Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+Population that Is Dependent onRETActivity

Abstract

Context: Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET protooncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. Objective: In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. Main Outcome Measures: 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 activation was assessed through RNA interference knockdown. Results: 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 -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 activity than epidermal growth factor or fibroblast growth factor. Conclusion: 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 receptor activity. These findings may provide new insights to develop more promising therapy for MTC. Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy Anat Ben-Shlomo, Herbert Schmid, Kolja Wawrowsky, Oxana Pichurin, Erika Hubina, Vera Chesnokova, Ning-Ai Liu, Michael Culler, and Shlomo Melmed (J Clin Endocrinol Metab, published October 9, 2009, 10.1210/jc.2009-1311) ABSTRACT Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933