Research Article Heterogeneity between triple negative breast cancer cells due to differential activation of Wnt and PI3K/AKT pathways Gabriela Martínez-Revollar a , Erika Garay b , Dolores Martin-Tapia a , Porfirio Nava a , Miriam Huerta c , Esther Lopez-Bayghen d , Noemí Meraz-Cruz e,f , José Segovia a , Lorenza González-Mariscal a,n a Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), México D.F., Mexico b National Laboratories of Genomics for Biodiversity, Center for Research and Advanced Studies (CINVESTAV), Irapuato, Mexico c Department of Biotechnology, Instituto Tecnológico de Monterrey (ITESM), Campus Puebla, Mexico d Department of Toxicology, Center for Research and Advanced Studies (CINVESTAV), México D.F., Mexico e Faculty of Medicine, UNAM, Mexico D.F., Mexico f National Institute of Genomic Medicine, Mexico D.F., Mexico article info Article history: Received 9 June 2015 Received in revised form 2 October 2015 Accepted 6 October 2015 Available online 21 October 2015 Keywords: Triple negative breast cancer β-catenin AKT Wnt Cancer cells heterogeneity abstract The lack of a successful treatment for triple-negative breast cancer demands the study of the hetero- geneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA- MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi- epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal trans- formation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of β-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of β- catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of β-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone. & 2015 Elsevier Inc. All rights reserved. 1. Introduction Breast cancer is the most common cause of cancer death among women and the most frequently diagnosed cancer among women in 140 of 184 countries worldwide [12]. Since 2008 estimates, breast cancer incidence has increased worldwide by more than 20% whereas mortality has increased by 14%. Breast cancer is of heterogeneous nature. Accordingly, gene expression studies using DNA microarrays [39] identified four common subtypes using biological markers, including the pre- sence ( þ ) or absence ( À ) of estrogen receptors (ER), progesterone receptors [9] and human epidermal growth factor receptor 2 (HER2): 1. Luminal A. The tumors are ER þ and/or PR þ and HER2 À . They constitute about 40% of breast cancers and have the most fa- vorable short-term prognosis. 2. Luminal B. The tumors are ER þ and/or PR þ and HER2 þ or have a high proliferation rate. They constitute about 10 to 20% of breast cancers. Luminal A and B breast cancers give a favorable response to hormonal therapy with tamoxifen [13]. 3. HER2 enriched. Tumors are ER- and PR- but generate an excess of HER2. Constitute about 10% of breast cancers, and can be treated with trastuzumab, an antibody against HER2 [14,42]. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/yexcr Experimental Cell Research http://dx.doi.org/10.1016/j.yexcr.2015.10.006 0014-4827/& 2015 Elsevier Inc. All rights reserved. Abbreviations: AJ, adherens junction; AKT, protein kinase B; ANOVA, analysis of variance; BRCA1, breast cancer 1; CD133, cluster of differentiation 133; CK-18, cy- tokeratin-18; Cpm, counts per minute; CSC, cancer stem cell; DKK1, dickkopf-re- lated protein 1; EGFR, epithelial growth factor receptor; EMT, epithelial me- senchymal transition; ER, estrogen receptor; F, fibroblast-like; GSK-3β, glycogen synthase kinase 3 beta; HER2, epidermal growth factor receptor 2; LRP6, low- density lipoprotein receptor-related protein 6; MMP2, 9, matrix metalloproteinase 2, 9; mTORC1, C2, mammalian target of rapamycin complex 1, 2; p70S6K, p70 ri- bosomal protein S6 kinase; p120cat, 120-kD protein catenin; PI3K, phosphatidyli- nositol-4,5-bisphosphate 3-kinase; PR, progesterone receptor; SE, semi-epithelial; Sox2, sry-related HMG box; Src, sarcoma non-receptor tyrosine kinase protein; TCF/ LEF, T-cell factor/lymphoid enhancing factor; TCN, triciribine; TJ, tight junction; Wnt, wingless-related integration site; ZO-1, -2, zonula occludens-1, -2 n Corresponding author. Fax: þ5255 57473996. E-mail address: lorenza@fisio.cinvestav.mx (L. González-Mariscal). Experimental Cell Research 339 (2015) 67–80