The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland.

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

Visceral adipose tissue adiponectin predicts excess weight loss after bariatric surgery in females with severe obesity.

OBJECTIVE: Bariatric surgery not always results in satisfactory excess weight loss (EWL) in severe obesity. Given the economic and clinical costs of bariatric surgery failure, defining predictors of successful EWL represents a relevant clinical issue for the health system to select patients benefiting from operation. METHODS: By ELISA and Western blot analyses, we assessed the predicting value of pre-operative adiponectin (APN) locally produced in abdominal visceral (VAT) and subcutaneous (SAT) adipose tissue versus plasma levels as a novel sex-linked biomarker of EWL at different time points of follow up (6-24 months) after bariatric surgery in 43 patients (56% females) affected by severe obesity undergoing a small pilot observational study. RESULTS: VAT-APN was lower in females and represented the only marker significantly correlated with EWL. In females, VAT-APN in the distribution upper quartile but not baseline BMI retained a statistically significant correlation with EWL at any time points (6-24 months) at multivariate analysis. The best VAT-APN cut-off value to predict 95% EWL at 12 months from surgery (98% accuracy, 100% sensitivity, 94% specificity, p = 0.010) was 5.1 µg/mg. CONCLUSIONS: In this very preliminary study, APN in VAT rather than its circulating or subcutaneous levels predicts EWL after bariatric surgery as an independent factor in the female sex only, thus contributing to identify those patients who could much benefit from surgery.

STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

Cachexia is a devastating pathology that worsens the quality of life and antineoplastic treatment outcomes of oncologic patients. Herein, we report that the secretome from murine colon carcinoma CT26 induces cachectic features in both murine and human adipocytes that are associated with metabolic alterations such as enhanced lactate production and decreased oxygen consumption. The use of oxamate, which inhibits lactate dehydrogenase activity, hinders the effects induced by CT26 secretome. Interestingly, the CT26 secretome elicits an increased level of lactate dehydrogenase and decreased expression of adiponectin. These modifications are driven by the STAT3 signalling cascade since the inhibition of STAT3 with WP1066 impedes the formation of the cachectic condition and the alteration of lactate dehydrogenase and adiponectin levels. Collectively, these findings show that STAT3 is responsible for the altered lactate dehydrogenase and adiponectin levels that, in turn, could participate in the worsening of this pathology and highlight a step forward in the comprehension of the mechanisms underlying the onset of the cachectic condition in adipocytes.

Intratumour microbiota modulates adrenocortical cancer responsiveness to mitotane.

The infiltrating microbiota represents a novel cellular component of the solid tumour microenvironment that can influence tumour progression and response to therapy. Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy for which mitotane (MTT) treatment represents the first-line therapy, though its efficacy is limited to a therapeutic window level (14-20 mg/L). Novel markers able to predict those patients who would benefit from MTT therapy are urgently needed to improve patient's management. The aim of our study was to evaluate the presence of intratumoural bacterial microbiota DNA in 26 human ACC tissues vs 9 healthy adrenals; moreover, the association between the relative bacterial composition profile, the tumour mass characteristics and MTT ability to reach high circulating levels in the early phase of treatment, were explored. We found the presence of bacterial DNA in all adrenal samples from both tumours and healthy cortex specimens, documenting significant differences in the microbial composition between malignancy and normal adrenals: in detail, the ACC tissues were characterised by a higher abundance of the Proteobacteria phylum (especially the Pseudomonas and Serratia genera). In addition, the Proteobacteria's low abundance was negatively associated with tumour size, Ki67 and cortisol secretion. MTT levels reached higher levels at 9 months in ACC patients with high abundance of Proteobacteria, Pseudomonas and Serratia and with low abundance of Bacteroidota, Firmicutes and Streptococcus. These findings are the first indication that human ACCs are characterised by infiltrating bacteria and their specific abundance profile seems to influence the increase in circulating MTT levels at 9 months.

A systematic benchmark of machine learning methods for protein-RNA interaction prediction.

