MINI REVIEW published: 15 March 2021 doi: 10.3389/fendo.2021.646210 Epithelial–Mesenchymal Transition in the Resistance to Somatostatin Receptor Ligands in Acromegaly Joan Gil 1, Mireia Jordà 1*†, Berta Soldevila 2 and Manel Puig-Domingo 1,2,3*† 1Endocrine Tumours Lab, Program of Predictive and Personalized Medicine of Cancer (PMPPC), Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain, 2 Department of Endocrinology and Nutrition, Germans Trias i Pujol University Hospital, Badalona, Spain, 3 Department of Medicine, Autonomous University of Barcelona, Bellaterra, Spain Epithelial-mesenchymal transition (EMT) is a dynamic process by which epithelial cells loss Edited by: their phenotype and acquire mesenchymal traits, including increased migratory and Manuel D. Gahete, invasive capacities. EMT is involved in physiological processes, such as embryogenesis Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Spain and wound healing, and in pathological processes such as cancer, playing a pivotal role in Reviewed by: tumor progression and metastasis. Pituitary tumors, although typically benign, can be Marta Korbonits, locally invasive. Different studies have shown the association of EMT with increased tumor Queen Mary University of London, size and invasion in pituitary tumors, and in particular with a poor response to United Kingdom Sabrina Chiloiro, Somatostatin Receptor Ligands (SRLs) treatment in GH-producing pituitary tumors, the Catholic University of the Sacred main cause of acromegaly. This review will summarize the current knowledge regarding Heart, Italy EMT and SRLs resistance in acromegaly and, based on this relation, will suggest new *Correspondence: Manel Puig-Domingo biomarkers and possible therapies to SRLs resistant tumors. mpuigd@igtp.cat Keywords: epithelial–menchymal transition, somatostatin analogs, pituitary, E-cadherin, somatotroph adenoma, Mireia Jordà growth hormone, PitNETs, endocrine tumors mjorda@igtp.cat † These authors have contributed equally to this work and share senior authorship INTRODUCTION Specialty section: Epithelial–mesenchymal transition (EMT) is a dynamic process that reorganizes the cell from an This article was submitted to epithelial to a mesenchymal phenotype leading to functional changes in cell invasion and migration Pituitary Endocrinology, capacities (1). This process is triggered by microenvironment signals that cells receive which a section of the journal produce changes in gene expression and post-translational regulation mechanisms leading to the Frontiers in Endocrinology loss of epithelial characteristics (cell polarity, stable epithelial cell-cell junctions and interactions Received: 25 December 2020 with extracellular matrix) and the acquisition of mesenchymal features (fibroblast-like morphology Accepted: 22 February 2021 and increased migratory and invasive properties). Although it has been considered as a binary Published: 15 March 2021 process for many years, EMT has been recently shown to occur through distinct transition cellular Citation: states that are driven by a network of transcription factors (EMT-TFs) (2, 3). SNAI1-2, TWIST, and Gil J, Jordà M, Soldevila B and ZEB protein families have been the most extensively studied EMT-TFs as they regulate the classical Puig-Domingo M (2021) Epithelial– EMT focused on the repression of E-cadherin, the prototypic adhesion molecule; however, the list of Mesenchymal Transition in the Resistance to Somatostatin EMT-TFs has largely grown in the last years (4). Receptor Ligands in Acromegaly. EMT was first described in embryonic development as a process that enables the correct Front. Endocrinol. 12:646210. morphogenetic events during migration of epithelial cells from the original position to their doi: 10.3389/fendo.2021.646210 ultimate destination. However, EMT also occurs in pathological situations such as cancer (2, 5). Frontiers in Endocrinology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 646210 Gil et al. EMT in Acromegaly During the progression of solid malignancies from benign all malignancies derived from epithelial cells, has also been tumors to locally invading tumors, and finally to metastatic reported in pituitary tumors. E-cadherin, a calcium-dependent neoplasms, EMT plays a key role. However, it seems that cell to cell adhesion transmembrane protein, is part of a cell cancer-associated EMT is only activated partially and adhesion complex where it is associated with other proteins (a−, transiently, in contrast to developmental EMT (3). This