Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly

Abstract

Background Acromegaly is produced by excess growth hormone secreted by a pituitary adenoma of somatotroph cells (ACRO). First-line therapy, surgery and adjuvant therapy with somatostatin analogs, fails in 25% of patients. There is no predictive factor of resistance to therapy. New therapies are investigated using few dispersed tumor cells in acute primary cultures in standard conditions where the cells do not grow, or using rat pituitary cell lines that do not maintain the full somatotroph phenotype. The RET/PIT1/p14ARF/p53 pathway regulates apoptosis in normal pituitary somatotrophs whereas the RET/GDNF pathway regulates survival, controlling PIT1 levels and blocking p14ARF (ARF) and p53 expression. Methods We investigated these two RET pathways in a prospective series of 32 ACRO and 63 non-functioning pituitary adenomas (NFPA), studying quantitative RNA and protein gene expression for molecular-clinical correlations and how the RET pathway might be implicated in therapeutic success. Clinical data was collected during post-surgical follow-up. We also established new'humanized’ pituitary cultures, allowing 20 repeated passages and maintaining the pituitary secretory phenotype, and tested five multikinase inhibitors (TKI: Vandetanib, Lenvatinib, Sunitinib, Cabozantinib and Sorafenib) potentially able to act on the GDNF-induced RET dimerization/survival pathway. Antibody arrays investigated intracellular molecular pathways. Findings In ACRO, there was specific enrichment of all genes in both RET pathways, especially GDNF. ARF and GFRA4 gene expression were found to be opposing predictors of response to first-line therapy. ARF cut-off levels, calculated categorizing by GNAS mutation, were predictive of good response (above) or resistance (below) to therapy months later. Sorafenib, through AMPK, blocked the GDNF/AKT survival action without altering the RET apoptotic pathway. Interpretation Tumor ARF mRNA expression measured at the time of the surgery is a prognosis factor in acromegaly. The RET inhibitor, Sorafenib, is proposed as a potential treatment for resistant ACRO. Fund This project was supported by national grants from Agencia Estatal de Investigación (AEI) and Instituto Investigación Carlos III, with participation of European FEDER funds, to IB (PI150056) and CVA (BFU2016-76973-R). It was also supported initially by a grant from the Investigator Initiated Research (IIR) Program (WI177773) and by a non-restricted Research Grant from Pfizer Foundation to IB. Some of the pituitary acromegaly samples were collected in the framework of the Spanish National Registry of Acromegaly (REMAH), partially supported by an unrestricted grant from Novartis to the Spanish Endocrine Association (SEEN). CVA is also supported from a grant of Medical Research Council UK MR/M018539/1.

GFRα 1-2-3-4 co-receptors for RET Are co-expressed in Pituitary Stem Cells but Individually Retained in Some Adenopituitary Cells

“…Further studies in individual GFRα co-expression with markers of differentiation would help to delineate the process of recruitment and differentiation leading to a wider comprehension of cell lineage differentiation within the pituitary and to a deeper knowledge of the differentiation process. This has implications in pituitary pathology as demonstrated for the role of RET and GFRα4 in acromegaly tumors (19).…”

“…Moreover, GFRα1 expression together with its ligand GDNF-remains relevant in somatotroph derived tumors causing acromegaly (18,19,45). The increased expression of GDNF, blocks the RET /PIT1/p14ARF/p53 apoptosis pathway inducing AKT activation and survival contributing to cell growth and tumor development (19,21,22). Pituitary stem cells are not the unique expressing GFRα1, since is expressed in Sertoli and Leydig cells in testis (Leydig cells also co-express GFRα2), where it arises as essential in spermatogonial stem cells (46).…”

“…GDNF the most abundant RET ligand in the pituitary was expressed exclusively at the AP, with no staining in the IL or NP (Figure 5F). It is known that GDNF is expressed by normal somatotrophs together with GFRα1 and its expression is preserved in somatotroph adenomas (18,19,22). SOX2 was intensely detected within the nuclei of the RC epithelium, as corresponds to a marker of GPS stem cells ( Figure 5G).…”

“…RNA was finally quantified by spectrophotometry (Nanodrop 2000, Thermo Fisher). Previous to cDNA synthesis, we performed a pre-treatment with DNase incubating 1 µg of RNA with 1 IU of RNase-free DNase I (EN0521, Thermo Fisher), (19,31,32). Negative controls of the reverse-transcription step (all reagents and RNA sample but without reverse transcriptase) and the PCR step (all reagents but no reverse-transcribed sample) were included in each assay plate.…”

“…On the other hand, GFRα4 was originally described as not expressed in human pituitary (30). However, GFRA4 mRNA has recently been shown to be expressed in somatotroph pituitary tumors causing acromegaly (19). GFRA4 was significantly correlated with poorer prognosis and resistance to first-line therapy.…”