Hotspots of Somatic Genetic Variation in Pituitary Neuroendocrine Tumors.

The most common genetic drivers of pituitary neuroendocrine tumors (PitNETs) lie within mutational hotspots, which are genomic regions where variants tend to cluster. Some of these hotspot defects are unique to PitNETs, while others are associated with additional neoplasms. Hotspot variants in GNAS and USP8 are the most common genetic causes of acromegaly and Cushing's disease, respectively. Although it has been proposed that these genetic defects could define specific clinical phenotypes, results are highly variable among studies. In contrast, DICER1 hotspot variants are associated with a familial syndrome of cancer predisposition, and only exceptionally occur as somatic changes. A small number of non-USP8-driven corticotropinomas are due to somatic hotspot variants in USP48 or BRAF; the latter is a well-known mutational hotspot in cancer. Finally, somatic variants affecting a hotspot in SF3B1 have been associated with multiple cancers and, more recently, with prolactinomas. Since the associations of BRAF, USP48, and SF3B1 hotspot variants with PitNETs are very recent, their effects on clinical phenotypes are still unknown. Further research is required to fully define the role of these genetic defects as disease biomarkers and therapeutic targets.

Clinical Spectrum of USP8 Pathogenic Variants in Cushing's Disease.

Cushing's disease (CD) is a life-threatening condition with a challenging diagnostic process and scarce treatment options. CD is caused by usually benign adrenocorticotrophic hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs), known as corticotropinomas. These tumors are predominantly of sporadic origin, and usually derive from the monoclonal expansion of a mutated cell. Somatic activating variants located within a hotspot of the USP8 gene are present in 11-62% of corticotropinomas, making USP8 the most frequent genetic driver of corticotroph neoplasia. In contrast, other somatic defects such as those affecting the glucocorticoid receptor gene (NR3C1), the BRAF oncogene, the deubiquitinase-encoding gene USP48, and TP53 are infrequent. Moreover, patients with familial tumor syndromes, such as multiple endocrine neoplasia, familial isolated pituitary adenoma, and DICER1 rarely develop corticotropinomas. One of the main molecular alterations in USP8-driven tumors is an overactivation of the epidermal growth factor receptor (EGFR) signaling pathway, which induces ACTH production. Hotspot USP8 variants lead to persistent EGFR overexpression, thereby perpetuating the hyper-synthesis of ACTH. More importantly, they condition a characteristic transcriptomic signature that might be useful for the clinical prognosis of patients with CD. Nevertheless, the clinical phenotype associated with USP8 variants is less well defined. Hereby we discuss the current knowledge on the molecular pathogenesis and clinical picture associated with USP8 hotspot variants. We focus on the potential significance of the USP8 mutational status for the design of tailored clinical strategies in CD.

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: human pituitary FSH18/21 and equine FSH (eqFSH) (hypo-glycosylated), and human FSH24 and chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 × 125 bp paired-end format, 10-15 × 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent differential regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways.