Impact of Morphology in the Genotype and Phenotype Correlation of Bilateral Macronodular Adrenocortical Disease (BMAD): A Series of Clinicopathologically Well-Characterized 35 Cases.

Bilateral macronodular adrenocortical disease (BMAD) is characterized by the development of adrenal macronodules resulting in a pituitary-ACTH independent Cushing's syndrome. Although there are important similarities observed between the rare microscopic descriptions of this disease, the small series published are not representative of the molecular and genetic heterogenicity recently described in BMAD. We analyzed the pathological features in a series of BMAD and determined if there is correlation between these criteria and the characteristics of the patients. Two pathologists reviewed the slides of 35 patients who underwent surgery for suspicion of BMAD in our center between 1998 and 2021. An unsupervised multiple factor analysis based on microscopic characteristics divided the cases into 4 subtypes according to the architecture of the macronodules (containing or not round fibrous septa) and the proportion of the different cell types: clear, eosinophilic compact, and oncocytic cells. The correlation study with genetic revealed subtype 1 and subtype 2 are associated with the presence of ARMC5 and KDM1A pathogenic variants, respectively. By immunohistochemistry, all cell types expressed CYP11B1 and HSD3B1. HSD3B2 staining was predominantly expressed by clear cells whereas CYP17A1 staining was predominant on compact eosinophilic cells. This partial expression of steroidogenic enzymes may explain the low efficiency of cortisol production in BMAD. In subtype 1, trabeculae of eosinophilic cylindrical cells expressed DAB2 but not CYP11B2. In subtype 2, KDM1A expression was weaker in nodule cells than in normal adrenal cells; alpha inhibin expression was strong in compact cells. This first microscopic description of a series of 35 BMAD reveals the existence of 4 histopathological subtypes, 2 of which are strongly correlated with the presence of known germline genetic alterations. This classification emphasizes that BMAD has heterogeneous pathological characteristics that correlate with some genetic alterations identified in patients.

Primary bilateral macronodular adrenal hyperplasia: definitely a genetic disease.

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is an adrenal cause of Cushing syndrome. Nowadays, a PBMAH diagnosis is more frequent than previously, as a result of progress in the diagnostic methods for adrenal incidentalomas, which are widely available. Although some rare syndromic forms of PBMAH are known to be of genetic origin, non-syndromic forms of PBMAH have only been recognized as a genetic disease in the past 10 years. Genomics studies have highlighted the molecular heterogeneity of PBMAH and identified molecular subgroups, allowing improved understanding of the clinical heterogeneity of this disease. Furthermore, the generation of these subgroups permitted the identification of new genes responsible for PBMAH. Constitutive inactivating variants in ARMC5 and KDM1A are responsible for the development of distinct forms of PBMAH. To date, pathogenic variants of ARMC5 are responsible for 20-25% of PBMAH, whereas germline KDM1A alterations have been identified in >90% of PBMAH causing food-dependent Cushing syndrome. The identification of pathogenic variants in ARMC5 and KDM1A demonstrated that PBMAH, despite mostly being diagnosed in adults aged 45-60 years, is a genetic disorder. This Review summarizes the important progress made in the past 10 years in understanding the genetics of PBMAH, which have led to a better understanding of the pathophysiology, opening new clinical perspectives.

Tumor suppressor gene ARMC5 controls adrenal redox state through NRF1 turnover.

ARMC5: is a tumor suppressor gene frequently mutated in primary bilateral macronodular adrenal hyperplasia (PBMAH), an adrenal cause of Cushing's syndrome. The function of ARMC5 is poorly understood, aside from the fact that it regulates cell viability and adrenal steroidogenesis by mechanisms still unknown. Tumor suppressor genes play an important role in modifying intracellular redox response, which in turn regulates diverse cell signaling pathways. In this study, we demonstrated that inactivation in adrenocortical cells increased the expression of actors scavenging reactive oxygen species, such as superoxide dismutases (SOD) and peroxiredoxins (PRDX) by increasing the transcriptional regulator NRF1. Moreover, ARMC5 is involved in the NRF1 ubiquitination and in its half-life. Finally, inactivation alters adrenocortical steroidogenesis through the activation of p38 pathway and decreases cell sensitivity to ferroptosis participation to increase cell viability. Altogether, this study uncovers a function of ARMC5 as a regulator of redox homeostasis in adrenocortical cells, controlling steroidogenesis and cell survival.

Identification of predictive criteria for pathogenic variants of primary bilateral macronodular adrenal hyperplasia (PBMAH) gene ARMC5 in 352 unselected patients.

