Medullary thyroid carcinoma cell lines contain a self-renewing CD133+ population that is dependent on ret proto-oncogene activity.

Abstract

Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET proto-oncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of ret proto-oncogene activation was assessed through RNA interference knockdown. CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133(+) cells in two MTC cell lines. The CD133(+) cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers beta-tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133(+) cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on ret proto-oncogene activity than epidermal growth factor or fibroblast growth factor. Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on ret proto-oncogene receptor activity. These findings may provide new insights to develop more promising therapy for MTC.