Abstract
The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.
Highlights
A local disease at the initial stage, a pernicious progression of cancer begins as the disease starts to metastasize to the distant organs as a result of an invasion of tumor cells into surrounding/distant tissues [1]
Our study demonstrated that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the salient genetic features of Triple-Negative Breast Cancer (TNBC) and established that WP signaling in TNBC is associated with metastasis and poor prognosis [45]
Since TNBC is an aggressive disease, we hypothesized that the upregulation of WP in this subtype has a functional relationship with integrin-directed metastasis-associated (ID-MA) phenotypes which led to our third report demonstrating that WP signals ID-MA tumor cell phenotypes [50]
Summary
A local disease at the initial stage, a pernicious progression of cancer begins as the disease starts to metastasize to the distant organs as a result of an invasion of tumor cells into surrounding/distant tissues [1]. Altered cell adhesion, increased cell migration, and invasion of tumor cells of a solid tumor are critical phenotypes during their spread to distant organs and are instrumental in the process of metastasis. Abnormal migration and invasion of tumor cells are key components of the metastatic phenotype [7] in solid tumors including breast cancers (BC) [8, 9]. These cells migrate in two distinct modes according to their cell type and their degrees of differentiation when invading the 3D environment, a rounded apolar ‘amoeboid’ movement or an elongated polar ‘mesenchymal’ movement [10] which involves cytoskeletal organization [11]. Understanding the mechanism of regulation of migration has the potency to refine targeted therapies for treating cancers in a metastatic setting
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