b−, g− and p120-catenins) through an intracellular domain (17). attribute and the fact that EMT programs have been associated Interestingly, E-cadherin can act as direct transcriptional with other cellular programs such as cell survival, stemness and regulator by nuclear translocation (18). In pituitary tumors, the resistance to drugs (4), makes EMT difficult to study by only loss of E-cadherin, specifically the loss of cytoplasmic E- analyzing the expression of EMT-TF network. cadherin, is frequently found concurrently with its detection in Recent studies suggest the involvement of EMT in first the nucleus (19). Importantly, nuclear staining E-cadherin is generation Somatostatin Receptor Ligands (SRLs) resistance in associated with tumor invasion, suggesting that cleavage of the GH-producing pituitary tumors (6–9). Here we review the role of extracellular domain of E-cadherin and nuclear translocation EMT in pituitary adenomas and discuss the relationship between may participate in local invasion in pituitary tumors. Similarly, EMT and SRLs resistance in GH-producing pituitary tumors as E-cadherin among other adhesion molecules was related to well as offer new potential biomarkers and therapeutic options. invasiveness and proliferative status of prolactinomas (20, 21). Other classical EMT markers, such as N-cadherin, SNAI1, SNAI2 and TWIST (21, 22) or b-catenin (23) have also been associated METHODS with a worse clinical course in pituitary tumors, especially indicating an invasive phenotype, although there is some We performed a systematic review with the aim of summarizing controversy regarding this subject in acromegaly (21, 24). the current knowledge of EMT in GH-secreting adenomas with a Furthermore, different other non-classical molecules related special focus on SRLs resistance. We performed a search in to EMT have been characterized as part of the mechanisms MEDLINE database using its PubMed tool of the literature allowing invasiveness in pituitary tumors, such as ADAM12 available until January 2021. We searched for the terms: (a disintegrin and metalloprotease 12), which has been postulated “Epithelial-mesenchymal transition” OR “EMT” AND as an EMT inducer in these tumors (25). ADAM12 overexpression “Acromegaly” OR “Pituitary adenoma” OR “Pituitary tumor” is associated with pituitary tumor invasiveness, while its silencing AND/OR “Somatostatin” OR “Somatostatin receptor ligands” prevents such biological behavior. Mechanistically, ADAM12 OR “Somatostatin analogs”. silencing impairs ectodomain shedding of epidermal growth factor receptor (EGFR) ligands and attenuated the EGFR/ERK signaling pathway. Inhibition of EGFR signaling resulted in EPITHELIAL–MESENCHYMAL EMT suppression similar to repression of ADAM12. Also, a TRANSITION IN PITUITARY TUMORS recent study by Falch at al (26). in non-functioning gonadotroph tumors reported that those tumors harboring invasive and rapid It is well known the importance of the expression of determined growing characteristics showed overexpression of genes involved in transcription factors during pituitary organogenesis to give the EMT, in particular SPAG9, SKIL, MTDH, HOOK1, CNOT6L final identity to every different hormone-secreting cell type (10), and PRKACB. and EMT plays an important role in this embryogenic process Surprisingly, pituitary tumor transforming gene 1 (PTTG1) (11). The PROP1 transcription factor, that is vital for the has been related to EMT in non-functioning pituitary adenomas ontogenesis of somatotroph cells, was discovered to promote (27), just the opposite role of PTTG2 (28). Other authors EMT during pituitary stem cell differentiation, making EMT an suggested that the mechanism triggering the EMT in pituitary important step to obtain fully-functional differentiated pituitary tumors is linked to the expression of S100A9, a member of the cells (12, 13). These results have been validated by single-cell S100 family of EF-hand motif Ca2+-binding proteins, mediated transcriptomic profiling of the different developmental lineages by activation of AKT1 (29). In addition, it has been showed that in human pituitary (14). the transcriptoma of USP8 wild-type corticotropinomas, EMT is not only linked to pituitary through development, as characterized by increased invasiveness, was enriched in EMT E-cadherin has been related to hormone secretion in mature signature (30). cells. E-cadherin reduces prolactin protein content through In another study in GH-secreting adenomas, cyclin B1 affecting trafficking of secretory granules (15). Furthermore, it has (CCNB1) knock-down was found to decrease the mesenchymal been also associated with follicle-stimulating hormone (FSH) marker N-cadherin and increase the epithelial markers E- content and subcellular localization in non-functioning pituitary cadherin and p120-catenin. Thus, inactivation of cyclin B1 tumors (16). results in a decreased proliferation and EMT, and an increased EMT also plays an important role in the aggressive biologic apoptosis (31). A similar approach was used in a different study behavior of pituitary tumors. Pituitary tumors are the second where the expression of SMAD4 was found to regulate EMT in most common primary brain tumors with invasive properties. somatotropinomas. SMAD4 was associated with invasion, The loss of E-cadherin, which is a key characteristic of EMT increased levels of vimentin and N-cadherin and, decreased associated with poor prognosis and high grade tumors in almost E-cadherin (32). Frontiers in Endocrinology | www.frontiersin.org 2 March 2021 | Volume 12 | Article 646210 Gil et al. EMT in Acromegaly Other genes have been related to the suppression of EMT and, by binding to different G protein-coupled receptors (SSTR1–5) therefore, invasion. It is the case of collagen type VI alpha 6 [reviewed in Ben-Shlomo and Melmed (47)]. As remnant of its (COL6A6) that inhibits cell proliferation, migration, invasion, somatroph origin, somatotropinomas express somatostatin and epithelial-mesenchymal transition (EMT) through the receptors, specially SSTR2 and SSTR5 (48). First-generation binding of P4HA3 resulting in PI3K-Akt axis inhibition in Somatostatin Receptor Ligands (SRLs), octreotide and pituitary adenomas (33). lanreotide, so far the accepted first-line medical therapy in Not only coding genes have been related to EMT in pituitary acromegaly despite that hormonal hypersecretion control of tumors, some microRNAs (miRNAs) and long non-coding the disease is generally reported to be lower than 50% and RNAs (lncRNAs) have been shown to modulate EMT. MiR- both show a high affinity for SSTR2 receptor (49). Molecular 149-5p and miR-99a-3p suppress the expression of EMT-related characterization of the tumors has unveiled several explanations genes (34). miR-132, miR-15a and miR-16 also inhibit EMT in for such uneven response (50). However, many studies have pituitary adenomas; in this case targeting SOX5 (35). Moreover, proved the involvement of many other players and nowadays we miR-424-3p inhibits EMT and invasion by targeting JAG1 (36). do not know the whole picture of SRLs resistance in acromegaly. On the other hand, lncRNAs seem to be related to EMT EMT and epithelial plasticity have been associated with enhancement rather than inhibition. For example, lncRNA resistance to conventional, targeted and immune therapies in SNHG6 induces EMT suppressing miR-944, which may inhibit many cellular and preclinical models in different tumor contexts, RAB11A (37). Furthermore, lncRNA PVT1 enhances EMT and although there is little evidence from clinical samples (51). In this migration by activating Wnt/ß-catenin (38). Finally, lncRNA line, different studies relate EMT and SRLs resistance in SNHG1 promotes EMT and invasion by activation of TGFBR2/ acromegaly. E-cadherin has been linked to SRLs response as an SMAD3 and RAB11A/Wnt/b-Catenin axis, and the inhibition of independent predictor by different studies and in different cohorts miRNAs such as miR-302/372/373/520 (39). (8, 52, 53). In a fair comparison between several known EMT is a dynamic process, not a binary process, with biomarkers of SRLs response, E-cadherin showed the greatest intermediary states (2). It is very unlikely that benign tumor performance in predicting postsurgical SRLs response, even cells undergo a complete mesenchymal transformation which is greater than SSTR2 or Ki-67 (8). There is a general consensus associated with metastatic tumors (5). Because of that, it is more that low levels of E-cadherin mRNA and protein indicate a poor likely that, as in the majority of neoplasms, pituitary tumors responsive tumor to SRLs (8, 52, 53). Furthermore, E-cadherin would exhibit partial EMT states (40). This would explain why in loss seems to be related to the granulation pattern of the tumor, transcriptomic analysis some EMT markers are up-regulated especially but not exclusively in GH-producing tumors (8, 53). It is while others do not, instead of showing a complete mesenchymal worth saying that the histological granulation pattern of profile (9, 41). the