Are there any completely sterile organs or tissues in the human body? Is there any sacred place?

The human microbiome comprises an ample set of organisms that inhabit and interact within the human body, contributing both positively and negatively to our health. In recent years, several research groups have described the presence of microorganisms in organs or tissues traditionally considered as 'sterile' under healthy and pathological conditions. In this sense, microorganisms have been detected in several types of cancer, including those in 'sterile' organs. But how can the presence of microorganisms be detected? In most studies, 16S and internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing has led to the identification of prokaryotes and fungi. However, a major limitation of this technique is that it cannot distinguish between living and dead organisms. RNA-based methods have been proposed to overcome this limitation, as the shorter half-life of the RNA would identify only the transcriptionally active microorganisms, although perhaps not all the viable ones. In this sense, metaproteomic techniques or the search for molecular metabolic signatures could be interesting alternatives for the identification of living microorganisms. In summary, new technological advances are challenging the notion of 'sterile' organs in our body. However, to date, evidence for a structured living microbiome in most of these organs is scarce or non-existent. The implementation of new technological approaches will be necessary to fully understand the importance of the microbiome in these organs, which could pave the way for the development of a wide range of new therapeutic strategies.

Antitumor effect of a small-molecule inhibitor of KRASG12D in xenograft models of mucinous appendicular neoplasms.

Pseudomyxoma peritonei (PMP) is a rare disease characterized by a massive accumulation of mucus in the peritoneal cavity. The only effective treatment is aggressive surgery, aimed at removing all visible tumors. However, a high percentage of patients relapse, with subsequent progression and death. Recently, there has been an increase in therapies that target mutated oncogenic proteins. In this sense, KRAS has been reported to be highly mutated in PMP, with KRASG12D being the most common subtype. Here, we tested the efficacy of a small-molecule KRASG12D inhibitor, MRTX1133, in a high-grade PMP xenograft mouse model carrying a KRASG12D mutation. The results obtained in this work showed a profound inhibition of tumor growth, which was associated with a reduction in cell proliferation, an increase in apoptosis, and a reduction in the MAPK and PI3K/AKT/mTOR signaling pathways. In conclusion, these results demonstrate the high potency and efficacy of MRTX1133 in KRASG12D-PMP tumors and provide a rationale for clinical trials.

Direct and systemic actions of growth hormone receptor (GHR)-signaling on hepatic glycolysis, de novo lipogenesis and insulin sensitivity, associated with steatosis.

BACKGROUND: Evidence is accumulating that growth hormone (GH) protects against the development of steatosis and progression of non-alcoholic fatty liver disease (NAFLD). GH may control steatosis indirectly by altering systemic insulin sensitivity and substrate delivery to the liver and/or by the direct actions of GH on hepatocyte function. APPROACH: To better define the hepatocyte-specific role of GH receptor (GHR) signaling on regulating steatosis, we used a mouse model with adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd). To prevent the reduction in circulating insulin-like growth factor 1 (IGF1) and the subsequent increase in GH observed after aHepGHRkd, subsets of aHepGHRkd mice were treated with adeno-associated viral vectors (AAV) driving hepatocyte-specific expression of IGF1 or a constitutively active form of STAT5b (STAT5bCA). The impact of hepatocyte-specific modulation of GHR, IGF1 and STAT5b on carbohydrate and lipid metabolism was studied across multiple nutritional states and in the context of hyperinsulinemic:euglycemic clamps. RESULTS: Chow-fed male aHepGHRkd mice developed steatosis associated with an increase in hepatic glucokinase (GCK) and ketohexokinase (KHK) expression and de novo lipogenesis (DNL) rate, in the post-absorptive state and in response to refeeding after an overnight fast. The aHepGHRkd-associated increase in hepatic KHK, but not GCK and steatosis, was dependent on hepatocyte expression of carbohydrate response element binding protein (ChREBP), in re-fed mice. Interestingly, under clamp conditions, aHepGHRkd also increased the rate of DNL and expression of GCK and KHK, but impaired insulin-mediated suppression of hepatic glucose production, without altering plasma NEFA levels. These effects were normalized with AAV-mediated hepatocyte expression of IGF1 or STAT5bCA. Comparison of the impact of AAV-mediated hepatocyte IGF1 versus STAT5bCA in aHepGHRkd mice across multiple nutritional states, indicated the restorative actions of IGF1 are indirect, by improving systemic insulin sensitivity, independent of changes in the liver transcriptome. In contrast, the actions of STAT5b are due to the combined effects of raising IGF1 and direct alterations in the hepatocyte gene program that may involve suppression of BCL6 and FOXO1 activity. However, the direct and IGF1-dependent actions of STAT5b cannot fully account for enhanced GCK activity and lipogenic gene expression observed after aHepGHRkd, suggesting other GHR-mediated signals are involved. CONCLUSION: These studies demonstrate hepatocyte GHR-signaling controls hepatic glycolysis, DNL, steatosis and hepatic insulin sensitivity indirectly (via IGF1) and directly (via STAT5b). The relative contribution of these indirect and direct actions of GH on hepatocytes is modified by insulin and nutrient availability. These results improve our understanding of the physiologic actions of GH on regulating adult metabolism to protect against NAFLD progression.

