Section 3 -

Animal Models of Pheochromocytoma Arthur S Tischler Professor of Pathology Tufts University School of Medicine Boston , Massachusetts

Recent advances in genetics and gene expression profiling have led to the accrual and cataloging of data providing clues to the pathobiology of pheochromocytomas and-extra-adrenal paragangliomas. Animal models are needed to decipher the biological significance of the catalogued information in order to understand the development and progression of neoplasia and to develop and test targeted treatments. Data from the Study of Human Tumors Genetic analyses have thus far revealed the basis for hereditary transmission of susceptibility to these tumors in Multiple Endocrine Neoplasia (MEN) 2A and 2B, von Hippel-Lindau disease, neurofibromatosis type 1 and familial paraganglioma/pheochromocytoma syndromes, due respectively to mutations of the RET proto-oncogene, VHL and NF1 tumor suppressor genes and SDHD, SDHB, and SDHC succinate dehydrogenase genes. Occult germline mutations in these genes are found in more than 20% of patients presenting with apparently sporadic tumors, bringing the percentage of tumors with a known genetic basis close to 30%. Risk of malignancy, frequency of extra-adrenal involvement and tumor phenotype vary according to the underlying genetic defect [1]. Somatic mutations of these genes are rare in tumors that are truly sporadic [2] While germline mutations predispose to tumor development, secondary genetic alterations are probably necessary to initiate tumor formation. Chromosomal losses involving 1p, 3p, 3q and 11q are frequent in both sporadic and familial pheochromocytomas or paragangliomas. The most prevalent abnormality identified is deletion of a portion of chromosome 1p that may contain an unidentified tumor suppressor gene. Particular secondary genetic changes tend to associate with particular germline mutations [2] and might contribute to differences in tumor phenotype. Chromosome 1p and 3q losses are common in MEN2, NF1 and sporadic tumors; while11p losses are more frequent in VHL. Microarray-based gene expression profiling studies complemented by immunohistochemical and/or biochemical analyses have revealed sets of markers that also cluster in tumors with specific genetic backgrounds, in subsets of sporadic tumors, and in benign versus malignant tumors. Genes associated with hypoxia-driven transcription pathways are preferentially expressed in VHL and in malignant tumors [1]. An additional distinctive characteristic of VHL tumors is that they usually produce exclusively norepinephrine (NE), while MEN2 tumors produce both epinephrine (E) and (NE) [3]. Limited studies of NF1 tumors show strong similarities to those in MEN2 [4]. Selection and Uses of Animal Models Appropriate selection and use of a pheochromocytoma model requires the investigator to be mindful of relevance to human disease, relevance of cell culture to in vivo data and relevance of cell lines to primary tumors. Species differences are a critical concern. In addition, technical pitfalls may complicate the integration of histological, immunohistochemical and molecular data with cell biology. Pheochromocytomas are rare in all species except rats. Their concurrence with other tumors in rats, bovines, horses and dogs sometimes appears to parallel hereditary human pheochromocytoma syndromes but a genetic basis for spontaneously occurring animal pheochromocytomas has not been established. Pheochromocytomas are inducible in rats by many non-genotoxic substances that may act indirectly by stimulating chromaffin cell proliferation. They are not known to be similarly inducible in other species but arise with increased frequency in transgenic and knockout mouse models that resemble human tumor syndromes to varying degrees [5]. The rat pheochromocytoma cell line PC12 has for 30 years served as a research tool for many aspects of normal and neoplastic chromaffin cell biology [6]. However PC12 cells differ in some respects from many human pheochromocytomas and from many spontaneous or drug-induced rat pheochromocytomas. Important differences include extremely low levels of the epinephrine –synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. In addition, the genetic basis of PC12 cells is unknown. Tumors and cell lines recently developed from neurofibromatosis knockout mice now supplement PC12 cells [7, 8]. Their advantages include direct genetic relevance to a known hereditary human pheochromocytoma and expression of substantial levels of both PNMT and Ret. In addition, preliminary evidence suggests the presence of somatic genetic changes homologous to those in human pheochromocytomas [9]. Disadvantages include an apparently less mature and less stable phenotype. It is difficult to establish pheochromocytoma cell lines from any species, although the tumor cells persist in culture for many months, and no