Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.
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.
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.
Pradip
P
De
Department of Molecular & Experimental Medicine, Avera Research Institute, Sioux Falls, SD, USA.
Jennifer H
JH
Carlson
Department of Hematology and Oncology, WCI, Emory University, Atlanta, GA, USA.
Hui
H
Wu
Department of Hematology and Oncology, WCI, Emory University, Atlanta, GA, USA.
Adam
A
Marcus
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.
Brian
B
Leyland-Jones
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.
Nandini
N
Dey
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology / Q000556:secondary; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D000072261:Dishevelled Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D016023:Integrins / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D009154:Mutation; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013467:Sulindac / Q000031:analogs & derivatives / Q000494:pharmacology; D064726:Triple Negative Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015854:Up-Regulation; D051153:Wnt Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000235:genetics*; D051176:beta Catenin / Q000235:genetics* / Q000378:metabolism
C532162:2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine; C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; C495270:CTNNB1 protein, human; C000606578:DVL1 protein, human; D000072261:Dishevelled Proteins; D005353:Fibronectins; D006575:Heterocyclic Compounds, 3-Ring; D007191:Indazoles; D016023:Integrins; D034741:RNA, Small Interfering; D013449:Sulfonamides; D051153:Wnt Proteins; C544261:XAV939; D051176:beta Catenin; D013467:Sulindac; C025462:sulindac sulfide; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7
LWnt3ACM stimulation; Wnt-pathway modulators; brain-metastasis specific TNBC cells; integrin-directed migration; matrigel-invasion