Intraoperative oxygen tension and redox homeostasis in Pseudomyxoma peritonei: A short case series.

Introduction: Pseudomyxoma peritonei (PMP) is a rare malignant disease characterized by a massive multifocal accumulation of mucin within the peritoneal cavity. The current treatment option is based on complete cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. However, the recurrence is frequent with subsequent progression and death. To date, most of the studies published in PMP are related to histological and genomic analyses. Thus, the need for further studies unveiling the underlying PMP molecular mechanisms is urgent. In this regard, hypoxia and oxidative stress have been extensively related to tumoral pathologies, although their contribution to PMP has not been elucidated. Methods: In this manuscript, we have evaluated, for the first time, the intratumoral real-time oxygen microtension (pO2mt) in the tumor (soft and hard mucin) and surrounding healthy tissue from five PMP patients during surgery. In addition, we measured hypoxia (Hypoxia Inducible Factor-1a; HIF-1α) and oxidative stress (catalase; CAT) markers in soft and hard mucin from the same five PMP patient samples and in five control samples. Results: The results showed low intratumoral oxygen levels, which were associated with increased HIF-1α protein levels, suggesting the presence of a hypoxic environment in these tumors. We also found a significant reduction in CAT activity levels in soft and hard mucin compared with healthy tissue samples. Discussion: In conclusion, our study provides the first evidence of low intratumoral oxygen levels in PMP patients associated with hypoxia and oxidative stress markers. However, further investigation is required to understand the potential role of oxidative stress in PMP in order to find new therapeutic strategies.

Integrative Clinical, Radiological, and Molecular Analysis for Predicting Remission and Recurrence of Cushing Disease.

CONTEXT: Adrenocorticotropin (ACTH)-secreting pituitary tumors (ACTHomas) are associated with severe comorbidities and increased mortality. Current treatments mainly focus on remission and prevention of persistent disease and recurrence. However, there are still no useful biomarkers to accurately predict the clinical outcome after surgery, long-term remission, or disease relapse. OBJECTIVES: This work aimed to identify clinical, biochemical, and molecular markers for predicting long-term clinical outcome and remission in ACTHomas. METHODS: A retrospective multicenter study was performed with 60 ACTHomas patients diagnosed between 2004 and 2018 with at least 2 years' follow-up. Clinical/biochemical variables were evaluated yearly. Molecular expression profile of the somatostatin/ghrelin/dopamine regulatory systems components and of key pituitary factors and proliferation markers were evaluated in tumor samples after the first surgery. RESULTS: Clinical variables including tumor size, time until diagnosis/first surgery, serum prolactin, and postsurgery cortisol levels were associated with tumor remission and relapsed disease. The molecular markers analyzed were distinctly expressed in ACTHomas, with some components (ie, SSTR1, CRHR1, and MKI67) showing instructive associations with recurrence and/or remission. Notably, an integrative model including selected clinical variables (tumor size/postsurgery serum cortisol), and molecular markers (SSTR1/CRHR1) can accurately predict the clinical evolution and remission of patients with ACTHomas, generating a receiver operating characteristic curve with an area under the curve of 1 (P < .001). CONCLUSION: This study demonstrates that the combination of a set of clinical and molecular biomarkers in ACTHomas is able to accurately predict the clinical evolution and remission of patients. Consequently, the postsurgery molecular profile represents a valuable tool for clinical evaluation and follow-up of patients with ACTHomas.

