Abstract
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, fibronection-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.
Highlights
The triple negative (TN) subtype of breast cancers (BC) represents 15-20% of breast tumors (BT) which are more commonly diagnosed in younger African American women with the prevalence of BRCA1/2 mutations [1,2,3]
Considering the fact that a significant percentage of the patients with triple-negative breast cancer (TNBC) develop brain metastasis [22], we extended our study towards testing the role of Wnt-beta-catenin pathway (WP) in the metastatic settings by using brain-metastasis specific TNBC cells [23], MDA-MB231BR and demonstrated that LWnt3ACM stimulated cell proliferation, clonogenicity and fibronectin-directed migration were abrogated in MDA-MB231BR cells by the blockade of WP signals following the treatment with sulindac sulfide, WP modulators, GDC-0941 and siRNA, which downregulated beta-catenin
We have previously demonstrated that the upregulation of Wnt signaling in TNBC patients is associated with metastasis [20] which in agreement with reports from Reis-Filho’s team and Khramtsov et al, clearly established that there is WP upregulation in TN subset of BC [19, 21]
Summary
The triple negative (TN) subtype of breast cancers (BC) represents 15-20% of breast tumors (BT) which are more commonly diagnosed in younger African American women with the prevalence of BRCA1/2 mutations [1,2,3]. Therapeutic options for patients with TNBC are limited because no pathway-specific targets and associated biomarkers have been established [5,6,7]. Such a situation is further challenged by the fact that the disease is heterogeneous [8,9,10]. Activation of the pathway leads to a context-dependent transcription of beta-catenin target genes including MMP7 and c-MYC to directly control cellular phenotypes including survival, proliferation, migration, polarity and matrix remodeling [18]
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