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From chromaffin cells to Phaeochromocytoma: insight into the sympathoadrenal cell lineage

Cleary, Susannah (2007) From chromaffin cells to Phaeochromocytoma: insight into the sympathoadrenal cell lineage. PhD thesis, Murdoch University.

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Chromaffin cells are a modified post-ganglionic sympathetic neuron, which synthesise and secrete catecholamines. The neoplastic transformation of chromaffin cells is demonstrated by the tumour phaeochromocytoma, a functional tumour that recapitulates the normal role of chromaffin cells by synthesising, storing and releasing excess catecholamines. Within this thesis we have explored several aspects of chromaffin cell and phaeochromocytoma tumour biology, including the specific expression of key sympathoadrenal markers such as the noradrenaline transporter (NAT), neuropeptide Y (NPY) and chromogranin A (CGA) in normal human and mouse chromaffin cells versus phaeochromocytomas of human and mouse origin.

Catecholamine-mediated signalling in chromaffin cells is terminated by the sequestration of extracellular catecholamines back into the cell via the noradrenaline transporter (NAT). Following observations that within the rat adrenal medulla, NAT is expressed in PNMT-positive chromaffin cells we explored whether this pattern of expression is also present in the human adrenal medulla. While we successfully established that NAT and PNMT are co localised, we also found that all human adrenal chromaffin cells are PNMT-positive. In the rat, NAT is also observed within the cytoplasm and has been suggested to be associated with secretory vesicles, thus using the secretory vesicle marker, CGA, we demonstrate that NAT is associated with secretory vesicles. However, in contrast to our findings within the normal chromaffin cells, in situ NAT expression in human phaeochromocytoma tumour samples was distorted, with observed changes including the level and type of staining observed, and disruptions to the strict NAT-CGA association observed in the normal adrenal.

Continuing our theme of NAT, we investigated if pre treating the phaeochromocytoma PC12 cell line with the chemotherapy drug cisplatin had an effect on the expression of NAT, to give an indication of the efficacy of this compound in the treatment of metastatic phaeochromocytoma with radiolabelled 131Iodometabenzylguanidine (131I-MIBG), a noradrenaline analogue which can be incorporated into phaeochromocytoma tumour cells though uptake through NAT. The premise of this study is derived from previous work in which neuroblastoma cells pre-treated with cisplatin were more responsive to (131I-MIBG) accumulation due to increased activity and expression of the transporter. Thus we treated PC12 cells for 24-hours in a range of cisplatin concentrations and measured the effect on NAT expression. However, unlike the findings in neuroblastoma cells, in our study, we did not observe an effect of cisplatin pretreatment on NAT activity or expression in PC12 cells.

Upto 30% of phaeochromocytoma arise as apart of a hereditary syndrome. The von Hippel-Lindau (VHL) syndrome, due to germline mutations to the VHL gene, and Multiple Endocrine Neoplasia type 2 (MEN 2), due to germline mutations to the RET gene represent two examples of hereditable endocrine disorders where phaeochromocytoma is a presenting feature. Notable differences in clinical presentation and tumour biology have been identified in phaeochromocytomas from patients with VHL and MEN 2. These differences prompted us to explore whether these observations extend to the chromaffin granule constituents, NPY and CGA.

Patients with MEN 2 disease have a greater incidence of hypertension than patients with VHL disease, MEN 2 are characterised by an adrenergic phenotype (produce predominantly-adrenaline), whereas VHL phaeochromocytomas are noradrenergic (produce predominantly-noradrenaline). Neuropeptide Y, which has powerful vasoactive properties capable of significantly elevating blood pressure, is stored and released with catecholamines and is thought to be associated with PNMT-positive chromaffin cells. Thus, we questioned whether the differences in the symptomatology between VHL and MEN 2 patients may be related to differences in NPY expression between the two groups, and whether any differences in NPY relate to adrenaline and/or PNMT content, or are linked to hereditary factors. Thus we compared tumour samples from four cohorts of patients: (i) adrenergic versus noradrenergic phenotype, (ii) hereditary versus no hereditary syndrome. Results demonstrated that although tumour NPY levels (mRNA and peptide) correlate with PNMT expression and/or adrenaline content, when NPY expression was compared between groups of patients (adrenergic vs noradrenergic; hereditary versus nonhereditary) difference in NPY levels were only significant between VHL and MEN 2 tumour and not between sporadic adrenergic and noradrenergic Immunohistochemistry also supported the above observations. Hence, we concluded that NPY expression in all groups of phaeochromocytoma examined in this study, this effect is not related to tumour biochemical phenotype but rather appears to be a specific unique trait of VHL phaeochromocytomas.

Continuing our theme of the possible differential expression of chromaffin granule constituents between VHL and MEN 2 patients, we also investigated CGA levels in plasma and tumour samples. Given, VHL tumours possess less chromaffin granules than MEN 2 tumours, and CGA has been implicated as a key director of secretory vesicle biogenesis we investigated whether CGA was differentially expressed between VHL and MEN 2 tumours. We found CGA expression was significantly greater in MEN 2 tumours (mRNA; 3-fold, and protein; 20-fold), with western blot confirming this trend. We also found that plasma CGA was greater in MEN 2 patients but not significantly, consequently, we explored the co-variables tumour size and tumour secretory activity (measured by plasma catecholamine concentrations), which influence plasma CGA and found that tumour size and plasma CGA are related but there was no influence of genotype on this relationship. In contrast, plasma CGA was significantly related to tumour secretory activity and the effect of genotype on this relationship narrowly missed significance, but when we expressed plasma CGA as a ratio of plasma catecholamines, plasma CGA was 2-fold greater in MEN 2 patients than VHL patients. Thus despite the tendency of phaeochromocytomas from VHL disease to readily and continuously release their catecholamine stores, plasma CGA levels still appeared to be higher in MEN 2 patients.

Finally, we examined whether the expression of NPY, Leu- enkephalin (Leu-Enk), NAT and the vesicular monoamine transporters type 1 and 2 (VMAT1 and VMAT2,), in normal mouse adrenal glands, and in histologically-confirmed adrenal phaeochromocytomas generated by injected nude mice with a phaeochromocytoma (MPC) cells line. The results of this study established that similar to the rat and human NAT expression is preferentially localised with PNMT within mouse chromaffin cells, while VMAT1 and NPY are found in both PNMT-negative and PNMT-positive cell populations, although expression of NPY was reduced in PNMT-negative cells. In contrast, both VMAT2 and Leu-Enk were found in PNMT-negative noradrenergic cells, and VMAT2 was present in all noradrenergic chromaffin cells while Leu-Enk was observed in a subpopulation of noradrenergic chromaffin cells. In contrast to the normal adrenal but similar to our findings in human phaeochromocytoma, the pattern of marker expression within adrenal phaeochromocytoma lesions of MPC-injected mice are severely disrupted related to both the level of expression of the respective markers, and association with PNMT within the tissue. Thus, the experimentally generated phaeochromocytoma mouse model provides a valuable tool in studying human phaeochromocytoma.

The data presented in this thesis further validate the heterogeneity observed in many aspects of phaeochromocytoma tumour biology, including the expression several chromaffin cell markers such as NAT, NPY and CGA. The altered expression of these markers may contribute to the clinical picture of these tumours, particularly relating to hereditary phaeochromocytoma from VHL and MEN 2 disease.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Biomedical Sciences
Supervisor(s): Phillips, Jacqueline
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