Objective: Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. It can be caused by ARMC5 germline pathogenic variants. In this study, we aimed to identify predictive criteria for ARMC5 variants. Methods: We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5 alteration. Clinical, biological and imaging data were collected retrospectively. Results: 52 patients (14.8%) carried ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-h urinary free cortisol 2.32 vs 1.11-fold ULN, respectively, P < 0.001) and adrenal morphology (maximal adrenal diameter 104 vs 83 mm, respectively, P < 0.001) and were more often surgically or medically treated (67.9 vs 36.8%, respectively, P < 0.001). ARMC5-mutated patients showed a constant, bilateral adrenal involvement and at least a possible autonomous cortisol secretion (defined by a plasma cortisol after 1 mg dexamethasone suppression above 50 nmol/L), while these criteria were not systematic in WT patients (78.3%). The association of these two criteria holds a 100% sensitivity and a 100% negative predictive value for ARMC5 pathogenic variant. Conclusion: We report the largest series of index patients investigated for ARMC5 and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype in most cases. To minimize negative ARMC5 screening, genotyping should be limited to clear bilateral adrenal involvement and autonomous cortisol secretion, with an optimum sensitivity for routine clinical practice. These findings will also help to better define PBMAH diagnostic criteria.

Transcriptome in paraffin samples for the diagnosis and prognosis of adrenocortical carcinoma.

Design: Molecular classification is important for the diagnosis and prognosis of adrenocortical tumors (ACT). Transcriptome profiles separate adrenocortical adenomas 'C2' from carcinomas, and identify two groups of carcinomas 'C1A' and 'C1B', of poor and better prognosis respectively. However, many ACT cannot be profiled because of improper or absent freezing procedures, a mandatory requirement so far. The main aim was to determine transcriptome profiles on formalin-fixed paraffin-embedded (FFPE) samples, using the new 3'-end RNA-sequencing technology. A secondary aim was to demonstrate the ability of this technique to explore large FFPE archives, by focusing on the rare oncocytic ACT variants. Methods: We included 131 ACT: a training cohort from Cochin hospital and an independent validation cohort from Wuerzburg hospital. The 3' transcriptome was generated from FFPE samples using QuantSeq (Lexogen, Vienna, Austria) and NextSeq500 (Illumina, San Diego, CA, USA). Results: In the training cohort, unsupervised clustering identified three groups: 'C1A' aggressive carcinomas (n = 28, 29%), 'C1B' more indolent carcinomas (n = 28, 29%), and 'C2' adenomas (n = 39, 41%). The prognostic value of FFPE transcriptome was confirmed in the validation cohort (5-year OS: 26% in 'C1A' (n = 26) and 100% in 'C1B' (n = 10), P = 0.003). FFPE transcriptome was an independent prognostic factor in a multivariable model including tumor stage and Ki-67 (OS HR: 7.5, P = 0.01). Oncocytic ACT (n = 19) did not form any specific cluster. Oncocytic carcinomas (n = 6) and oncocytic ACT of uncertain malignant potential (n = 4) were all in 'C1B'. Conclusions: The 3' RNA-sequencing represents a convenient solution for determining ACT molecular class from FFPE samples. This technique should facilitate routine use and large retrospective studies.

KDM1A inactivation causes hereditary food-dependent Cushing syndrome.

PURPOSE: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. METHODS: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). RESULTS: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10-12 and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS. CONCLUSION: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.

Genomic classification of benign adrenocortical lesions.

Benign adrenal tumors cover a spectrum of lesions with distinct morphology and steroid secretion. Current classification is empirical. Beyond a few driver mutations, pathophysiology is not well understood. Here, a pangenomic characterization of benign adrenocortical tumors is proposed, aiming at unbiased classification and new pathophysiological insights. Benign adrenocortical tumors (n = 146) were analyzed by transcriptome, methylome, miRNome, chromosomal alterations and mutational status, using expression arrays, methylation arrays, miRNA sequencing, SNP arrays, and exome or targeted next-generation sequencing respectively. Pathological and hormonal data were collected for all tumors. Pangenomic analysis identifies four distinct molecular categories: (1) tumors responsible for overt Cushing, gathering distinct tumor types, sharing a common cAMP/PKA pathway activation by distinct mechanisms; (2) adenomas with mild autonomous cortisol excess and non-functioning adenomas, associated with beta-catenin mutations; (3) primary macronodular hyperplasia with ARMC5 mutations, showing an ovarian expression signature; (4) aldosterone-producing adrenocortical adenomas, apart from other benign tumors. Epigenetic alterations and steroidogenesis seem associated, including CpG island hypomethylation in tumors with no or mild cortisol secretion, miRNA patterns defining specific molecular groups, and direct regulation of steroidogenic enzyme expression by methylation. Chromosomal alterations and somatic mutations are subclonal, found in less than 2/3 of cells. New pathophysiological insights, including distinct molecular signatures supporting the difference between mild autonomous cortisol excess and overt Cushing, ARMC5 implication into the adreno-gonadal differentiation faith, and the subclonal nature of driver alterations in benign tumors, will orient future research. This first genomic classification provides a large amount of data as a starting point.