Epigenetic and post-transcriptional regulation of somatostatin receptor subtype 5 (SST5 ) in pituitary and pancreatic neuroendocrine tumors.

Somatostatin receptor subtype 5 (SST5 ) is an emerging biomarker and actionable target in pituitary (PitNETs) and pancreatic (PanNETs) neuroendocrine tumors. Transcriptional and epigenetic regulation of SSTR5 gene expression and mRNA biogenesis is poorly understood. Recently, an overlapping natural antisense transcript, SSTR5-AS1, potentially regulating SSTR5 expression, was identified. We aimed to elucidate whether epigenetic processes contribute to the regulation of SSTR5 expression in PitNETs (somatotropinomas) and PanNETs. We analyzed the SSTR5/SSTR5-AS1 human locus in silico to identify CpG islands. SSTR5 and SSTR5-AS1 expression was assessed by quantitative real-time PCR (qPCR) in 27 somatotropinomas, 11 normal pituitaries (NPs), and 15 PanNETs/paired adjacent (control) samples. We evaluated methylation grade in four CpG islands in the SSTR5/SSTR5-AS1 genes. Results revealed that SSTR5 and SSTR5-AS1 were directly correlated in NP, somatotropinoma, and PanNET samples. Interestingly, selected CpG islands were differentially methylated in somatotropinomas compared with NPs. In PanNETs cell lines, SSTR5-AS1 silencing downregulated SSTR5 expression, altered aggressiveness features, and influenced pasireotide response. These results provide evidence that SSTR5 expression in PitNETs and PanNETs can be epigenetically regulated by the SSTR5-AS1 antisense transcript and, indirectly, by DNA methylation, which may thereby impact tumor behavior and treatment response.

Towards Understanding the Direct and Indirect Actions of Growth Hormone in Controlling Hepatocyte Carbohydrate and Lipid Metabolism.

Growth hormone (GH) is critical for achieving normal structural growth. In addition, GH plays an important role in regulating metabolic function. GH acts through its GH receptor (GHR) to modulate the production and function of insulin-like growth factor 1 (IGF1) and insulin. GH, IGF1, and insulin act on multiple tissues to coordinate metabolic control in a context-specific manner. This review will specifically focus on our current understanding of the direct and indirect actions of GH to control liver (hepatocyte) carbohydrate and lipid metabolism in the context of normal fasting (sleep) and feeding (wake) cycles and in response to prolonged nutrient deprivation and excess. Caveats and challenges related to the model systems used and areas that require further investigation towards a clearer understanding of the role GH plays in metabolic health and disease are discussed.

Growth hormone-releasing hormone-secreting pulmonary neuroendocrine tumor associated with pituitary hyperplasia and somatotropinoma.

Acromegaly caused by ectopic growth hormone-releasing hormone (GHRH)-secreting tumor is exceedingly rare. We report a case of acromegaly secondary to GHRH secretion by an incidentally diagnosed pulmonary neuroendocrine tumor (NET) and review 47 similar cases in literature. A 22-year-old male patient presented with symptoms of pituitary apoplexy. Magnetic resonance imaging (MRI) showed apoplexy of a pituitary adenoma. Routinely prior to surgery, a chest radiography was performed which revealed a mass in the left lung. During investigation, the patient was diagnosed with metastatic GHRH-secreting pulmonary NET. In retrospect, it was noted that the patient had pituitary hyperplasia 20 months prior to the MRI which showed the presence of a pituitary adenoma. The histological findings confirmed somatotroph hyperplasia adjacent to somatotropinoma. This case suggests that GHRH secretion can be associated with pituitary hyperplasia, which may be followed by pituitary adenoma formation.

