Malignant potential of breast cancer stem cells is associated with environment- dependent upregulation of the Oct3/4 network

Abstract

Introduction: The pioneer transcriptional factors (PTFs) of the Oct3/4 network including Oct3/4, Nanog, Sox2, Sall4 (ONSS), have been associated with breast cancer. Regulation of ONSS and other factors in this network were assessed for their role in malignancy. Methods: Triple negative breast cancer cell line (MDA-MB-231) transfected with human Oct3/4-GFP promoter was sorted using FACS. Differentially expressed genes (DEGs) were identified using qPCR and microarray. 3D mammospheres (CSC) from Oct3/4(+) cells were assessed for stable Oct3/4 expression. Tumor seeding and lung metastatic potential of Oct3/4(+) cells were assessed in immunocompromised mice. DEGs in the tumors were assessed with respect to implanted tissue (SQ, lungs or brain), recurrence, and metastases. Expression of CD44+/CD24- was evaluated using flow cytometry. Resistance of Oct3/4(+) cells to paclitaxel was assessed using MTS assay. Results: Oct3/4-GFP expression was stable in mammospheres. Oct3/4(+) cells showed 25 DEGs and significant resistance to paclitaxel when compared to non-transfected cells. Upregulated growth and developmental genes included Gata6, FoxA2, Sall4, Zic2, H2afJ, Stc1 and Bmi1. The Oct3/4(+) cells also showed enhanced tumorigenic potential and aggressive growth in immunocompromised mice. Additionally, this modulated transcriptome of the Oct3/4 (+) cells showed further upregulation of several genes in metastatic lung lesions in mice (> 5 fold) compared to orthotopic tumors including Oct4A, Bmi1, Ezh2, Klf5, Hox7B, Gja1, Stc1, Amigo2 and Dkk1. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted Sall4, c-Myc, Mmp1, Mmp9 and Dkk1 genes in maintaining an upregulated expression specifically in metastatic lesions and a 2-fold higher expression of stem cell phenotype markers (CD44+/CD24-). Overall Oct3/4 expression in tumors in lungs, brain and metastases were significantly higher than orthotopic mammary fat pad tumors. Additionally, the transcriptome was most upregulated in brain except for Gja1 and H2faJ, indicating tissue-specific regulation of this transcriptome. Conclusion: ONSS and other Oct3/4 related factors may drive the differentiation and maintenance of breast cancer stem cells and may promote their tumorigenic potential and resistance to drugs such as paclitaxel. However, there is tissue-specific heterogeneity in the differential upregulation of this transcriptome as well stemness phenotype of tumors in these tissues.