De Pradip, Carlson Jennifer H, Wu Hui, Marcus Adam, Leyland-Jones Brian, Dey Nandini
Department of Molecular & Experimental Medicine, Avera Research Institute, Sioux Falls, SD, USA.
Department of Internal Medicine, SSOM, University of South Dakota, Sioux Falls, SD, USA.
Oncotarget. 2016 Jul 12;7(28):43124-43149. doi: 10.18632/oncotarget.8988.
Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.
肿瘤细胞在发生基因改变后会获得转移相关（MA）表型，这些基因改变会导致不同信号通路失调。此前，我们报道Wnt-β-连环蛋白通路（WP）的上调是三阴性乳腺癌（TNBC）的重要遗传学特征之一，并且WP信号传导与TNBC的转移相关。利用cBioPortal，我们发现乳腺浸润性癌中WP基因CTNNB1、APC和DVL1的改变总和为21%，而在PAM50基底样型中为56%。为了了解WP在异质性/转移性TNBC细胞生物学中的功能相关性，我们使用15种细胞系进行了这项全面研究，在整合素依赖性MA表型的背景下研究了WP的作用。通过共聚焦免疫荧光显微镜和定量共聚焦视频显微镜观察肿瘤细胞的定向运动，同时通过MMP7特异性酪蛋白酶谱法研究基质胶侵袭情况。WntC59、XAV939、舒林酸硫化物和β-连环蛋白小干扰RNA（1）抑制纤连蛋白介导的迁移，（2）降低伪足参数和运动描述符，（3）改变丝状肌动蛋白，（4）减少基质胶侵袭，（5）抑制细胞增殖以及三维克隆生长。舒林酸硫化物和β-连环蛋白小干扰RNA降低了β-连环蛋白/活性β-连环蛋白和MMP7。WP调节剂、舒林酸硫化物和GDC-0941干扰了LWnt3ACM刺激的增殖、克隆形成、纤连蛋白介导的迁移和基质胶侵袭。我们通过刺激脑转移特异性MDA-MB231BR细胞研究了WP在转移中的直接作用，结果表明，在使用舒林酸硫化物、GDC-0941和敲低β-连环蛋白后，LWnt3ACM刺激的增殖、克隆形成和迁移受到了抑制。我们首次提供证据表明WP激活与整合素依赖性MA表型之间存在直接功能关系。通过证明WP激活与MA表型之间的功能关系，我们的数据从机制上解释了（1）为什么TNBC中WP的不同组分会上调，（2）WP激活如何与转移相关，以及（3）如何通过减轻WP来调节整合素依赖性MA表型。