GH directly inhibits steatosis and liver injury in a sex-dependent and IGF1-independent manner.

A reduction in hepatocyte growth hormone (GH)-signaling promotes non-alcoholic fatty liver disease (NAFLD). However, debate remains as to the relative contribution of the direct effects of GH on hepatocyte function vs indirect effects, via alterations in insulin-like growth factor 1 (IGF1). To isolate the role of hepatocyte GH receptor (GHR) signaling, independent of changes in IGF1, mice with adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd) were treated with a vector expressing rat IGF1 targeted specifically to hepatocytes. Compared to GHR-intact mice, aHepGHRkd reduced circulating IGF1 and elevated GH. In male aHepGHRkd, the shift in IGF1/GH did not alter plasma glucose or non-esterified fatty acids (NEFA), but was associated with increased insulin, enhanced systemic lipid oxidation and reduced white adipose tissue (WAT) mass. Livers of male aHepGHRkd exhibited steatosis associated with increased de novo lipogenesis, hepatocyte ballooning and inflammation. In female aHepGHRkd, hepatic GHR protein levels were not detectable, but moderate levels of IGF1 were maintained, with minimal alterations in systemic metabolism and no evidence of steatosis. Reconstitution of hepatocyte IGF1 in male aHepGHRkd lowered GH and normalized insulin, whole body lipid utilization and WAT mass. However, IGF1 reconstitution did not reduce steatosis or eliminate liver injury. RNAseq analysis showed IGF1 reconstitution did not impact aHepGHRkd-induced changes in liver gene expression, despite changes in systemic metabolism. These results demonstrate the impact of aHepGHRkd is sexually dimorphic and the steatosis and liver injury observed in male aHepGHRkd mice is autonomous of IGF1, suggesting GH acts directly on the adult hepatocyte to control NAFLD progression.

Growth hormone-releasing hormone-secreting pulmonary neuroendocrine tumor associated with pituitary hyperplasia and somatotropinoma

SUMMARY Acromegaly caused by ectopic growth hormone-releasing hormone (GHRH)-secreting tumor is exceedingly rare. We report a case of acromegaly secondary to GHRH secretion by an incidentally diagnosed pulmonary neuroendocrine tumor (NET) and review 47 similar cases in literature. A 22-year-old male patient presented with symptoms of pituitary apoplexy. Magnetic resonance imaging (MRI) showed apoplexy of a pituitary adenoma. Routinely prior to surgery, a chest radiography was performed which revealed a mass in the left lung. During investigation, the patient was diagnosed with metastatic GHRH-secreting pulmonary NET. In retrospect, it was noted that the patient had pituitary hyperplasia 20 months prior to the MRI which showed the presence of a pituitary adenoma. The histological findings confirmed somatotroph hyperplasia adjacent to somatotropinoma. This case suggests that GHRH secretion can be associated with pituitary hyperplasia, which may be followed by pituitary adenoma formation.

Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3.

Glioblastomas remain the deadliest brain tumour, with a dismal ∼12-16-month survival from diagnosis. Therefore, identification of new diagnostic, prognostic and therapeutic tools to tackle glioblastomas is urgently needed. Emerging evidence indicates that the cellular machinery controlling the splicing process (spliceosome) is altered in tumours, leading to oncogenic splicing events associated with tumour progression and aggressiveness. Here, we identify for the first time a profound dysregulation in the expression of relevant spliceosome components and splicing factors (at mRNA and protein levels) in well characterized cohorts of human high-grade astrocytomas, mostly glioblastomas, compared to healthy brain control samples, being SRSF3, RBM22, PTBP1 and RBM3 able to perfectly discriminate between tumours and control samples, and between proneural-like or mesenchymal-like tumours versus control samples from different mouse models with gliomas. Results were confirmed in four additional and independent human cohorts. Silencing of SRSF3, RBM22, PTBP1 and RBM3 decreased aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere-formation, etc.) and induced apoptosis, especially SRSF3. Remarkably, SRSF3 was correlated with patient survival and relevant tumour markers, and its silencing in vivo drastically decreased tumour development and progression, likely through a molecular/cellular mechanism involving PDGFRB and associated oncogenic signalling pathways (PI3K-AKT/ERK), which may also involve the distinct alteration of alternative splicing events of specific transcription factors controlling PDGFRB (i.e. TP73). Altogether, our results demonstrate a drastic splicing machinery-associated molecular dysregulation in glioblastomas, which could potentially be considered as a source of novel diagnostic and prognostic biomarkers as well as therapeutic targets for glioblastomas. Remarkably, SRSF3 is directly associated with glioblastoma development, progression, aggressiveness and patient survival and represents a novel potential therapeutic target to tackle this devastating pathology.

