There is increasing evidence that the Hippo pathway plays a functional role in the pituitary gland, though it is strongly associated with the stem cell state. Pituitary stem cells are able to give rise to all endocrine cell types of the anterior pituitary gland and their dysregulation can lead to tumorigenesis
[17]. It has been shown that the stem cell transcription factor SOX2 + interferes with the tumor suppressive Hippo pathway, leading to high YAP function and the repression of the differentiated state in the cancer stem cells in osteosarcomas
[111]. The role of the Hippo pathway in pituitary development and stem cell regulation was shown for the first time by Lodge and her colleagues
[71][109]. They found that the YAP and TAZ were active and primarily localized in the nucleus in SOX2 + pituitary stem cells throughout the development and at the postnatal stages in mice
[109]. Subsequently, in a preliminary study, Xekouki et al., showed evidence of an immunohistochemical expression of the YAP/TAZ in fetal and adult human pituitary cells as well as an increased expression in the poorly differentiated pituitary tumors (null cell adenomas, ACPs and PCPs), and all tumors with a large undifferentiated compartment
[16]. Consistent with the previous mouse data where the absence of LATS1 resulted in anterior pituitary hyperplasia and decreased the serum levels of GH, LH, and PRL
[112], the knockdown of LATS1 in the rat GH3 mammosomatotropinoma cells repressed the GH and PRL promoter activity, further supporting the role of the Hippo dysregulation in pituitary tumorigenesis
[16].
2.4. Wnt Pathway
Wingless/Int (Wnt) signaling is involved in pituitary organogenesis and controls the cell activity in the adult gland. The Wnt pathway has a pivotal role both in the differentiation of the pluripotent cells and in the proliferation of the mature pituitary cells, as well as in pituitary tumorigenesis. The most crucial component in the intracellular Wnt signaling pathway is β-catenin, an oncogenic protein encoded by the CTNNB1 gene. The Wnt proteins are the crucial regulators of this pathway, which interact with Frizzled (Fzd) receptor and facilitate the transcription of the cell proliferation and differentiation genes. In the inactive state (absence of the Wnt ligand), β-catenin is phosphorylated by the protein complex consisting of AXIN, glycogen synthase kinase-3β (GSK-3β), adenomatosis polyposis coli (APC), and casein kinase 1 alpha (CK1α), leading to its ubiquitination and degradation. In the active state (presence of the Wnt ligand), the regulatory complex axin/APC/GSK-3β/CK1α is inactivated by the disheveled (Dsh) protein, so β-catenin is not phosphorylated and enters the nucleus, acting as a transcription factor for the cell proliferation genes (cyclin D and c-Myc)
[113].
There is increasing evidence that Wnt signaling is implicated in the PitNets. It has been shown that Wnt4 was highly expressed in human pituitary tumors expressing GH, PRL, and TSH, all of which belong to the Pit1 cell lineage. Its presence was correlated with the Fzd6 expression, suggesting that the activation of the Wnt4/Fzd6 signaling contributed to tumorigenesis, but there was no change in the β-catenin distribution. β-catenin was localized only at the cell membrane in all the pituitary tumors and the normal pituitary glands. These findings indicated that the Wnt4/Fzd6 signaling was activated via a β-catenin-independent pathway
[114]. Another study investigated 47 pituitary tumors in which β-catenin was localized in the cell membrane with no difference between the PitNETs and normal controls. Still, they found a high nuclear accumulation of the Wnt target genes Cyclin D1 and c-Myc in the tumor tissue, indicating a β-catenin-independent activation of the Wnt pathway
[115]. Contrary to the previous studies, Semba et al. found a nuclear accumulation of β-catenin in 57% of the investigated PitNets, but they did not compare their findings to the normal pituitary gland
[116].
3. Tumor Suppressor Genes/Oncogenes
3.1. Menin Gene
Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant disorder with a high penetrance that is present in endocrine and non-endocrine tumors. Only 10% of patients are identified with de novo mutations. The patients are predisposed to the formation of the PitNETs, parathyroid hyperplasia, and gastroenteropancreatic neuroendocrine tumors (GEP-NETs)
[117]. Parathyroid tumors are the most common in approx. 95% of patients, followed by GEP-NETs in approx. 40%. These include gastrinomas, insulinomas, pancreatic polypeptidomas (PPomas), glucagonomas, and vasoactive intestinal polypeptidomas (VIPomas). Anterior pituitary tumors occur in about 30–40% of patients and the most prevalent type is lactortroph tumors (28–80%), followed by NF-PitNETs (15–48.1%), somatotroph tumors (5–15%), co-secreting tumors (9.1%), and rarely corticotroph tumors (5%), depending on the different series
[117][118][119]. Overall, MEN1 is responsible for less than 3% of patients with anterior pituitary tumors
[120].
