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Progress Report 2013

KFO252_long

Progress Report 2013: Project 2 - Biochemical and molecular profiling of chromaffin cell tumours


Background

This project has clinical and basic components involving patient-oriented studies directed at genotype-phenotype relationships in patients with pheochromocytomas and paragangliomas (PPGLs) and use of chromaffin cell model systems to explore the mechanisms linking germ line mutations to tumorigenesis and distinct clinical presentations. A key focus is directed at links between hypoxia pathways (particularly related to HIF2α) with mitochondrial energy and catecholamine biosynthetic and secretory pathways. The underlying hypothesis is that the variable biology and presentations of chromaffin cell tumors reflect their development from distinct chromaffin progenitor cells with different susceptibilities to underlying mutations. Direction of the project has been influenced by recent key findings concerning Myc-associated factor X (MAX) and HIF2αas novel PPGL susceptibility genes.


Achievements

Several different LC-MS/MS based analytical methods have been developed enabling targeted analyses of plasma and urine monoamine-related biomarkers, precise and sensitive measurements of phenylethanolamine N-methyltransferase (PNMT) and profiling of mitochondrial energy pathways, all required methodologies for the project. The clinical component of the project depends on a Dresden-based international multicenter trial (https://pmt-study.pressor.org), which has accrued to date biomaterial and data from 1044 enrolled patients, including over 100 with PPGLs.  Several clinical sub-studies have been completed evaluating the various biomarkers for diagnosis or stratification of disease related to malignancy or mutation status. Among these is a mitochondrial energy pathway profiling study that establishes utility of tumor levels of Krebs cycle metabolites as biomarkers for identification and stratification of PPGLs due to of mutations of succinate dehydrogenase (SDH) genes. Mechanistic studies have taken advantage of two pheochromocytoma cell lines: PC12 cells derived from natural mutations of MAX and MPC cells derived from engineered mutations of the neurofibromatosis gene.  Stable HIF2α overexpression in MPC cells increased proliferation and blocked the effect of dexamethasone to increase PNMT expression. In contrast, silencing HIF1α or MAX decreased PNMT expression. Stable MAX overexpression in PC12 cells inhibited cell cycle progression and led to expression of active PNMT. These data support a mechanism in which HIF1α and HIF2α alter MAX/MYC binding, acting in opposing directions to regulate chromaffin cell proliferation and differentiation, with resulting distinct catecholamine phenotypic features of PPGLs. Expression of HIF2α in tumors due to mutations of SDH and VHL genes explains why these tumors maintain an undifferentiated phenotype without expression of PNMT, even after stimulation with adrenal steroids. A first draft of the manuscript has been prepared and other data related to the underlying hypothesis is reviewed elsewhere. All supported personnel have published.


Completion of objectives

1. The multicenter PMT clinical trial is close to half complete with several sub-studies satisfying objective 1 completed and published.

2. Chromaffin cell model systems have been established that mechanistically link hypoxia and catecholamine secretory pathways according to manipulations of key components (e.g., MAX re-expression in PC12 and HIF2α and HIF1α silencing or over-expression).

3. new tumor biomarkers have been identified (e.g., succinate:fumarate ratios) and new engineered cell lines established for therapeutic targeting.