RNA-binding proteins (RBPs) are central actors of RNA post-transcriptional regulation. Experiments to profile-binding sites of RBPs in vivo are limited to transcripts expressed in the experimental cell type, creating the need for computational methods to infer missing binding information. While numerous machine-learning based methods have been developed for this task, their use of heterogeneous training and evaluation datasets across different sets of RBPs and CLIP-seq protocols makes a direct comparison of their performance difficult. Here, we compile a set of 37 machine learning (primarily deep learning) methods for in vivo RBP-RNA interaction prediction and systematically benchmark a subset of 11 representative methods across hundreds of CLIP-seq datasets and RBPs. Using homogenized sample pre-processing and two negative-class sample generation strategies, we evaluate methods in terms of predictive performance and assess the impact of neural network architectures and input modalities on model performance. We believe that this study will not only enable researchers to choose the optimal prediction method for their tasks at hand, but also aid method developers in developing novel, high-performing methods by introducing a standardized framework for their evaluation.

FSCN1 as a new druggable target in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high risk of relapse and metastatic spread. The actin-bundling protein fascin (FSCN1) is overexpressed in aggressive ACC and represents a reliable prognostic indicator. FSCN1 has been shown to synergize with VAV2, a guanine nucleotide exchange factor for the Rho/Rac GTPase family, to enhance the invasion properties of ACC cancer cells. Based on those results, we investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade on the invasive properties of ACC cells, both in vitro and in an in vivo metastatic ACC zebrafish model. Here, we showed that FSCN1 is a transcriptional target for ß-catenin in H295R ACC cells and that its inactivation resulted in defects in cell attachment and proliferation. FSCN1 knock-out modulated the expression of genes involved in cytoskeleton dynamics and cell adhesion. When Steroidogenic Factor-1 (SF-1) dosage was upregulated in H295R cells, activating their invasive capacities, FSCN1 knock-out reduced the number of filopodia, lamellipodia/ruffles and focal adhesions, while decreasing cell invasion in Matrigel. Similar effects were produced by the FSCN1 inhibitor G2-044, which also diminished the invasion of other ACC cell lines expressing lower levels of FSCN1 than H295R. In the zebrafish model, metastases formation was significantly reduced in FSCN1 knock-out cells and G2-044 significantly reduced the number of metastases formed by ACC cells. Our results indicate that FSCN1 is a new druggable target for ACC and provide the rationale for future clinical trials with FSCN1 inhibitors in patients with ACC.

Prognostic Value of Microscopic Tumor Necrosis in Adrenal Cortical Carcinoma.

Adrenal cortical carcinoma (ACC) is an uncommon neoplasm with variable prognosis. Several histologic criteria have been identified as predictors of malignancy in adrenal cortical tumors. The Weiss score is the system most widely employed for diagnostic purposes, but also possesses prognostic value. We aim to determine the relative impact of each Weiss parameter on ACC patient survival. A multicenter retrospective analysis was conducted on a series of 79 conventional ACCs surgically treated at the Florence and Utrecht centers of the European Network for the Study of Adrenal Tumors (ENSAT). Weiss classification was recapitulated using principal component analysis (PCA). The Kaplan-Meier and Cox multivariate regression analyses were applied in order to estimate the prognostic power of Weiss versus other clinical parameters. PCA reduced the nine Weiss parameters to the best fitting 4-component model, each parameter clustering with a single component. Necrosis and venous invasion clustered together with the highest scores, thus establishing the most relevant component (Component 1) to explain Weiss distribution variability. Only Component 1 significantly predicted overall survival (OS, log-rank = 0.008) and disease-free survival (DFS, log-rank < 0.001). When considering the prognostic power of Weiss parameters, necrosis alone could independently assess OS (log-rank = 0.004) and DFS (log-rank < 0.001) at both the Kaplan-Meier and multivariate Cox regression analyses [hazard ratio (HR) = 7.8, 95% confidence interval [CI] = 1.0-63.5, p = 0.05, and HR = 12.2, 95% CI = 1.6-95.0, p = 0.017, respectively]. The presence of necrosis significantly shortened time to survival (TtS) and time to recurrence (TtR), 57.5 [31.5-103.5] vs 34 [12-78] months (p = 0.05) and 57.5 [31.5-103.5] vs 7 [1.0-31.5] months (p < 0.001), respectively. Our study suggests that, of the Weiss parameters, necrosis is the most powerful adverse factor and the best predictor of OS and DFS in ACC patients.

A computational map of the human-SARS-CoV-2 protein-RNA interactome predicted at single-nucleotide resolution.

RNA-binding proteins (RBPs) are critical host factors for viral infection, however, large scale experimental investigation of the binding landscape of human RBPs to viral RNAs is costly and further complicated due to sequence variation between viral strains. To fill this gap, we investigated the role of RBPs in the context of SARS-CoV-2 by constructing the first in silico map of human RBP-viral RNA interactions at nucleotide-resolution using two deep learning methods (pysster and DeepRiPe) trained on data from CLIP-seq experiments on more than 100 human RBPs. We evaluated conservation of RBP binding between six other human pathogenic coronaviruses and identified sites of conserved and differential binding in the UTRs of SARS-CoV-1, SARS-CoV-2 and MERS. We scored the impact of mutations from 11 variants of concern on protein-RNA interaction, identifying a set of gain- and loss-of-binding events, as well as predicted the regulatory impact of putative future mutations. Lastly, we linked RBPs to functional, OMICs and COVID-19 patient data from other studies, and identified MBNL1, FTO and FXR2 RBPs as potential clinical biomarkers. Our results contribute towards a deeper understanding of how viruses hijack host cellular pathways and open new avenues for therapeutic intervention.

Hypogonadism and sexual function in men affected by adrenocortical carcinoma under mitotane therapy.

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive tumor. ACC male patients under adjuvant mitotane therapy (AMT) frequently develop hypogonadism, however sexual function has never been assessed in this setting. The aim of this retrospective study was to evaluate in AMT treated ACC patients the changes in Luteinizing hormone (LH), Sex Hormone Binding Globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART). Methods: LH, SHBG, TT and cFT were assessed in ten ACC patients at baseline (T0) and six (T1), twelve (T2), and eighteen (T3) months after AMT. At T3, ART was initiated in eight hypogonadal patients, and LH, SHBG, TT and cFT levels were evaluated after six months (T4). In six patients, sexual function was evaluated before (T3) and after (T4) ART using the International Index of Erectile Function-15 (IIEF-15) questionnaire. Results: Under AMT we observed higher SHBG and LH and lower cFT levels at T1-T3 compared to T0 (all p<0.05). At T3, hypergonadotropic hypogonadism and erectile dysfunction (ED) were detected in 80% and 83.3% of cases. At T4, we observed a significant cFT increase in men treated with T gel, and a significant improvement in IIEF-15 total and subdomains scores and ED prevalence (16.7%) in men under ART. Conclusion: AMT was associated with hypergonatropic hypogonadism and ED, while ART led to a significant improvement of cFT levels and sexual function in the hypogonadal ACC patients. Therefore, we suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal ACC patients with sexual dysfunction.

Novel Germline PHD2 Variant in a Metastatic Pheochromocytoma and Chronic Myeloid Leukemia, but in the Absence of Polycythemia.

Background: Pheochromocytoma (Pheo) and paraganglioma (PGL) are rare tumors, mostly resulting from pathogenic variants of predisposing genes, with a genetic contribution that now stands at around 70%. Germline variants account for approximately 40%, while the remaining 30% is attributable to somatic variants. Objective: This study aimed to describe a new PHD2 (EGLN1) variant in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) without polycythemia and to emphasize the need to adopt a comprehensive next-generation sequencing (NGS) panel. Methods: Genetic analysis was carried out by NGS. This analysis was initially performed using a panel of genes known for tumor predisposition (EGLN1, EPAS1, FH, KIF1Bß, MAX, NF1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, and VHL), followed initially by SNP-CGH array, to exclude the presence of the pathogenic Copy Number Variants (CNVs) and the loss of heterozygosity (LOH) and subsequently by whole exome sequencing (WES) comparative sequence analysis of the DNA extracted from tumor fragments and peripheral blood. Results: We found a novel germline PHD2 (EGLN1) gene variant, c.153G>A, p.W51*, in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) in the absence of polycythemia. Conclusions: According to the latest guidelines, it is mandatory to perform genetic analysis in all Pheo/PGL cases regardless of phenotype. In patients with metastatic disease and no evidence of polycythemia, we propose testing for PHD2 (EGLN1) gene variants. A possible correlation between PHD2 (EGLN1) pathogenic variants and CML clinical course should be considered.

The function of Wtap in N6-adenosine methylation of mRNAs controls T cell receptor signaling and survival of T cells.

