Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma

Abstract

Phaeochromocytomas and paragangliomas (PPGLs) are neuroendocrine catecholamine-producing tumours that may progress into inoperable metastatic disease. Treatment options for metastatic disease are limited, indicating a need for functional studies to identify pharmacologically targetable pathophysiological mechanisms, which require biologically relevant experimental models. Recently, a human progenitor phaeochromocytoma cell line named “hPheo1” was established, but its genotype has not been characterised. Performing exome sequencing analysis, we identified a KIF1B T827I mutation, and the oncogenic NRAS Q61K mutation. While KIF1B mutations are recurring somatic events in PPGLs, NRAS mutations have hitherto not been detected in PPGLs. Therefore, we aimed to assess its implications for the hPheo1 cell line, and possible relevance for the pathophysiology of PPGLs. We found that transient downregulation of NRAS in hPheo1 led to elevated expression of genes associated with cell adhesion, and enhanced adhesion to hPheo1 cells’ extracellular matrix. Analyses of previously published mRNA data from two independent PPGL patient cohorts (212 tissue samples) revealed a subcluster of PPGLs featuring hyperactivated RAS pathway-signalling and under-expression of cell adhesion-related gene expression programs. Thus, we conclude that NRAS activity in hPheo1 decreases adhesion to their own extracellular matrix and mirrors a transcriptomic RAS-signalling-related phenomenon in PPGLs.

Genetic impairment of succinate metabolism disrupts bioenergetic sensing in adrenal neuroendocrine cancer

“…Recent studies have identified ancillary driver mutations in ATRX (Fishbein et al, 2015), KIF1B, and NF1 (Evenepoel et al, 2017) in patients harboring SDHB mutations. This is congruent with recently discovered mutations in KIF1B and NRAS genes in hPheo1 cells (Rossitti et al, 2020). In addition to the germline driver SDHB mutations, ancillary mutations in hPheo1 create the opportunity to study malignant PC behavior.…”

Case report: Two sisters with a germline CHEK2 variant and distinct endocrine neoplasias

“…Indeed, subsequent evaluation of DNA from the patient’s PCC tumor and hPheo1 cell line detected the pathogenic mutation in the NF1 gene, NM_001042492.3 (NF1): c.4330A>G (p.Lys1444Glu) ( Figure 3B ). A second variant identified in primary PCC and hPheo1 was KIF1B variation T827I (c.2480C > T, rs121908162), which is predicted to be benign by PolyPhen-2 ( 24 ). Other mutation, previously reported for hPheo1 line, in addition to KIF1B , also includes NRAS Q61K ( 24 ) which was not detected in the patient’s tumor DNA, and may represent an adaptation of the cell line in culture (data not shown).…”

“…A second variant identified in primary PCC and hPheo1 was KIF1B variation T827I (c.2480C > T, rs121908162), which is predicted to be benign by PolyPhen-2 ( 24 ). Other mutation, previously reported for hPheo1 line, in addition to KIF1B , also includes NRAS Q61K ( 24 ) which was not detected in the patient’s tumor DNA, and may represent an adaptation of the cell line in culture (data not shown).…”

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS

“…The “trypsin-sensitive cells” was generated by transferring cells detached by 1 min trypsinization to a new dish. The “trypsin-resistant cells” were generated by continuing the culturing of the starting population for 30 min, washing with PBS and discarding the detached cells [ 33 , 50 ].…”