tumor has been related to SRLs response for many years It is noteworthy to highlight the importance of the tumor (54, 55). Interestingly, some studies have shown the association microenvironment in mediating EMT and, therefore, the between E-cadherin downregulation and E-cadherin promoter aggressive behavior of pituitary adenomas. The alteration of hypermethylation in GH-secreting tumors, suggesting the the tumor microenvironment seems to be triggered by tumor involvement of epigenetic mechanisms (56–58). Another study chemokines that attract immune cells (42). Additionally, IL-6 pointed to the presence of progenitor mesenchymal cells and CCL2 produced by tumor associated fibroblasts have been derived from cancer stem cells as the cause of E-cadherin associated with EMT-like morphological changes and aggressive decrease and EMT induction (through TGFbRII increase) in behavior trough E-cadherin downregulation and ZEB1 somatotropinomas (59). upregulation in an in vitro study (43). Taking into account that E-cadherin is routinely assessed in It is really important to confirm the link between EMT and pathology departments as diagnostic tool for other cancer types AIP, since AIP-ZAC1 pathway is one of the main molecular (60), it would be easy to implement it as biomarker of response to mechanisms described for SRLs resistance (44). SRLs can activate SRLs to better define acromegaly treatment (61). AIP which inhibits adenylate cyclase, reducing cyclic AMP levels. On the other hand, AIP activates ZAC1. This molecule binds directly p53 and activates gene transcription; moreover, p53 BEYOND E-CADHERIN LOSS: arrests cell cycle, through p21 interaction, and increases INVOLVEMENT OF OTHER EPITHELIAL– apoptosis (45). Deeper explanation of the pathway could be found in some other reviews (45, 46). MESENCHYMAL TRANSITION MOLECULES IN SRL RESISTANCE IN ACROMEGALY E-CADHERIN LOSS IS AN OUTSTANDING Since E-cadherin loss is a marker of advanced EMT, some BIOMARKER FOR SRL RESISTANCE IN authors have further investigated this phenomena in GH- ACROMEGALY producing tumors. Lekva and colleagues analyzed the transcriptome of tumors with very high and very low levels of Somatostatin is secreted by the hypothalamus and inhibits E-cadherin and identified several EMT-related genes. hormone secretion and to a lesser extent pituitary cell growth Interestingly, in vitro, the expression of these genes were not Frontiers in Endocrinology | www.frontiersin.org 3 March 2021 | Volume 12 | Article 646210 Gil et al. EMT in Acromegaly regulated by E-cadherin but by Epithelial Splicing Regulatory For more detailed explanation of the cytoskeleton involvement Protein1 (ESRP1) (6). ESRP1 has been characterized as an in SRLs resistance, we recommend the review by Peverelli et al. important contributor to EMT by mediating alternative published in 2015 in this same journal (74). splicing in EMT affecting the maintenance of epithelial features (62). It is important to mention that several studies have proved the relation of splicing and SRLs resistance in acromegaly (7, 63– EPITHELIAL–MESENCHYMAL 65). ESRP1, thus, may be a master regulator of the EMT, SRLs TRANSITION-RELATED THERAPIES response and other pathological processes in acromegaly (7). Lekva and colleagues also investigated genes that were The involvement of EMT in acromegaly pathogenesis and SRLs differentially expressed upon treatment with SRLs in different resistance offers new therapeutic approaches that should be EMT contexts. They found that RAR Related Orphan Receptor C explored. As an example, CCNB1 overexpression in acromegaly (RORC) was overexpressed in phenotypically epithelial tumors can be targeted with resveratrol, inhibiting CCNB1 and reverting but not in mesenchymal ones (9). Moreover, RORC expression its effects on invasion (31). Interestingly, Pasireotide, a second was associated with SRLs response, a result that has been generation SRL, has been associated with a reduction of EMT- confirmed by finding that RORC is a biomarker of SRLs associated chemokines in tumor associated fibroblasts, suggesting response improvement after surgical debulking (66). an anti-tumor effect targeting the microenvironment (43). In On the other hand, patients harboring AIP-mutated contrast, other first generation SRLs do not appear to affect somatotropinomas tend to be diagnosed at a younger age with EMT (59). larger, more aggressive, and SRLs resistance tumors (44). Some Several EMT regulating TFs (SNAI1, SNAI2, TWIST…) can studies have shown that AIP is an important mediator of SRLs induce a therapy-resistant intrinsic mechanism (overexpression response (45), and AIP expression has been found to be a potent of drug efflux pumps) as well as an extrinsic one (gaining SRLs response predictor (44). In this context, it was interesting to resistance to apoptosis inducing agents). This explains why prove that the transcriptome of ten somatotropinomas and five EMT is often related to drug resistance in tumors (2). normal pituitaries revealed EMT as one of the most significantly However, EMT features are emerging as novel therapeutic altered pathways in AIP-mutated tumors. Furthermore, the cell- targets in cases of resistance to current therapies (75). Some of conditioned media of AIP-knockdown cells increases migration of the drugs proposed to inhibit EMT in clinical phases are well- macrophages (41), reinforcing the role of tumor microenvironment known for endocrinologists such as metformin (76). Others have in inducing EMT and a more aggressive phenotype. been proposed to be useful in acromegaly to target GDNF-RET/ PIT1/p14ARF/p53 pathway, like Sorafenib (77). EMT offers target opportunities in different levels: inhibiting stimuli from the tumor microenvironment, inhibiting extracellular CYTOSKELETON, EPITHELIAL– mediators and their corresponding receptors, inhibiting or MESENCHYMAL TRANSITION, AND SRL activating intracellular signaling pathways, and inhibiting RESISTANCE IN ACROMEGALY transcription factors that indirectly induce EMT (78). On this last regard, the usage of an inhibitor of STAT3 could very much benefit One of the main characteristics of EMT is the reorganization of acromegaly therapy since it would act reverting EMT process (79– cell polarity through changes in the cytoskeleton, which is 81) and directly inhibiting GH hypersecretion (82). More than a composed of the actin cytoskeleton, the microtubule network dozen of different therapies targeting EMT are being tested in and the intermediate filaments that provide structural design and clinical trials, however the vast majority are used in combination mechanical strength. The cytoskeleton is known to play an with regular chemotherapy since it is expected to recover sensitivity important role in EMT during cancer progression (67). of more conventional drugs upon EMT inhibition (78). In Concretely, refilin proteins perform their function through acromegaly, the main concern rather than the proliferation and filamin A (FLNA) to regulate the actin cytoskeleton formation of metastasis is the normalization of hormone levels; this reorganization. RefilinA promotes the conversion of FLNA is the reason why rather than the expected antiproliferative effects of from an actin branching protein into an F-actin bundler, and these drugs, we would expect a resensitization to SRLs. However, RefilinB combined with FLNA organize a unique perinuclear nowadays it is unknown if this effect would be achieved and which actin network at the apical surface during the EMT (68, 69). molecules should be targeted. Interestingly, in SNAI1-induced EMT, it has been proved that the changes in nuclear morphology and in the cytoskeleton structure correlate with decreased expression of FLNA (70). DISCUSSION FLNA plays an important role in GH-producing tumors since it has been related to pituitary tumors migration and invasion It is very likely that EMT plays an important role in acromegaly (71) and, more importantly, also to SRLs resistance (72). pathogenesis, but also in the modulation of pharmacologic Additionally, it has been proved that FLNA mediates response, thus inducing SRLs resistance in particular. However, octreotide-induced SSTR2 trafficking through endosomal most of this relationship is unknown since the molecular proteins in acromegaly. Moreover, FLNA influences the pathways relating EMT and SRLs signaling are not really number of available SSTR2 at the surface of the cell (73). understood and sufficiently explored. We are only beginning to Frontiers in Endocrinology | www.frontiersin.org 4 March 2021 | Volume 12 | Article 646210 Gil et al. EMT in Acromegaly unveil this relationship and we have, by now, been able to find In conclusion, EMT is a process that plays an important role some of the key molecules, but the whole picture remains elusive. in the heterogeneity of pituitary adenomas and is associated with To add a little more complexity to the acromegaly and EMT a more aggressive phenotype. Furthermore, it has been linked to relationship, it is worth to mention that one of the surprising SRLs response in somatotropinomas. Thus, EMT-related effects of GH is the induction of EMT (83), closing the circle therapies may be taken into consideration in the treatment of around EMT and acromegaly. The induction of EMT by GH acromegaly, especially in SRLs non-responder patients. This seems to be mediated by the tumor microenvironment involving could be an opportunity to find new therapies for pituitary not only tumor cells but multiple non-tumoral cell types (84, 85). adenomas; however, the increasing therapeutic options for Importantly, the study of EMT has provided some interesting acromegaly may overwhelm clinicians making more difficult the biomarkers to predict SRLs response in acromegaly, for example, choice of the best molecule(s) to target. In this regard, some E-cadherin and RORC. Furthermore, as we could only glimpse authors have developed a universal and quantitative EMT scoring for now, EMT in acromegaly is involved in a lot of processes like based on transcriptomic data that allows the prediction of stemness, apoptosis, secretory vesicles trafficking, cytoskeleton response to different pharmacological treatments (86). We organization, invasion capacities and aberrant splicing. All of think that this type of tools should be the basis of the future them are, to some extent, individually related to SRLs resistance medicine in acromegaly; the “trial‐and‐error” approach to decide and that makes very difficult to delimitate the action of EMT. It the appropriate drug would no longer be an option (87–90). forces to contemplate EMT as a dynamic process with deep connections with a multitude of different cellular programs. Moreover, the presence of intermediate EMT states in tumors, AUTHOR CONTRIBUTIONS which generates tumor heterogeneity, is probably key in the contribution of EMT to treatment resistance. All authors listed have made a substantial, direct, and intellectual Further studies of the EMT process would not only provide contribution to the work and approved it for publication. in-deep knowledge about the dedifferentiation of GH-secreting tumors and the SRLs desensitization, but will certainly offer alternative treatments to the SRLs. Several EMT inhibitors are FUNDING currently been tested in clinical trials for other malignancies. The plastic and dynamic nature of EMT increases the difficulty in Work in our laboratory is supported by the Instituto de salud determining the appropriate therapeutic and diagnostic Carlos III (PM 15/00027) and Novartis through the REMAH windows. However, targeting EMT blockade as an adjuvant (Registro Español Molecular de Adenomas Hipofisarios) therapy could potentially increase the effectiveness of the GH- consortium to the SEEN (Sociedad Española de Endocrinologı́a secreting tumors to SRLs. y Nutrició n). somatotroph adenomas following treatment with somatostatin analogs is REFERENCES associated with poor clinical recovery. PloS One (2013) 8:e66927. 1. Yang J, Antin P, Berx G, Blanpain C, Brabletz T, Bronner M, et al. Guidelines doi: 10.1371/journal.pone.0066927 and definitions for research on epithelial–mesenchymal transition. 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Clinical significance of filamin A in patients with The remaining authors declare that the research was conducted in the absence of acromegaly and its association with somatostatin and dopamine receptor any commercial or financial relationships that could be construed as a potential profiles. Sci Rep (2019) 9:1122. doi: 10.1038/s41598-018-37692-3 conflict of interest. 73. Treppiedi D, Mangili F, Giardino E, Catalano R, Locatelli M, Lania AG, et al. Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Copyright © 2021 Gil, Jordà, Soldevila and Puig-Domingo. This is an open-access Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 article distributed under the terms of the Creative Commons Attribution License Sorting Endosomes in Tumor Somatotroph Cells. Neuroendocrinology (CC BY). The use, distribution or reproduction in other forums is permitted, provided (2020) 110:642–52. doi: 10.1159/000503791 the original author(s) and the copyright owner(s) are credited and that the original 74. Peverelli E, Treppiedi D, Giardino E, Vitali E, Lania AG, Mantovani G. publication in this journal is cited, in accordance with accepted academic practice. No Dopamine and Somatostatin Analogues Resistance of Pituitary Tumors: use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Endocrinology | www.frontiersin.org 7 March 2021 | Volume 12 | Article 646210