Cullin 3 targets the tumor suppressor gene ARMC5 for ubiquitination and degradation.

ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.

Link between steroidogenesis, the cell cycle, and PKA in adrenocortical tumor cells.

Adrenocortical tumors (ACTs) frequently cause steroid excess and present cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in cell-cycle regulation. We investigated, by cell synchronization in the different phases of the cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical cells (H295R and culture of primary pigmented nodular adrenocortical disease cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in cells in G1, and triggered cell-cycle progression in all groups. These findings provide evidence for a tight association between steroidogenesis and cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and cell-cycle progression in adrenocortical cells.

EZH2 cooperates with E2F1 to stimulate expression of genes involved in adrenocortical carcinoma aggressiveness.

BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.

Value of Molecular Classification for Prognostic Assessment of Adrenocortical Carcinoma.

IMPORTANCE: The risk stratification of adrenocortical carcinoma (ACC) based on tumor proliferation index and stage is limited. Adjuvant therapy after surgery is recommended for most patients. Pan-genomic studies have identified distinct molecular groups closely associated with outcome. OBJECTIVE: To compare the molecular classification for prognostic assessment of ACC with other known prognostic factors. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective biomarker analysis, ACC tumor samples from 368 patients who had undergone surgical tumor removal were collected from March 1, 2005, to September 30, 2015 (144 in the training cohort and 224 in the validation cohort) at 21 referral centers with a median follow-up of 35 months (interquartile range, 18-74 months). Data were analyzed from March 2016 to March 2018. EXPOSURES: Meta-analysis of pan-genomic studies (transcriptome, methylome, chromosome alteration, and mutational profiles) was performed on the training cohort. Targeted biomarker analysis, including targeted gene expression (BUB1B and PINK1), targeted methylation (PAX5, GSTP1, PYCARD, and PAX6), and targeted next-generation sequencing, was performed on the training and validation cohorts. MAIN OUTCOMES AND MEASURES: Disease-free survival. Cox proportional hazards regression and C indexes were used to assess the prognostic value of each model. RESULTS: Of the 368 patients (mean [SD] age, 49 [16] years), 144 were in the training cohort (100 [69.4%] female) and 224 were in the validation cohort (142 [63.4%] female). In the training cohort, pan-genomic measures classified ACC into 3 molecular groups (A1, A2, and A3-B), with 5-year survival of 9% for group A1, 45% for group A2, and 82% for group A3-B (log-rank P < .001). Molecular class was an independent prognostic factor of recurrence in stage I to III ACC after complete surgery (hazard ratio, 55.91; 95% CI, 8.55-365.40; P < .001). The combination of European Network for the Study of Adrenal Tumors (ENSAT) stage, tumor proliferation index, and molecular class provided the most discriminant prognostic model (C index, 0.88). In the validation cohort, the molecular classification, determined by targeted biomarker measures, was confirmed as an independent prognostic factor of recurrence (hazard ratio, 5.96 [95% CI, 1.81-19.58], P = .003 for the targeted classifier combining expression, methylation, and chromosome alterations; and 2.61 [95% CI, 1.31-5.19], P = .006 for the targeted classifier combining methylation, chromosome alterations, and mutational profile). The prognostic value of the molecular markers was limited for patients with stage IV ACC. CONCLUSIONS AND RELEVANCE: The findings suggest that in localized ACC, targeted classifiers may be used as independent markers of recurrence. The determination of molecular class may improve individual prognostic assessment and thus may spare unnecessary adjuvant treatment.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

MET overexpression and activation favors invasiveness in a model of anaplastic thyroid cancer.

