RET Expression and Neuron-like Differentiation of Pheochromocytoma and Normal Chromaffin Cells

Abstract

Receptor tyrosine kinase RET is normally expressed at low levels in chromaffin cells and high levels in sympathetic neurons. Paradoxically, it is overexpressed in subsets of pheochromocytomas. The overexpressed protein is usually wild-type, except in multiple endocrine neoplasia type 2. Possible explanations for overexpression include tumor origin from RET-expressing sympathoadrenal progenitors that escape developmental culling during embryogenesis, or reactivation of signaling pathways related to neuronal differentiation. Normal adult chromaffin and pheochromocytoma cells can undergo neuron-like differentiation in cell culture. In this investigation, cultured cells from two normal human adrenal medullas, two of three human pheochromocytomas, and one extra-adrenal paraganglioma showed RET induction corresponding with extensive nerve growth factor-induced outgrowth of neurite-like processes, while one pheochromocytoma showed neither processes nor RET induction. RET was similarly upregulated in parallel with process outgrowth in cultures of normal rat chromaffin cells and PC12 rat pheochromocytoma cells. In contrast, mouse pheochromocytoma cells that constitutively express high levels of wild-type RET together with other neuronal progenitor markers showed no further RET increase after cyclic AMP-induced process outgrowth. The RET-activating ligand GDNF was anti-apoptotic for mouse pheochromocytoma but not for PC12 cells. The findings suggest that overexpression of RET in pheochromocytomas could result either from a secondary event that activates signaling pathways mediating adult chromaffin cell plasticity or as a component of a persistent sympathoadrenal progenitor phenotype. Whether wild-type RET contributes to tumor development or is merely a lineage marker for cells at various stages of neuronal differentiation may vary, with other tumor characteristics.

Key words

adrenal medulla - neuroendocrine tumor - paraganglioma - nerve growth factor

References

• 1 Powers JF, Brachold JM, Tischler AS. Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma.  Endocr Pathol. 2003;  14 351-361
• 2 Takaya K, Yoshimasa T, Arai H, Tamura N, Miyamoto Y, Itoh H, Nakao K. Expression of the ret proto-oncogene in normal human tissues, pheochromocytomas, and other tumors of neural crest origin.  J Mol Med. 1996;  74 617-621
• 3 Lee S, Nakamura E, Yang H, Wei W, Linggi MS, Sajan MP, Farese RV, Freeman RS, Carter BD, Kaelin  Jr  WG, Schlisio S. Neuronal apoptosis linked to egln3 prolyl hydroxylase and familial pheochromocytoma genes: Developmental culling and cancer.  Cancer Cell. 2005;  8 155-167
• 4 Lai AZ, Gujral TS, Mulligan LM. Ret signaling in endocrine tumors: Delving deeper into molecular mechanisms.  Endocr Pathol. 2007;  18 57-67
• 5 Krieglstein K, Deimling F, Suter-Crazzolara C, Unsicker K. Expression and localization of GDNF in developing and adult adrenal chromaffin cells.  Cell Tissue Res. 1996;  286 263-268
• 6 Forander P, Broberger C, Stromberg I. Glial-cell-line-derived neurotrophic factor induces nerve fibre formation in primary cultures of adrenal chromaffin cells.  Cell Tissue Res. 2001;  305 43-51
• 7 Tischler AS, Greene LA. Phenotypic plasticity of pheochromocytoma and normal adrenal medullary cells.  Adv Biochem Psychopharmacol. 1980;  25 61-68
• 8 Tischler AS, DeLellis RA, Biales B, Nunnemacher G, Carabba V, Wolfe HJ. Nerve growth factor-induced neurite outgrowth from normal human chromaffin cells.  Lab Invest. 1980;  43 399-409
• 9 Powers JF, Shahsavari M, Tsokas P, Tischler AS. Nerve growth factor receptor signaling in proliferation of normal adult rat chromaffin cells.  Cell Tissue Res. 1999;  295 21-32
• 10 Marek L, Levresse V, Amura C, Zentrich E, Van Putten V, Nemenoff RA, Heasley LE. Multiple signaling conduits regulate global differentiation-specific gene expression in pc12 cells.  J Cell Physiol. 2004;  201 459-469
• 11 Batistatou A, Greene LA. Internucleosomal DNA cleavage and neuronal cell survival/death.  J Cell Biol. 1993;  122 523-532
• 12 Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.  Proc Natl Acad Sci USA. 1976;  73 2424-2428
• 13 Powers JF, Evinger MJ, Tsokas P, Bedri S, Alroy J, Shahsavari M, Tischler AS. Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.  Cell Tissue Res. 2000;  302 309-320
• 14 Powers JF, Evinger MJ, Zhi J, Picard KL, Tischler AS. Pheochromocytomas in nf1 knockout mice express a neural progenitor gene expression profile.  Neuroscience. 2007;  147 928-937
• 15 Powers JF, Schelling KH, Brachold JM, Tischler AS. Plasticity of pheochromocytoma cell lines from neurofibromatosis knockout mice.  Ann N Y Acad Sci. 2002;  971 371-378
• 16 Evinger MJ, Powers JF, Tischler AS. Transcriptional silencing of glucocorticoid-inducible phenylethanolamine N-methyltransferase expression by sequential signaling events.  Exp Cell Res. 2007;  313 772-781
• 17 Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(adp-ribose) polymerase: An early marker of chemotherapy-induced apoptosis.  Cancer Res. 1993;  53 3976-3985
• 18 Rossel M, Pasini A, Chappuis S, Geneste O, Fournier L, Schuffenecker I, Takahashi M, van Grunsven LA, Urdiales JL, Rudkin BB, Lenoir GM, Billaud M. Distinct biological properties of two ret isoforms activated by MEN 2a and MEN 2b mutations.  Oncogene. 1997;  14 265 275

Correspondence