Statins Directly Regulate Pituitary Cell Function and Exert Antitumor Effects in Pituitary Tumors.

INTRODUCTION: Pituitary neuroendocrine tumors (PitNETs), the most abundant of all intracranial tumors, entail severe comorbidities. First-line therapy is transsphenoidal surgery, but subsequent pharmacological therapy is often required. Unfortunately, many patients are/become unresponsive to available drugs (somatostatin analogues [SSAs]/dopamine agonists), underscoring the need for new therapies. Statins are well-known drugs commonly prescribed to treat hyperlipidemia/cardiovascular diseases, but can convey additional beneficial effects, including antitumor actions. The direct effects of statins on normal human pituitary or PitNETs are poorly known. Thus, we aimed to explore the direct effects of statins, especially simvastatin, on key functional parameters in normal and tumoral pituitary cells, and to evaluate the combined effects of simvastatin with metformin (MF) or SSAs. METHODS: Effects of statins in cell proliferation/viability, hormone secretion, and signaling pathways were evaluated in normal pituitary cells from a primate model (Papio anubis), tumor cells from corticotropinomas, somatotropinomas, nonfunctioning pituitary tumors, and PitNET cell-lines (AtT20/GH3-cells). RESULTS: All statins decreased AtT20-cell proliferation, simvastatin showing stronger effects. Indeed, simvastatin reduced cell viability and/or hormone secretion in all PitNETs subtypes and cell-lines, and ACTH/GH/PRL/FSH/LH secretion (but not expression), in primate cell cultures, by modulating MAPK/PI3K/mTOR pathways and expression of key receptors (GH-releasing hormone-receptor/ghrelin-R/Kiss1-R) regulating pituitary function. Addition of MF or SSAs did not enhance simvastatin antitumor effects. CONCLUSION: Our data reveal direct antitumor effects of simvastatin on PitNET-cells, paving the way to explore these compounds as a possible tool to treat PitNETs.

A New Generation Somatostatin-Dopamine Analogue Exerts Potent Antitumoral Actions on Pituitary Neuroendocrine Tumor Cells.

BACKGROUND: Pituitary neuroendocrine tumors (PitNETs) represent approximately 15% of all intracranial tumors and usually are associated with severe comorbidities. Unfortunately, a relevant number of patients do not respond to currently available pharmacological treatments, that is, somatostatin analogs (SSAs) or dopamine-agonists (DA). Thus, novel, chimeric somatostatin/dopamine compounds (dopastatins) that could improve medical treatment of PitNETs have been designed. OBJECTIVE: This study aims to determine the direct therapeutic effects of a new-generation dopastatin, BIM-065, on primary cell cultures from different PitNETs subtypes. METHODS: Thirty-one PitNET-derived cell cultures (9 corticotropinomas, 9 somatotropinomas, 11 nonfunctioning pituitary adenomas [NFPAs], and 2 prolactinomas), were treated with BIM-065, and key functional endpoints were assessed (cell viability, apoptosis, hormone secretion, expression levels of key genes, free cytosolic [Ca2+]i dynamics, etc.). AtT-20 cell line was used to evaluate signaling pathways in response to BIM-065. RESULTS: This chimeric compound decreased cell viability in all corticotropinomas and somatotropinomas tested, but not in NFPAs. BIM-065 reduced ACTH, GH, chromogranin-A and PRL secretion, and increased apoptosis in corticotropinomas, somatotropinomas, and NFPAs. These effects were possibly mediated through modulation of pivotal signaling cascades like [Ca2+]i kinetic and Akt- or ERK1/2-phosphorylation. CONCLUSIONS: Our results unveil a robust antitumoral effect in vitro of the novel chimeric compound BIM-065 on the main PitNET subtypes, inform on the mechanisms involved, and suggest that BIM-065 could be an efficacious therapeutic option to be considered in the treatment of PitNETs.

