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Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties

Oncogene volume 31, pages 1896–1909 (2012)Cite this article

Abstract

Emerging evidence suggests that cancers arise in stem/progenitor cells. Yet, the requirements for transformation of these primitive cells remains poorly understood. In this study, we have exploited the ‘mammosphere’ system that selects for primitive mammary stem/progenitor cells to explore their potential and requirements for transformation. Introduction of Simian Virus 40 Early Region and hTERT into mammosphere-derived cells led to the generation of NBLE, an immortalized mammary epithelial cell line. The NBLEs largely comprised of bi-potent progenitors with long-term self-renewal and multi-lineage differentiation potential. Clonal and karyotype analyses revealed the existence of heterogeneous population within NBLEs with varied proliferation, differentiation and sphere-forming potential. Significantly, injection of NBLEs into immunocompromised mice resulted in the generation of invasive ductal adenocarcinomas. Further, these cells harbored a sub-population of CD44+/CD24− fraction that alone had sphere- and tumor-initiating potential and resembled the breast cancer stem cell gene signature. Interestingly, prolonged in vitro culturing led to their further enrichment. The NBLE cells also showed increased expression of stemness and epithelial to mesenchymal transition markers, deregulated self-renewal pathways, activated DNA-damage response and cancer-associated chromosomal aberrations—all of which are likely to have contributed to their tumorigenic transformation. Thus, unlike previous in vitro transformation studies that used adherent, more differentiated human mammary epithelial cells our study demonstrates that the mammosphere-derived, less-differentiated cells undergo tumorigenic conversion with only two genetic elements, without requiring oncogenic Ras. Moreover, the striking phenotypic and molecular resemblance of the NBLE-generated tumors with naturally arising breast adenocarcinomas supports the notion of a primitive breast cell as the origin for this subtype of breast cancer. Finally, the NBLEs represent a heterogeneous population of cells with striking plasticity, capable of differentiation, self-renewal and tumorigenicity, thus offering a unique model system to study the molecular mechanisms involved with these processes.

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Acknowledgements

We thank Dr Robert A Weinberg (Whitehead Institute) for hTERT cDNA, HMLE and HMLER cells and Dr Inder M Verma (Salk Institute) for pCSCG lentiviral vector. We acknowledge Swati Maiti for insightful discussions, and thank D Alaguraj, Drs Omana Joy, William Surin (IISc) and H Krishnamurthy (NCBS) for help with flow cytometry. We also thank Dr Paturu Kondaiah and Imran Khan (IISc), and Mohammed Aiyaz (Genotypic India Pvt. Ltd) for help with microarray experiments and data analysis. This work was largely supported by research grants from DBT to AR for equipment and consumables. We acknowledge the confocal, IRIS, FACS and animal facilities at IISc, and grants from UGC to the Department of MRDG, IISc.

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Authors and Affiliations

  1. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India

    A N Paranjape, T Mandal & A Rangarajan

  2. Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore, Karnataka, India

    A N Paranjape, G Mukherjee & M V Kumar

  3. Department of Biology, Indian Institute of Science Education and Research, Pune, Maharashtra, India

    K Sengupta

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  1. A N Paranjape
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Correspondence to A Rangarajan.

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Paranjape, A., Mandal, T., Mukherjee, G. et al. Introduction of SV40ER and hTERT into mammospheres generates breast cancer cells with stem cell properties. Oncogene 31, 1896–1909 (2012). https://doi.org/10.1038/onc.2011.378

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