T cell antigen-receptor (TCR) signaling controls the development, activation and survival of T cells by involving several layers and numerous mechanisms of gene regulation. N6-methyladenosine (m6A) is the most prevalent messenger RNA modification affecting splicing, translation and stability of transcripts. In the present study, we describe the Wtap protein as essential for m6A methyltransferase complex function and reveal its crucial role in TCR signaling in mouse T cells. Wtap and m6A methyltransferase functions were required for the differentiation of thymocytes, control of activation-induced death of peripheral T cells and prevention of colitis by enabling gut RORγt+ regulatory T cell function. Transcriptome and epitranscriptomic analyses reveal that m6A modification destabilizes Orai1 and Ripk1 mRNAs. Lack of post-transcriptional repression of the encoded proteins correlated with increased store-operated calcium entry activity and diminished survival of T cells with conditional genetic inactivation of Wtap. These findings uncover how m6A modification impacts on TCR signal transduction and determines activation and survival of T cells.

GLP1 Exerts Paracrine Activity in the Intestinal Lumen of Human Colon.

GLP1 produced in the upper part of the gut is released after food intake and acts by activating insulin secretion, but the role of GLP1 in the colon, where it is predominantly produced, remains unknown. Here we characterized the apical versus basolateral secretion of GLP1 and PYY and the paracrine mechanisms of action of these enterohormones in the human colon. We stimulated human colon tissue in different ex vivo models with meat peptone and we used immunofluorescence to study the presence of canonical and non-canonical receptors of GLP1. We found that PYY and GLP1 are secreted mainly at the gut lumen in unstimulated and stimulated conditions. We detected DPP4 activity and found that GLP1R and GCGR are widely expressed in the human colon epithelium. Unlike GLP1R, GCGR is not expressed in the lamina propria, but it is located in the crypts of Lieberkühn. We detected GLP1R expression in human colon cell culture models. We show that the apical secretion of PYY and GLP1 occurs in humans, and we provide evidence that GLP1 has a potential direct paracrine function through the expression of its receptors in the colon epithelium, opening new therapeutic perspectives in the use of enterohormones analogues in metabolic pathologies.

Stimulated Expression of CXCL12 in Adrenocortical Carcinoma by the PPARgamma Ligand Rosiglitazone Impairs Cancer Progression.

Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis when metastatic and scarce treatment options in the advanced stages. In solid tumors, the chemokine CXCL12/CXCR4 axis is involved in the metastatic process. We demonstrated that the human adrenocortex expressed CXCL12 and its cognate receptors CXCR4 and CXCR7, not only in physiological conditions, but also in ACC, where the receptors' expression was higher and the CXCL12 expression was lower than in the physiological conditions. In a small pilot cohort of 22 ACC patients, CXCL12 negatively correlated with tumor size, stage, Weiss score, necrosis, and mitotic activity. In a Kaplan-Meier analysis, the CXCL12 tumor expression significantly predicted disease-free, progression-free, and overall survival. In vitro treatment of the primary ACC H295R and of the metastatic MUC-1 cell line with the PPARγ-ligand rosiglitazone (RGZ) dose-dependently reduced proliferation, resulting in a significant increase in CXCL12 and a decrease in its receptors in the H295R cells only, with no effect on the MUC-1 levels. In ACC mouse xenografts, tumor growth was inhibited by the RGZ treatment before tumor development (prevention-setting) and once the tumor had grown (therapeutic-setting), similarly to mitotane (MTT). This inhibition was associated with a significant suppression of the tumor CXCR4/CXCR7 and the stimulation of human CXCL12 expression. Tumor growth correlated inversely with CXCL12 and positively with CXCR4 expression, suggesting that local CXCL12 may impair the primary tumor cell response to the ligand gradient that may contribute to driving the tumor progression. These findings indicate that CXCL12/CXCR4 may constitute a potential target for anti-cancer agents such as rosiglitazone in the treatment of ACC.

Circulating Fascin 1 as a Promising Prognostic Marker in Adrenocortical Cancer.