A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors.

PURPOSE: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. EXPERIMENTAL DESIGN: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. RESULTS: We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. CONCLUSIONS: This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Splicing Machinery is Dysregulated in Pituitary Neuroendocrine Tumors and is Associated with Aggressiveness Features.

Pituitary neuroendocrine tumors (PitNETs) constitute approximately 15% of all brain tumors, and most have a sporadic origin. Recent studies suggest that altered alternative splicing and, consequently, appearance of aberrant splicing variants, is a common feature of most tumor pathologies. Moreover, spliceosome is considered an attractive therapeutic target in tumor pathologies, and the inhibition of SF3B1 (e.g., using pladienolide-B) has been shown to exert antitumor effects. Therefore, we aimed to analyze the expression levels of selected splicing-machinery components in 261 PitNETs (somatotropinomas/non-functioning PitNETS/corticotropinomas/prolactinomas) and evaluated the direct effects of pladienolide-B in cell proliferation/viability/hormone secretion in human PitNETs cell cultures and pituitary cell lines (AtT-20/GH3). Results revealed a severe dysregulation of splicing-machinery components in all the PitNET subtypes compared to normal pituitaries and a unique fingerprint of splicing-machinery components that accurately discriminate between normal and tumor tissue in each PitNET subtype. Moreover, expression of specific components was associated with key clinical parameters. Interestingly, certain components were commonly dysregulated throughout all PitNET subtypes. Finally, pladienolide-B reduced cell proliferation/viability/hormone secretion in PitNET cell cultures and cell lines. Altogether, our data demonstrate a drastic dysregulation of the splicing-machinery in PitNETs that might be associated to their tumorigenesis, paving the way to explore the use of specific splicing-machinery components as novel diagnostic/prognostic and therapeutic targets in PitNETs.

E-cadherin expression is associated with somatostatin analogue response in acromegaly.

Acromegaly is a rare disease resulting from hypersecretion of growth hormone (GH) and insulin-like growth factor 1 (IGF1) typically caused by pituitary adenomas, which is associated with increased mortality and morbidity. Somatostatin analogues (SSAs) represent the primary medical therapy for acromegaly and are currently used as first-line treatment or as second-line therapy after unsuccessful pituitary surgery. However, a considerable proportion of patients do not adequately respond to SSAs treatment, and therefore, there is an urgent need to identify biomarkers predictors of response to SSAs. The aim of this study was to examine E-cadherin expression by immunohistochemistry in fifty-five GH-producing pituitary tumours and determine the potential association with response to SSAs as well as other clinical and histopathological features. Acromegaly patients with tumours expressing low E-cadherin levels exhibit a worse response to SSAs. E-cadherin levels are associated with GH-producing tumour histological subtypes. Our results indicate that the immunohistochemical detection of E-cadherin might be useful in categorizing acromegaly patients based on the response to SSAs.

Biguanides Exert Antitumoral Actions in Pituitary Tumor Cells Through AMPK-Dependent and -Independent Mechanisms.