The causative defect is the germline heterozygous mutation
in the MEN1 gene, a tumor suppressor gene localized on chromosome 11q13
[121]. Until recently, more than 1200 germline mutations have been identified in the MEN1 gene. In the majority of patients, the tumor formation follows the Knudson’s “two hit model” having one germline mutation in the MEN1 gene while a loss of heterozygosity (LOH) or somatic mutations occurs in the MEN1 alleles of the tumor
[122]. Menin is a nuclear protein with a ubiquitous expression, which is expressed differently from tissue to tissue
[123]. The cytoplasmic expression, as well as in the cell membrane, has also been described but to a lesser extent. Menin can regulate the gene transcription either positively or negatively. Recent studies suggest that it may act as a scaffold protein that controls the gene expression and cell signaling
[123]. Menin binds with the transcription factor JunD, one of the AP-1 transcription factors, and blocks its phosphorylation and activation from the c-Jun N-terminal kinase (JNK). Menin and JunD suppress the expression of the gastrin gene by binding to its promoter
[123]. On the other hand, menin activates the gene transcription by forming complexes with the transcription activator mixed lineage leukemia protein 1 (MLL1), a methyltransferase which functions as an oncogenic co-factor to promote the gene transcription and leukemogenesis
[123][124].
3.2. CDKN1B Gene
Not all patients with a MEN1-like phenotype harbor mutations in menin. About 10–15% have mutations in different genes and 3% of them carry germline mutations in the
CDKN1B gene, classified as MEN4
[125]. The
CDKN1B gene is a tumor suppression gene located on chromosome 12p13.1, encoding for the protein p27Kip1 (known as p27 or as KIP1)
[126]. The protein p27 is a member of the CDKI family, which binds to the cyclin/cyclin-dependent kinase complexes, preventing the cell cycle progression. In most cases there are germline heterozygous nonsense mutations, which lead to a reduced expression of p27, thereby resulting in an uncontrolled cell cycle proliferation
[127]. MEN4 patients usually exhibit parathyroid tumors and primary hyperparathyroidism. However, neuroendocrine tumors such as PitNETs, adrenal, and enteropancreatic tumors, testicular and papillary thyroid cancer, as well as non-endocrine tumors such as cervical carcinoma, colon cancer, and meningiomas, have also been reported
[127][128].
3.3. CABLES1 (CDK5 and ABL Enzyme Substrate 1)
The
CABLES1 gene mapped in the chromosome locus 18q11.2 counteracts the cell cycle progression that is activated in the corticotroph cells in response to glucocorticoids in the adrenal–pituitary negative feedback. The loss-of-function mutations of this tumor suppressor gene leads to an uncontrolled cell proliferation in corticotropinomas
[129]. The original description of the CABLES1 protein viewed it as an interacting partner and a substrate of the cyclin-dependent kinase-3 (CDK3)
[130]. In addition, it stabilizes the regulators of the cell cycle, such as CDKN1A (P21), CDK5R1 (P35), and TP63, preventing their degradation
[131][132].
3.4. PitNETs Related to Succinate Dehydrogenase (SDHx) Mutations
The SDHx gene mutations are known for their implication in pheochromocytomas and paragagliomas tumor formation
[133]. However, in 2012, Xekouki et al. described a patient with an acromegaly and concomitant presence of paragangliomas (PGLs) and pheochromocytomas (PHEOs) carrying a germline
SDHD mutation while he exhibited loss of heterozygosity at the SDHD locus in the pituitary tumor, and increased transcription hypoxia-inducible factor α(HIF-1α) levels similar to the PHEO/PGLs
[134]. Subsequently, the same group described the 3PAs syndrome characterized by the presence of the PHEOs and/or PGLs, and pituitary adenoma in the same patient
[135]. Although the SDHx mutations are common in the 3PAs familiar cases (62.5–75%), they are quite rare in the sporadic setting of the syndrome (0.3–1.8%)
[135][136].
The SDHx genes are tumor suppressor genes, encoding for the different subunits of the mitochondrial enzyme SDH, also named complex II or succinate:quinone oxidoreductase
[137]. SDH is located in the inner mitochondrial membrane and has a critical role in the oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycles, two major mechanisms in the metabolism and energy production within the cells
[138]. SDH consists of four subunits, SDHA-D. SDHA and B constitute the catalytic domain, which is extrinsic on the matrix side, while SDHC and D comprise the anchor subunits, which are intrinsic transmembrane proteins. The catalytic subunits catalyze the oxidation of the succinate to fumarate while the anchor subunits contribute to the transfer of the electrons from the succinate in the mitochondrial matrix to the ubiquinone in the inner membrane
[138].
The PitNETs in the 3PAs are more common among familial cases and they are usually macroadenomas secreting PRL or GH, while less frequently, they can be non-functioning and secrete ACTH
[139]. Most of the described cases required more than one type of treatment as they exhibited a more aggressive behavior and resistance to SSAs. Interestingly, the PitNETs in the context of the 3PAs were present at a younger age, in contrast to non-syndromic pituitary tumors, while the co-existence with the PHEO/PGLs was compatible with a more aggressive pituitary tumor, which implies a critical role of these tumors in the phenotype of the disease
[139][140].
