Molecular Endocrine Pathology
Moderators: Dr. Ricardo Lloyd and Dr. George Kontogeorgos
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 .
Somatic mutations of these genes are rare in tumors that are truly sporadic 
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  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 .
An additional distinctive characteristic of VHL tumors is that they usually produce exclusively
norepinephrine (NE), while MEN2 tumors produce both epinephrine (E) and (NE)
. Limited studies of NF1
tumors show strong similarities to those in MEN2 .
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 .
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 . 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
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 . 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 . 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
. 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  . 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  or involvement of
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 , 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
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