CONTEXT: Pituitary neuroendocrine tumors (PitNETs) are a commonly underestimated pathology in terms of incidence and associated morbimortality. Currently, an appreciable subset of patients are resistant or poorly responsive to the main current medical treatments [i.e., synthetic somatostatin analogs (SSAs) and dopamine agonists]. Thus, development and optimization of novel and available medical therapies is necessary. Biguanides (metformin, buformin, and phenformin) are antidiabetic drugs that exert antitumoral actions in several tumor types, but their pharmacological effects on PitNETs are poorly known. OBJECTIVE: We aimed to explore the direct effects of biguanides on key functions (cell viability, hormone release, apoptosis, and signaling pathways) in primary cell cultures from human PitNETs and cell lines. Additionally, we evaluated the effect of combined metformin with SSAs on cell viability and hormone secretion. DESIGN: A total of 13 corticotropinomas, 13 somatotropinomas, 13 nonfunctioning PitNETs, 3 prolactinomas, and 2 tumoral pituitary cell lines (AtT-20 and GH3) were used to evaluate the direct effects of biguanides on cell viability, hormone release, apoptosis, and signaling pathways. RESULTS: Biguanides reduced cell viability in all PitNETs and cell lines (with phenformin being the most effective biguanide) and increased apoptosis in somatotropinomas. Moreover, buformin and phenformin, but not metformin, reduced hormone secretion in a cell type-specific manner. Combination metformin/SSA therapy did not increase SSA monotherapy effectiveness. Effects of biguanides on PitNETs could involve the modulation of AMP-activated protein kinase-dependent ([Ca2+]i, PI3K/Akt) and independent (MAPK) mechanisms. CONCLUSION: Altogether, our data unveil clear antitumoral effects of biguanides on PitNET cells, opening avenues to explore their potential as drugs to treat these pathologies.

Clinical significance of filamin A in patients with acromegaly and its association with somatostatin and dopamine receptor profiles.

Filamin-A (FLNA) plays a crucial role in somatostatin receptor (sst) subtype-2 signaling in somatotropinomas. Our objective was to investigate the in vivo association between FLNA and sst2 expression, sst5 expression, dopamine receptor subtype-2 (D2) expression, somatostatin receptor ligand (SRL) responsiveness and tumor invasiveness in somatotropinomas. Quantitative real-time PCR was used to evaluate the absolute mRNA copy numbers of FLNA/sst2/sst5/D2 in 96 somatotropinomas. FLNA, sst2 and sst5 protein expression levels were also evaluated using immunohistochemistry. The Knosp-Steiner criteria were used to evaluate tumor invasiveness. Median FLNA, sst2, sst5 and D2 copy numbers were 4,244, 731, 156 and 3,989, respectively. Thirty-one of the 35 available tumors (89%) were immune positive for FLNA in the cytoplasm and membrane but not in the nucleus. FLNA and sst5 expression were positively correlated at the mRNA and protein levels (p < 0.001 and p = 0.033, respectively). FLNA was positively correlated with sst2 mRNA in patients who were responsive to SRL (p = 0.014, R = 0.659). No association was found between FLNA and tumor invasiveness. Our findings show that in somatotropinomas FLNA expression positively correlated with in vivo sst5 and D2 expression. Notably, FLNA was only correlated with sst2 in patients who were controlled with SRL. FLNA was not associated with tumor invasiveness.

Molecular determinants of the response to medical treatment of growth hormone secreting pituitary neuroendocrine tumors.

Acromegaly is a chronic systemic disease mainly caused by a growth hormone (GH)-secreting pituitary neuroendocrine tumor (PitNETs), which is associated with many health complications and increased mortality when not adequately treated. Transsphenoidal surgery is considered the treatment of choice in GH-secreting PitNETs, but patients in whom surgery cannot be considered or with persistent disease after surgery require medical therapy. Treatment with available synthetic somatostatin analogues (SSAs) is considered the mainstay in the medical management of acromegaly which exert their beneficial effects through the binding to a family of G-protein coupled receptors encoded by 5 genes (SSTR1-5). However, although it has been demonstrated that the SST1-5 receptors are physically present in tumor cells, SSAs are in many cases ineffective (i.e. approximately 10-30% of patients with GH-secreting PitNET are unresponsive to SSAs), suggesting that other cellular/molecular determinants could be essential for the response to the pharmacological treatment in patients with GH-secreting PitNETs. Therefore, the scrutiny of these determinants might be used for the identification of subgroups of patients in whom an appropriate pharmacological treatment can be successfully employed (responders vs. non-responders). In this review, we will describe some of the existing, classical and novel, genetic and molecular determinants involved in the response of patients with GH-secreting PitNETs to the available therapeutic treatments, as well as new molecular/therapeutic approaches that could be potentially useful for the treatment of GH-secreting PitNETs.