Rose Michael, Kloten Vera, Noetzel Erik, Gola Lukas, Ehling Josef, Heide Timon, Meurer Steffen K, Gaiko-Shcherbak Aljona, Sechi Antonio S, Huth Sebastian, Weiskirchen Ralf, Klaas Oliver, Antonopoulos Wiebke, Lin Qiong, Wagner Wolfgang, Veeck Jürgen, Gremse Felix, Steitz Julia, Knüchel Ruth, Dahl Edgar
Institute of Pathology, Medical Faculty of the RWTH Aachen University, Aachen, Germany.
Institute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Jülich, Germany.
Mol Cancer. 2017 Feb 23;16(1):44. doi: 10.1186/s12943-017-0610-2.
Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.
ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.
ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.
Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.
细胞外基质（ECM）已知可维持上皮细胞的完整性。在致癌过程中，ECM降解通过控制迁移和分化（包括癌症干细胞（CSC）特征）来触发转移。ECM调节剂α-胰蛋白酶抑制剂重链家族成员5（ITIH5）最近被鉴定为可能参与抑制乳腺癌进展的肿瘤抑制因子，但其功能的分子机制仍不清楚。
使用公共的TCGA数据库分析ITIH5的表达。基于T47D和MDA-MB-231细胞系建立ITIH5过表达的单细胞克隆。在体外研究了集落形成、生长、凋亡、迁移、基质粘附、牵引力分析和肿瘤细胞的极化。通过建立转移小鼠模型分析肿瘤起始特征。为了确定受ITIH5影响的通路，我们利用了全基因组基因表达和DNA甲基化谱。靶向ITIH5下游调控基因DAPK1的RNA干扰用于确认其功能参与。
在预后不良的乳腺癌亚型（如基底型肿瘤）中，ITIH5的缺失很明显。在功能上，在两种细胞系中重新表达ITIH5后，细胞和集落形成均受到损害。在转移小鼠模型中，表达ITIH5的MDA-MB-231细胞几乎完全无法引发肺转移。在这些转移细胞中，ITIH5调节细胞-基质粘附动力学并改变生物力学信号。整合素受体的谱向β1整合素转移，同时在表达ITIH5的克隆中Rac1活性降低，RhoA活性增加，而细胞极化和单细胞迁移受到损害。相反，ITIH5的表达触发了上皮样细胞簇的形成，这些细胞簇经历了表观遗传重编程。由于ITIH5的表达，214个可能标记有H3K4和/或H3K27甲基化的启动子区域显示出高甲基化或低甲基化的DNA构型，最终导致肿瘤抑制因子DAPK1的重新表达。反过来，在表达ITIH5的MDA-MB-231单细胞克隆中，RNAi介导的DAPK1敲低明显恢复了细胞运动性。
我们的结果提供了证据，即ITIH5通过影响已知肿瘤抑制基因（如DAPK1）的全局表观遗传变化，触发具有已知干细胞CSC特性的乳腺癌细胞重编程为上皮样表型。因此，ITIH5可能代表乳腺上皮组织中的一种ECM调节剂，介导对肿瘤起始癌细胞特征的抑制，这些特征被认为是乳腺癌转移的原因。