usable human pheochromocytoma lines are currently available. Understanding of factors that permit pheochromocytoma cells to proliferate might itself provide important insights for tumor biology. Puzzles and Pitfalls Microarray-based gene expression profiling studies show very little overlap between human, rat and mouse pheochromocytomas in the expression of "interesting" genes that might relate specifically to tumorigenesis. This may in part be due to artefactual differences between array systems [10]. In addition, several studies of human pheochromocytomas compare different groups of tumors to each other but not to normal adrenal medulla, so that common denominators in tumorigenesis might be overlooked. Nonetheless, intrinsic differences between species and between models in any given species are substantial and dictate that models should be employed selectively. Mouse pheochromocytomas and MPC cell lines overexpress a number of neural progenitor and stem cell genes and may offer unique opportunities to study the functions of those genes and their roles in neoplasia. One such gene that does frequently appear to be a common denominator overexpressed in different models is the receptor tyrosine kinase Ret [11]. However, with the exception of the pheochromocytomas in MEN2, it is not clear whether Ret is causally involved in tumorigenesis or merely serves as a cell lineage marker. Additional puzzles and pitfalls pertain to correlations of mRNA and protein expression. In a significant percentage of cases lack of correlation results from spurious microarray data- the gold standard for validation of microarrays is quantitative PCR [12] . For some important genes, e.g., PNMT, regulation of mRNA and protein expression is truly discordant and may serve a physiological purpose. Possible mechanisms include lack of protein stabilization by other molecules [13] or involvement of microRNAs. The analysis of protein expression in animal models of pheochromocytoma suffers both from the general malady of poor quality commercial antibodies and an odd phenomenon of non-specific staining. Both the rat and mouse adrenal contain separate populations of epinephrine and norepinephrine cells. The former often show technically perfect-appearing but non-specific staining with normal rabbit serum [14], making the staining of E-producing cells for any marker difficult to interpret. This artefact is usually not eliminated by buffers containing high salt or protein concentrations or Fc fragments or by endogenous biotin blocking. Immunohistochemical staining should therefore be validated with immunoblots and antigen adsorption controls. References Eisenhofer G, Bornstein SR, Brouwers FM, et al.: Malignant pheochromocytoma: current status and initiatives for future progress. Endocr Relat Cancer 11:423-36, 2004 Dannenberg H, Komminoth P, Dinjens WN, Speel EJ, de Krijger RR: Molecular genetic alterations in adrenal and extra-adrenal pheochromocytomas and paragangliomas. Endocr Pathol 14:329-50, 2003 Eisenhofer G, Huynh TT, Pacak K, et al.: Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome. Endocr Relat Cancer 11:897-911, 2004 Dahia PL, Ross KN, Wright ME, et al.: A HIF1alpha Regulatory Loop Links Hypoxia and Mitochondrial Signals in Pheochromocytomas. PLoS Genet 1:e8, 2005 Tischler AS, Powers JF, Alroy J: Animal models of pheochromocytoma. Histol Histopathol 19:883-95, 2004 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 U S A 73:2424-8, 1976 Jacks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, Weinberg RA: Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet 7:353-61., 1994 Powers JF, Evinger MJ, Tsokas P, et al.: Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice. Cell Tissue Res 302:309-20., 2000 Powers JF, Tischler AS, Mohammed M, Naeem R: Microarray-based comparative genomic hybridization of pheochromocytoma cell lines from neurofibromatosis knockout mice reveals genetic alterations similar to those in human pheochromocytomas. Cancer Genet Cytogenet 159:27-31, 2005 Marshall E: Getting the noise out of gene arrays. Science 306:630-631, 2004 Powers JF, Schelling K, Brachold JM, et al.: High-level expression of receptor tyrosine kinase ret and responsiveness to ret-activating ligands in pheochromocytoma cell lines from neurofibromatosis knockout mice. Mol Cell Neurosci 20:382-9., 2002 Dallas PB, Gottardo NG, Firth MJ, et al.: Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate? BMC Genomics 6:59, 2005 Ciaranello RD, Wong DL, Berenbeim DM: Regulation of phenylethanolamine N-methyltransferase synthesis and degradation. II. Control of the thermal stability of the enzyme by an endogenous stabilizing factor. Mol Pharmacol 14:490-501, 1978 Tischler AS, Tsokas P, Shahsavari M, Powers JF: Immunoreactivity of normal rabbit serum with epinephrine (E) cells of the rat adrenal medulla after microwave antigen retrieval. Cell Tissue Res 293:563-6, 1998