3.5. DICER1, Ribonuclease III
DICER1 is a predisposition syndrome for the different types of tumors characterized by germline or mosaic loss-of-function (LOF) mutations in the
DICER1 gene mapped on the chromosome locus 14q32.13
[141]. It encodes a ubiquitously expressed endonuclease, a member of the ribonuclease (RNase) III family, required for the biogenesis of microRNA (miRNA) and small interfering RNA V (siRNA). However, the specific role of the
DICER1 gene in pituitary tumorigenesis is still under investigation
[141][142]. The most characteristic tumor in DICER1 patients is pleuropulmonary blastoma (PBB), a rare, early childhood pulmonary mesenchyma tumor. The other tumors include cystic nephroma, Wilms tumors, ovarian sex cord-stromal tumors (OSCSTs), especially Sertoli–Leydig cell tumors (SLCTs), and childhood embryonal rhabdomyosarcomas (ERMS)
[143]. Pituitary blastoma, a very rare embryonal aggressive pituitary tumor, can be part of DICER1 expressed with an ACTH-dependent hypercortisolemia (Cushing disease) and neuro-ophthalmopathy. Apart from surgery, polychemotherapy (cyclophosphamide, vincristine, methotrexate, carboplatin, and etoposide used in DICER1 patients) and adjuvant radiotherapy may be needed. However, the clinical experience with such tumors is very limited
[144].
4. Stem Cells in the Pituitary Gland and Tumorigenesis
Nowadays, it is well established that cancer stem cells (CSCs) stimulate tumor initiation, progression, recurrence, metastasis, and/or therapy resistance in different types of tumors. CSCs are characterized by persistent self-renewal and a multipotent differentiation capacity, representing a tumor-initiating cell population with intra-tumor heterogeneity [145]. Additionally, CSCs have high levels of plasticity with the ability to dedifferentiate. Similarly, CSCs have been identified in PitNETs. Several studies have isolated CSCs from human pituitary tumors with a clonogenic, sphere-forming potential in cultures that expressed pituitary-specific markers, such as Pit1, and markers of stemness, such as OCT4, Notch1 and 4, CD15, CD90, CD133, NESTIN, NANOG, CXCR4, and KLF4 [146][147][148][149][150][151]. Additionally, the regulatory signaling pathways that are essential for self-renewal and the differentiation of normal stem cells, such as Notch, Sonic hedgehog, Wnt, and Hippo are associated with cancer stem cells and pituitary oncogenesis as well [73].
Moreover, recent studies suggested that human pituitary adenoma stem cells (hPASCs) express DRD2, SSTR2, and SSTR5, whose activation using current treatment strategies such as DAs and SSAs seem to have promising results [147][148]. For example, Würth and his colleagues showed a decreased cell survival in hPASC cultures when incubated using the somatostatin/dopamine chimera BIM-23A760 [147]. Similarly, another study demonstrated that the DRD2 agonist BIM53097 and SSTR2 agonist BIM23120 had antiproliferative effects on both the spheres and tumor tissues in about half of the studied NF-PitNETs. In addition, the reduction in the proliferation ability of sphere-forming cells was confirmed by an increased CDKI p27 expression and a decrease in the cyclin D3 expression [148]. It is important to note that there was no difference in the frequency of the sphere formation between the NF-PitNETs that were in vitro resistant or sensitive to DRD2 and the SSTR2 agonists. However, the spheres that came from the tumors resistant to the DRD2 and SSTR2 agonists were larger compared to those derived from the sensitive NF-PitNETs [148].
5. MicroRNAs
MicroRNAs are short protein non-coding RNAs that act as regulatory proteins and control the post-transcriptional expression of specific genes through RNA interference and mRNA destabilization. They can induce a rapid degradation of the target messenger or inhibit its translation into a protein, and their expression can be regulated at different levels
[152]. In 2005, their expression was described for the first time in the pituitary gland. Since then, several studies have shown that miRNAs are involved in many mechanisms regulating the pituitary hormone production, tumor formation, progression, and aggressiveness
[153][154][155]. MiRNAs may play an important role in the pathogenesis and progression of PitNETs and may provide new molecular targets for their diagnosis and treatment.
It is estimated that miRNAs may control up to 50% of all the protein-coding genes
[156]. Several miRNAs are found to be involved in cell proliferation and apoptosis through an interference with the different pathways. For instance, the miR-187-3p elevation seems to promote the cell cycle progression and inhibit the proliferation of pituitary tumor cells via the NF-κB signaling pathway
[157]. Furthermore, the upregulation of several miRNAs (miR-17-5p, miR-20a, miR-106b, miR-21, miR200c, and miR-128) in pituitary tumors may inhibit the tumor suppressor signaling pathway PIK3/AKT, including PTEN, enabling a more aggressive behavior of these tumors
[152][158]. On the other hand, another group of miRNAs (miR-132, miR-15a, and miR-16) has the ability to inhibit the cell invasion and metastasis in several PitNETs by targeting SOX5, rendering these miRNAs as potential therapeutic targets for more aggressive pituitary tumors
[159].