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?-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells.

ABSTRACT: Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether ?-catenin was necessary for tumorigenic behaviors in vivo and in vitro. ?-catenin expression level was significantly reduced in two human triple-negative breast cancer cell lines, MDA-MB-231 and HCC38, using lentiviral delivery of ?-catenin-specific small hairpin RNAs (shRNAs). Upon implantation of the cells in the mammary fat pad of immunocompromised mice, we found that ?-catenin shRNA HCC38 cells formed markedly smaller tumors than control cells and grew much more slowly. In in vitro assays, ?-catenin silencing significantly reduced the percentage of Aldefluor-positive cells, a read-out of the stem-like cell population, as well as the expression of stem cell-related target genes including Bmi-1 and c-Myc. ?-catenin-knockdown cells were also significantly impaired in their ability to migrate in wound-filling assays and form anchorage-independent colonies in soft agar. ?-catenin-knockdown cells were more sensitive to chemotherapeutic agents doxorubicin and cisplatin. Collectively, these data suggest that ?-catenin is required for triple-negative breast cancer development by controlling numerous tumor-associated properties, such as migration, stemness, anchorage-independent growth and chemosensitivity.

SUBMITTER: Xu J

PROVIDER: S-EPMC4319896 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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β-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells.

Xu Jinhua J Prosperi Jenifer R JR Choudhury Noura N Olopade Olufunmilayo I OI Goss Kathleen H KH

PloS one 20150206 2

Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether β-catenin was necessary for tumorigenic behaviors in vivo and in vitro. β-catenin expression level was significantly reduced in two human triple-negative breast cancer cell li ...[more]

PMID: 25658419

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Project description:PurposeLipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-?-cyclodextrin (M?CD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells.MethodsWe measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively.ResultsThe present study found that the cholesterol-depleting agent M?CD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1-0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. M?CD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following M?CD treatment (48 hours). M?CD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that M?CD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that M?CD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines.ConclusionM?CD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Project description:BackgroundMetastatic breast cancer is responsible for the death of the majority of breast cancer patients. In fact, metastatic BC is the 2nd leading cause of cancer-related deaths in women in the USA and worldwide. Triple negative breast cancer (TNBC), which lacks expression of hormone receptors (ER-α and PR) and ErbB2/HER2, is especially lethal due to its highly metastatic behavior, propensity to recur rapidly, and for its resistance to standard of care therapies, through mechanisms that remain incompletely understood. WAVE3 has been established as a promoter of TNBC development and metastatic progression. In this study, we investigated the molecular mechanisms whereby WAVE3 promotes therapy-resistance and cancer stemness in TNBC, through the regulation of β-catenin stabilization.MethodsThe Cancer Genome Atlas dataset was used to assess the expression of WAVE3 and β-catenin in breast cancer tumors. Kaplan-Meier Plotter analysis was used to correlate expression of WAVE3 and β-catenin with breast cancer patients' survival probability. MTT assay was used to quantify cell survival. CRISPR/Cas9-mediated gene editing, 2D and 3D tumorsphere growth and invasion assays, Immunofluorescence, Western blotting, Semi-quantitative and real-time quantitative PCR analyses were applied to study the WAVE3/β-catenin oncogenic signaling in TNBC. Tumor xenograft assays were used to study the role of WAVE3 in mediating chemotherapy resistance of TNBC tumors.ResultsGenetic inactivation of WAVE3 in combination of chemotherapy resulted in inhibition of 2D growth and 3D tumorsphere formation and invasion of TNBC cells in vitro, as well as tumor growth and metastasis in vivo. In addition, while re-expression of phospho-active WAVE3 in the WAVE3-deficient TNBC cells restored the oncogenic activity of WAVE3, re-expression of phospho-mutant WAVE3 did not. Further studies revealed that dual blocking of WAVE3 expression or phosphorylation in combination with chemotherapy treatment inhibited the activity and expression and stabilization of β-catenin. Most importantly, the combination of WAVE3-deficiency or WAVE3-phospho-deficiency and chemotherapy suppressed the oncogenic behavior of chemoresistant TNBC cells, both in vitro and in vivo.ConclusionWe identified a novel WAVE3/β-catenin oncogenic signaling axis that modulates chemoresistance of TNBC. This study suggests that a targeted therapeutic strategy against WAVE3 could be effective for the treatment of chemoresistant TNBC tumors.

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?-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells.

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β-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells.

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