Hotspots of Somatic Genetic Variation in Pituitary Neuroendocrine Tumors

Abstract

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.

Keywords: druggable target; genetic driver; mutational hotspot; pituitary neuroendocrine tumor; somatic variant.

Figure 1

The RAS-RAF-MEK-ERK signaling pathway in corticotroph cells. Under physiological conditions, this pathway is activated in response to the interaction of extracellular ligands such as growth factors, hormones, or cytokines with a tyrosine kinase receptor. The receptor-like growth factor receptor-binding protein 2 (GRB2) binds to the activated receptor and interacts with the proline-rich sequence at the C-terminus of the son of sevenless (SOS) protein to form the receptor-GRB2-SOS complex, which in turn promotes the GTP-mediated activation of RAS. Activated RAS protein binds to and recruits BRAF to the inner side of the cell membrane, where it is phosphorylated by tyrosine kinases. The C-terminal catalytic domain of BRAF interacts with and phosphorylates MEK1 and 2 into their catalytic VIII subregion. In turn, MEK1 and 2 phosphorylate and thus activate ERK1 and 2 (also known as mitogen-activated protein kinases (MAPK) 3 and 1). In addition to phosphorylating cytoplasmic targets, active ERK1 and 2 enter the nucleus and phosphorylate multiple transcription factors, such as ELK1, ETS, FOS, JUN, and MYC, thereby inducing the expression of their target genes. Via the phosphorylation of RPS6KA1, ERK1 and 2 also activate the transcription factor cAMP response element-binding protein (CREB). The activation of this pathway leads to tissue-specific molecular consequences, although in the pituitary gland and in many other tissues it results in increased cell proliferation and survival [18,23,58,59]. In corticotroph cells, this pathway also activates POMC transcription, although the membrane receptor triggering this response in physiological conditions and in corticotropinomas remains unclear [40]. The BRAF p.V600E variant leads to the overactivation of this signaling pathway.

Figure 2

The cAMP pathway in somatotroph cells. G proteins are composed of three subunits, and the α subunit contains high-affinity binding sites for guanine nucleotides. The GDP-bound form binds tightly to βγ and is inactive, whereas the GTP-bound form dissociates from βγ and is the active form. GPCRs cause the activation of G proteins by facilitating the exchange of GTP for GDP on the α subunit, which in turn activates ACs. These enzymes use ATP as a substrate to produce cAMP. The latter binds to the regulatory subunits (R) of PKA, allowing for the release of the catalytic subunits (C). Active PKA catalyzes the serine/threonine phosphorylation of target molecules, including the transcription factors CREB, CRE modulator (CREM), and activating transcription factor 1 (ATF1). In complex with co-activators such as CREB-binding protein (CBP) and members of the cAMP-regulated transcriptional co-activators (CRTC), these transcription factors bind the 8 bp palindromic sequence known as cAMP response element (CRE) in the promoter region of target genes to increase their transcription. In somatotrophs, the GH-releasing hormone receptor (GHRHR) is the main GPCR activating this pathway, promoting both cell proliferation and GH transcription [66,67,68,69,121]. GNAS hotspot variants result in the constitutive activation of this pathway.

Figure 3

RNA processing pathways involved in PitNETs. (a) Biogenesis of small RNAs. In the nucleus, RNA polymerases II and III (RNA Pol II and III) generate primary miRNA transcripts (pri-miRNAs) from miRNA-encoding genes, which are then processed by the microprocessor complex, including the DROSHA RNaseIII. This initial step renders ~60-nucleotide-long hairpin-folded pre-miRNAs, which are in turn exported to the cytoplasm via exportin 5 (XPO5)/Ran-GTP. In the cytoplasm, DICER1 cleaves pre-miRNAs and long dsRNAs into mature miRNAs and siRNAs, respectively, both of which are 20–22 nucleotide-long double-stranded RNAs. The DICER1-dsRNA complex is then bound by a member of the AGO protein family (AGO2 is the best characterized of them) and TARBP2 to form the RISC-loading complex. This complex in turn loads dsRNAs into the RISC, which is required to produce single-stranded small RNAs that serve as a guide to recognize complementary RNA sequences (located in the 3′ untranslated region of mRNAs). The small RNA-loaded RISC can either block translation and promote degradation or directly cleave (via AGO proteins) target mRNAs. Additional roles for DICER1 in the responses to DNA damage (nuclear) and viral infections (cytoplasmic) have recently been described. In PitBs, this abnormal repertoire of small RNAs results in PRAME dysregulation, among other transcriptional alterations [131,133,155,156,157]. DICER1 variants result in abnormal processing of small RNAs, thereby impairing their ability to regulate gene expression. (b) Processing of mRNAs by the spliceosome. The spliceosome is a large complex of snRNPs and other proteins that carries out the removal of introns and the ligation of exons from mRNA precursors (pre-mRNAs), rendering mature mRNAs. Two types of spliceosomes, U2-dependent and U12-dependent, are recognized in eukaryotes, the former being the predominant one. The U2-dependent spliceosome is composed of U1, U2, U5, and U4/U6 snRNP, as well as other proteins. This process beings when the U1 snRNP binds to the 5′ SS to form the E complex. Then, the non-ribonucleoprotein complex components SF1, U2AF2, and U2AF1 bind the BS (18–40 nucleotides upstream from the 3′ SS), the polypyrimidine tract (a sequence immediately downstream from the BS), and an AG dinucleotide at the intron-exon junction, respectively. The U2 snRNP in turn replaces SF1, forming the A complex, and the U5, and U4/U6 snRNPs are then recruited to form the precatalytic B complex. Rearrangements in RNA–RNA and RNA–protein interactions ultimately lead to dissociation of the U1 and U4 snRNPs, thereby producing the active B complex. The latter is activated by the pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16, thereby generating the B∗ complex, which catalyzes the first step of splicing. The C complex is then formed, triggering the second step of splicing. Finally, the spliceosome is removed and recycled. SF3B1 hotspot variants lead to the use of cryptic pre-mRNA 3′ SSs, and aberrantly spliced mRNAs are degraded via NMD [158,159,160,161,162,163,164]. The repertoire of aberrantly spliced mRNAs involved in lactotroph tumorigenesis remains unknown.

Figure 4

Roles of USP8 and USP48 in deubiquitination in corticotroph cells. DUBs counteract ubiquitination of specific targets, thereby preventing their proteasomal or lysosomal degradation [212]. Upon ligand binding, EGFR is internalized, and then ubiquitinated, and directed for degradation by the lysosomal pathway. USP8 deubiquitinates EGFR both at the cell membrane and in the lysosomes, reducing its degradation and favoring its recycling. In corticotroph cells, EGFR signaling promotes cell proliferation, POMC expression, and ACTH secretion. Another important regulator of corticotroph tumorigenesis is the SHH pathway. Its effector, GLI1, is a substrate for USP48 that under physiological conditions leads to increased POMC expression. Another USP48 target, NFKB, has the opposite effect [40,41,197,201]. USP8 and USP48 hotspot variants associated with corticotropinomas lead to enhanced EGFR and GLI signaling, respectively. USP48 variants also inhibit the function of NFKB. Additional effects of hotspot variants affecting these DUBs are still incompletely described.