Further delineation of molecular alterations in adreno-medullary tumors

Abstract

Pheochromocytomas, abdominal paragangliomas and neuroblastomas are tumors of the sympathetic nervous system. Anatomically pheochromocytomas and abdominal paragangliomas arise from sympathoadrenal paraganglia, within and outside the adrenal medulla, respectively. On the other hand, neuroblastomas may present both in the adrenal medulla and along the sympathetic trunk. While pheochromocytomas and abdominal paragangliomas are mostly adult cancers originating from chromaffin cells, neuroblastoma is a pediatric tumor, deriving from immature sympathetic nerve cells. During cancer development, neoplastic cells accumulate a perplexing variety of genetic and epigenetic changes, enabling them to escape control mechanisms of proliferation and cell death.

The overall objective of this thesis is to shed further light on the genetic and epigenetic pathways that contribute to the molecular pathogenesis of these tumors.

In Paper I promoter specific and genome wide methylation changes were quantitatively assessed in relation to clinical features in a panel pheochromocytomas / abdominal paragangliomas. Based on methylation levels in the tumor suppressor genes, p16INK4A, CDH1, DCR2, RARB, RASSF1A, NORE1A, TP73, APC, DAPK1, p14ARF and PTEN, a CpG island methylator phenotype (CIMP) was defined as concerted hypermethylation in three or more genes. A subset of abdominal paragangliomas displayed CIMP phenotype, which was associated with malignant behavior and young age at presentation. This observation raises a prospective for potential benefits of epigenetically acting drugs for a subgroup of young abdominal paraganglioma patients with adverse prognosis. In addition genome wide hypomethylation was seen in tumors as compared to normal adrenal samples providing further support for the general significance of global hypomethylation in neuroendocrine cancers.

In Paper II the RIZ1 tumor suppressor in 1p36.2 was assessed as a candidate target for the distal 1p deletions in these tumors. Intragenic LOH and suppressed mRNA expression was detected in a substantial proportion of the tumors. This was not associated, however with RIZ1 promoter methylation. The recurrent inactivation RIZ1 suggests that this event may be a significant contributing factor to tumorigenesis in pheochromocytomas and abdominal paragangliomas.

NORE1A and RASSF1A are proapoptotic RAS effectors with tumor suppressor functions. In paper III their role was explored in pheochromocytomas and abdominal paraganliomas. Suppressed NORE1A and RASSF1A mRNA levels were detected in tumors compared to normal adrenal medulla. Methylation of the RASSF1A promoter was significantly associated with malignant behavior, while NORE1A methylation was uncommon. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A transfected PC12 pheochromocytoma cells. Ectopic expression of Nore1a resulted in enhanced apoptosis and impaired colony formation in soft agar suggesting that suppression of NORE1A contributes to the transformed phenotype in these tumors.

In paper IV the role of NORE1A was investigated in neuroblastoma tumors and cell lines. Supression of NORE1A mRNA expression was evident in cell lines and tumors, particularly in cases without MYCN amplification. Methylation of the NORE1A promoter was not a characteristic event contrasting RASSF1A, which showed frequent hypermethylation. Transient expression of Nore1a in SK-N-BE(2) cells resulted in enhanced apoptosis and delayed cell cycle progression, suggesting that suppression of NORE1A may contribute to tumorigenesis.

In Paper V, to further substantiate the general role of NORE1A and RASSF1A in cancer, we extended the analysis of these molecules to follicular thyroid tumors. Substantially reduced NORE1A mRNA expression was seen in all PAX8-PPARγ positive tumors while RAS mutation and PAX8-PPARγ fusions were mutually exclusive. RASSF1A expression was reduced in the majority of tumors analyzed.

Studies in knock-out models indicate that inactivation of products of the CDKN2A locus, p16INK4A and p14ARF, promotes pheochromocytoma development. In Paper VI, the involvement of CDKN2A in human pheochromocytomas and abdominal paragangliomas was studied, with regard to promoter methylation, expression and sequence alterations. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and it was associated with malignant disease. Frequent suppression of p16INK4A mRNA and protein expression was seen in tumors. By contrast no significant methylation or reduced mRNA expression was evident for p14ARF. Sequence variations were observed in four tumors including a missense mutation. These results suggest that p16INK4A, and not p14ARF is a frequent target of inactivating events in human pheochromocytoma and abdominal paraganglioma.