Fascin-1 (FSCN1) is an actin-bundling protein associated with an invasive and aggressive phenotype of several solid carcinomas, as it is involved in cell cytoskeleton rearrangement and filopodia formation. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy characterized by poor prognosis, particularly when metastatic at diagnosis. Radical resection is the only therapeutic option for ACC patients in addition to the adjuvant treatment with mitotane. Novel specific biomarkers suggestive of tumor progression to refine diagnosis and prognosis of patients with advanced ACC are urgently needed. ACC intratumoral FSCN1 has previously been suggested as a valid prognostic marker. In the present study, we identified FSCN1 in the bloodstream of a small cohort of ACC patients (n = 27), through a specific ELISA assay for human FSCN1. FSCN1 can be detected in the serum, and its circulating levels were evaluated in pre-surgery samples, which resulted to be significantly higher in ACC patients from stage I/II and stage III/IV compared with nontumoral healthy controls (HC, n = 4, FI: 5.5 ± 0.8, P<0.001, and 8.0 ± 0.5, P < 0.001 for stage I/II and stage III/IV group vs HC, respectively). In particular, FSCN1 levels were significantly higher in advanced stage versus stage I/II (22.8 ± 1.1 vs 15.8 ± 1.8 ng/ml, P < 0.005, respectively). Interestingly, circulating levels of pre-surgical FSCN1 can significantly predict tumor progression/recurrence (Log rank = 0.013), but not the overall survival (Log rank=0.317), in patients stratified in high/low PreS FSCN1. In conclusion, these findings-though very preliminary-suggest that circulating FSCN1 may represent a new minimally-invasive prognostic marker in advanced ACC, in particular when measured before surgery enables histological diagnosis.

Prognostic and Monitoring Value of Circulating Tumor Cells in Adrenocortical Carcinoma: A Preliminary Monocentric Study.

Adrenocortical carcinoma (ACC), a rare and aggressive neoplasia, presents poor prognosis when metastatic at diagnosis and limited therapies are available. Specific and sensitive markers for early diagnosis and a monitoring system of therapy and tumor evolution are urgently needed. The liquid biopsy represents a source of tumor material within a minimally invasive blood draw that allows the recovery of circulating tumor cells (CTCs). CTCs have been recently shown to be detectable in ACC. In the present paper, we evaluated the prognostic value of CTCs obtained by size-filtration in a small pilot cohort of 19 ACC patients. We found CTCs in 68% of pre-surgery and in 38% of post-surgery blood samples. In addition, CTC clusters (CTMs) and cancer associated macrophages (CAMLs) were detectable in some ACC patients. The median number of CTCs significantly decreased after the mass removal. Finally, stratifying patients in high and low pre-surgery CTC number groups, assuming the 75th percentile CTC value as cut-off, CTCs significantly predicted patients' overall survival (log rank = 0.005), also in a multivariate analysis adjusted for age and tumor stage. In conclusion, though preliminary and performed in a small cohort of patients, our study suggests that CTC number may represent a promising marker for prognosis and disease monitoring in ACC.

SDHx and Non-Chromaffin Tumors: A Mediastinal Germ Cell Tumor Occurring in a Young Man with Germline SDHB Mutation.

Background: Mutations in genes encoding one of the subunits of succinate dehydrogenase (SDH) are involved in pheochromocytoma (PHEO) and paraganglioma (PGL) development. Over the last few years, such mutations have also been associated with non-chromaffin tumors. However, immunohistochemistry (IHC) on the tumor tissue and a study on the loss of heterozygosity (LOH) aimed at demonstrating the pathogenic role of SDHx genes have only been employed in a few cases. Case report: We describe the case of a 19-year-old Caucasian man with a germline SDHB mutation, who presented with acne vulgaris resistant to medical treatment. His follow-up for chromaffin tumors was negative, while hormonal tests revealed suppressed gonadotropins with testosterone in the upper range of normality and elevated ß-human chorionic gonadotropin (ß-hCG). At the whole-body enhanced CT scan, a mediastinal lesion suggestive of a germ cell tumor (GCT) was detected. 18FDG-PET (fluorodeoxyglucose-positron emission tomography) imaging showed low glucose metabolism at the mediastinal site. Surgical removal of the mass was uneventful. Pathology confirmed the diagnosis of GCT consisting of cystic teratoma (95%) and seminoma (5%). IHC for SDHB showed normal protein expression, and genetic analysis of the tumor tissue revealed the absence of SDHB LOH. Normalization of the hormonal tests and acne attenuation were achieved after surgery. Conclusion: We report an incidental association of a germinal SDHB mutation and mediastinal GCT in a young Caucasian man. Our paper highlights the importance of IHC and genetic analysis in confirming the etiologic role of SDHx genes in nonchromaffin tumors, thus excluding incidental associations.

