will illustrate the role of CALR in specification of the BCSC phenotype and reveal the regulatory role of HIFs in CALR expression, which may provide potential targets for breast cancer therapy and it’s of great importance both theoretically and practically.

前期研究发现HIFs在BCSC表型维持中发挥重要作用，而CALR与HIF靶基因的表达水平正相关，已有文献报道CALR在CSCs中高表达。据此提出假说：HIFs可调控CALR以维持BCSC表型。本研究先检测CALR在乳腺癌组织中表达情况，分析其表达水平与患者临床病理特征及生存的相关性；通过乳腺癌干细胞成球实验及ALDH实验流式细胞分选募集BCSCs，对比CALR在不同细胞亚群中的表达水平，证实CALR与BCSCs的关系；进而下调CALR，检测BCSCs的比例变化、癌细胞体内致瘤能力及其向远处转移能力的改变，验证CALR在BCSC表型调控中的作用；通过上调以及下调HIFs，观察CALR表达水平的变化，并进一步通过ChIP及荧光素酶报告基因实验明确HIFs对CALR的直接调控作用，最终阐明CALR在BCSC表型调控中的作用及上游机制。研究结果可为乳腺癌的治疗提供新靶点，具有重要的理论和实践意义。

钙网蛋白是一种多功能的内质网蛋白，参与钙稳态、细胞粘附、恶性肿瘤的形成和发展等过程。然而钙网蛋白在乳腺癌中的作用仍不清楚。公共数据集的数据表明，与正常组织相比钙网蛋白在乳腺癌组织中呈现高表达，且其表达水平与患者死亡率和干细胞指数相关。体外研究表明，在乳腺癌干细胞球培养物、乳腺癌干细胞富集的CD24-CD44+亚群和ALDH+亚群中钙网蛋白表达增加。在敲低钙网蛋白后，乳腺癌干细胞比例下降，且体内试验发现shCALR组肿瘤的发生、转移减少，同时增强了在体内的化疗敏感性。染色质免疫沉淀和双荧光素酶报告实验显示缺氧可通过缺氧诱导因子1（HIF-1）直接激活缺氧乳腺癌细胞中的钙网蛋白的转录来增强钙网蛋白的表达水平，证实钙网蛋白是HIF-1的靶基因。RNA测序分析表明钙网蛋白通过Wnt/β-catenin通路激活促进乳腺癌干细胞表型。Wnt/β-catenin通路激活剂可以消除钙网蛋白基因敲除对乳腺微球体形成的抑制作用。综上，我们的研究结果表明钙网蛋白通过Wnt/β-catenin通路以HIF-1依赖的方式促进乳腺癌干细胞表型，从而促进乳腺癌的侵袭和远处转移。这提示靶向钙网蛋白可能为乳腺癌的治疗提供有效的手段。