In thyroid cancers, MET receptor overexpression has been associated with higher risk of metastatic progression. In this study, it was shown that the anaplastic thyroid cancer (ATC)-derived TTA1 cell line overexpressed MET. By using FISH and relative quantification by qPCR, it was demonstrated that this overexpression resulted from a MET amplification with more than 20 copies. As expected, MET overexpression led to its constitutive activation and upregulated signaling towards the MAPK, PI3K/AKT, STAT3 and NF-κB pathways. Since the usual feature of MET-amplified cell lines is the "MET addiction" for their cell proliferation, the effect of the highly selective ATP competitive MET inhibitor PHA665752 was analyzed. While PHA665752 strongly inhibited the MAPK pathway, it did not reduce cell proliferation in TTA1 cells (IC50 = 4100 nM). This resistance to PHA665752 of the TTA1 cell line was demonstrated to be related to EGFR-MET functional cross-talk and PI3K/AKT and NF-κB signaling. Nevertheless, PHA665752 suppressed the anchorage-independent growth capacity of the TTA1 cell line and reduced cell migration and invasion in a transwell assay. The role of activated MET in these neoplastic properties of the TTA1 cells was also proved with si-MET-RNA targeting. Thus, this work highlights the TTA1 cell line as the first model of MET amplification in an ATC cell line, which leads to MET constitutive activation and underlies its neoplastic properties. Besides being a useful model for MET inhibitors screening, the TTA1 cell line also supports the argument for searching for MET amplification in ATC, as it could have therapeutic implications.

Fascin-1 Is a Novel Prognostic Biomarker Associated With Tumor Invasiveness in Adrenocortical Carcinoma.

CONTEXT: Novel tumor markers are urgently needed to better stratify adrenocortical cancer (ACC) patients and improve therapies for this aggressive neoplasm. OBJECTIVE: To assess the diagnostic and prognostic value of the actin-bundling protein fascin-1 (FSCN1) in adrenocortical tumors. DESIGN, SETTING AND PARTICIPANTS: A local series of 37 malignant/37 benign adrenocortical tumors at Careggi University Hospital and two independent validation ACC cohorts (Cochin, TCGA) from the European Network for the Study of Adrenal Tumors were studied. MAIN OUTCOME MEASURES: FSCN1 expression was quantified by immunohistochemistry, Western blot and quantitative RT-PCR in ACC specimens; overall and disease-free survival associated with FSCN1 expression were assessed by Kaplan-Meier analysis and compared with that of Ki67 labeling index and tumor stage. RESULTS: Despite the low diagnostic power, in the Florence ACC series, FSCN1 immunohistochemical detection appeared as an independent prognostic factor, also refining results obtained with staging and Ki67 labeling index. The robust prognostic power of FSCN1 levels was further confirmed in two independent ACC cohorts. A positive correlation was found between FSCN1 and steroidogenic factor-1 (SF-1), with a substantially higher expression of both factors in ACCs at advanced stages and with at least one of the three Weiss score parameters associated with invasiveness. Moreover, we demonstrated FSCN1 role in promoting cell invasion in a human ACC cell line only in the case of increased SF-1 dosage. CONCLUSIONS: These findings show that FSCN1 is a novel independent prognostic marker in ACC and may serve as a potential therapeutic target to block tumor spread.

AXIN deficiency in human and mouse hepatocytes induces hepatocellular carcinoma in the absence of ß-catenin activation.

BACKGROUND & AIMS: The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/ß-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/ß-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/ß-catenin program. METHODS: We evaluated two independent human HCC datasets for the expression of a 23-ß-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling. RESULTS: Based on gene expression, we defined three levels of ß-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of ß-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures. CONCLUSIONS: AXIN1-mutated HCCs occur independently of the Wnt/ß-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations. LAY SUMMARY: Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/ß-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/ß-catenin pathway.

Assessment of VAV2 Expression Refines Prognostic Prediction in Adrenocortical Carcinoma.

Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with overall poor prognosis. The Ki67 labeling index (LI) has a major prognostic role in localized ACC after complete resection, but its estimates may suffer from considerable intra- and interobserver variability. VAV2 overexpression induced by increased Steroidogenic Factor-1 dosage is an essential factor driving ACC tumor cell invasion. Objective: To assess the prognostic role of VAV2 expression in ACC by investigation of a large cohort of patients. Design, Setting, and Participants: A total of 171 ACC cases (157 primary tumors, six local recurrences, eight metastases) from seven European Network for the Study of Adrenal Tumors centers were studied. Outcome Measurements: H-scores were generated to quantify VAV2 expression. VAV2 expression was divided into two categories: low (H-score, <2) and high (H-score, ≥2). The Ki67 LI retrieved from patients' pathology records was also categorized into low (<20%) and high (≥20%). Clinical and immunohistochemical markers were correlated with progression-free survival (PFS) and overall survival (OS). Results: VAV2 expression and Ki67 LI were significantly correlated with each other and with PFS and OS. Heterogeneity of VAV2 expression inside the same tumor was very low. Combined assessment of VAV2 expression and Ki67 LI improved patient stratification to low-risk and high-risk groups. Conclusion: Combined assessment of Ki67 LI and VAV2 expression improves prognostic prediction in ACC.

Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.

Growth factor receptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is associated with hepatocellular carcinoma.

Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition-mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1-S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level. CONCLUSION: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368).

PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progression.

Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.