The Role of Metabolic Changes in Shaping the Fate of Cancer-Associated Adipose Stem Cells.

Adipose tissue in physiological and in metabolically altered conditions (obesity, diabetes, metabolic syndrome) strictly interacts with the developing tumors both systemically and locally. In addition to the cancer-associated fibroblasts, adipose cells have also recently been described among the pivotal actors of the tumor microenvironment responsible for sustaining tumor development and progression. In particular, emerging evidence suggests that not only the mature adipocytes but also the adipose stem cells (ASCs) are able to establish a strict crosstalk with the tumour cells, thus resulting in a reciprocal reprogramming of both the tumor and adipose components. This review will focus on the metabolic changes induced by this interaction as a driver of fate determination occurring in cancer-associated ASCs (CA-ASCs) to support the tumor metabolic requirements. We will showcase the major role played by the metabolic changes occurring in the adipose tumor microenvironment that regulates ASC fate and consequently cancer progression. Our new results will also highlight the CA-ASC response in vitro by using a coculture system of primary ASCs grown with cancer cells originating from two different types of adrenal cancers [adrenocortical carcinoma (ACC) and pheochromocytoma]. In conclusion, the different factors involved in this crosstalk process will be analyzed and their effects on the adipocyte differentiation potential and functions of CA-ASCs will be discussed.

Cancer-testis Antigen FATE1 Expression in Adrenocortical Tumors Is Associated with A Pervasive Autoimmune Response and Is A Marker of Malignancy in Adult, but Not Children, ACC.

The SF-1 transcription factor target gene FATE1 encodes a cancer-testis antigen that has an important role in regulating apoptosis and response to chemotherapy in adrenocortical carcinoma (ACC) cells. Autoantibodies directed against FATE1 were previously detected in patients with hepatocellular carcinoma. In this study, we investigated the prevalence of circulating anti-FATE1 antibodies in pediatric and adult patients with adrenocortical tumors using three different methods (immunofluorescence, ELISA and Western blot). Our results show that a pervasive anti-FATE1 immune response is present in those patients. Furthermore, FATE1 expression is a robust prognostic indicator in adult patients with ACC and is associated with increased steroidogenic and decreased immune response gene expression. These data can open perspectives for novel strategies in ACC immunotherapy.

The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model.

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.

Glucagon modulates proliferation and differentiation of human adipose precursors.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs), which are currently used for the treatment of type 2 diabetes, have recently been proposed as anti-obesity drugs, due to their relevant effects on weight loss. Furthermore, dual agonists for both GLP-1R and glucagon receptor (GCGR) are under investigation for their promising action on adiposity, although underlying mechanisms still need to be clarified. We have recently demonstrated that GLP-1 and liraglutide interfere with the proliferation and differentiation of human adipose precursors, supporting the hypothesis of a peripheral action of GLP-1RA on weight. Here, we investigated glucagon activity in an in vitro model of primary human adipose-derived stem cells (ASCs). Glucagon significantly inhibited ASC proliferation in a dose- and time-dependent manner, as evaluated by cell count and thymidine incorporation. When added during in vitro-induced adipogenesis, glucagon significantly reduced adipocyte differentiation, as demonstrated by the evaluation of intracellular fat content and quantitative expression of early and mature adipocyte markers (PPARγ and FABP4, HSL). Notably, the inhibitory effect of glucagon on cell proliferation and adipogenesis was reversed by specific GLP-1R (exendin-9) and GCGR (des-His1-Glu9-glucagon(1-29)) antagonists. The presence of both receptors was demonstrated by Western blot, immunofluorescence and cytofluorimetric analysis of ASCs. In conclusion, we demonstrated a direct inhibitory action of glucagon on the proliferation and differentiation of human adipose precursors, which seems to involve both GLP-1R and GCGR. These findings suggest that the adipose stem compartment is a novel target of glucagon, possibly contributing to the weight loss obtained in vivo with dual GLP-1R/glucagon agonists.