N-type calcium channel v2.2 is a target of TCF21 in adrenocortical carcinomas.

Transcription factor 21 (TCF21) directly binds and regulates SF1 mRNA expression in tumor and normal adrenocortical cells, and both are involved in the development and steroidogenesis of the adrenal cortex. TCF21 is a tumor suppressor gene and its expression is reduced in malignant tumors. In adrenocortical tumors, it is less expressed in adrenocortical carcinomas (ACC) than in adrenocortical adenomas (ACA) and normal tissues. However, a comprehensive analysis to identify TCF21 targets has not yet been conducted in any type of cancer. In this study, we performed Chromatin Immunoprecipitation and Sequencing (ChIP-Seq) in an adrenocortical carcinoma cell line (NCI-H295R) overexpressing TCF21, with the aim of identifying TCF21 new targets. The five most frequently identified sequences corresponded to the PRDM7, CNTNAP2, CACNA1B, PTPRN2, and KCNE1B genes. Validation experiments showed that, in NCI-H295R cells, TCF21 negatively regulates the expression of the CACNA1B gene. Recently, it was observed that the N-type calcium channel v2.2 (Cav2.2) encoded by the CACNA1B gene is important in Angiotensin II signal transduction for corticosteroid biosynthesis in NCI-H295R adrenocortical carcinoma cells. Indeed, TCF21 inhibits CACNA1B and Cav2.2 expression in NCI-H295R. In addition, in a cohort of 55 adult patients with adrenocortical tumors, CACNA1B expression was higher in ACC than ACA and was related to poor disease-free survival in ACC patients. These results suggest a mechanism of steroidogenesis control by TCF21 in adrenocortical tumor cells, in addition to the control observed through SF1 inhibition. Importantly, steroid production could impair tumor immunogenicity, contributing to the immune resistance described in adrenal cancer.

Stathmin 1 is highly expressed and associated with survival outcome in malignant adrenocortical tumours.

Adrenocortical carcinoma (ACC) is an aggressive endocrine cancer with few molecular predictors of malignancy and survival, especially in paediatric patients. Stathmin 1 (STMN1) regulates microtubule dynamics and has been involved in the malignant phenotype of cancer cells. Recently, it was reported that STMN1 is highly expressed in ACC patients, and STMN1 silencing reduces the clonogenicity and migration of ACC cell lines. However, the prognostic significance of STMN1 and its therapeutic potential remain undefined in ACC. In the present study, STMN1 mRNA levels were significantly higher (p < 0.05) in ACC patients, especially in an advanced stage, and correlated with BUB1B and PINK1 expression, the prognostic-related genes in ACC. In paediatric tumours, high STMN1 expression was observed in both adrenocortical carcinoma and adrenocortical adenoma patients. Among the adult malignant tumours, STMN1 level was an independent predictor of survival outcomes (overall survival: hazard ratio = 6.08, p = 0.002; disease-free survival: hazard ratio = 4.65, p < 0.0001). Paclitaxel, a microtubule-stabilizing drug, reduces the activation of STMN1 and significantly decreases cell migration and invasion in ACC cell lines and ACC cells from secondary cell culture (all p < 0.0001). In summary, STMN1 expression may be of great value to clinical and pathological findings in therapeutic trials and deserves future studies in ACC.

The tyrosine kinase inhibitor nilotinib is more efficient than mitotane in decreasing cell viability in spheroids prepared from adrenocortical carcinoma cells.

BACKGROUND: New drugs for adrenocortical carcinoma (ACC) are needed because most patients undergo rapid disease progression despite surgery and adjuvant therapy with mitotane. In this study, we aimed to investigate the in vitro effects of different chemotherapy drugs, alone or combined with mitotane, on the viability of adrenocortical carcinoma cells. METHODS: Everolimus, sunitinib, zoledronic acid, imatinib and nilotinib cytotoxicity, alone or combined with mitotane were tested on ACC H295R cells in monolayer or spheroid cultures using MTS assays and confocal microscopy. Moreover, the nilotinib effects were investigated in spheroids cultured from patient tumor-derived ACC-T36 cells. RESULTS: Morphological characterization of H295R cell spheroids using histochemistry was performed and showed that dense, homogenously sized, multicellular spheroids were obtained. We observed that sunitinib and nilotinib alone were equally effective in a monolayer preparation, whereas mitotane was the most effective even at a low dose. A combination of sunitinib and mitotane was the most effective treatment, with only 23.8% of cells in the monolayer remaining viable. Spheroid preparations showed resistance to different drugs, although the poor effect produced by mitotane alone was surprising, with a cell viability of 84.6% in comparison with 13.1% in monolayer cells. The most ineffective drugs in spheroid preparations were everolimus, zoledronic acid and imatinib. In both cell types, nilotinib, either alone or in combination with mitotane induced more significant cell viability inhibition in monolayer and spheroid preparations. In addition, the mechanism of nilotinib activity involves the ERK1/2 pathway. CONCLUSION: Taken together, our data identified nilotinib as a cytotoxic drug that combined with ERK inhibitors deserves to be tested as a novel therapy for adrenocortical carcinoma.

Protective effect of metformin in an aberrant crypt foci model induced by 1,2-dimethylhydrazine: Modulation of oxidative stress and inflammatory process.

Colorectal Cancer (CRC) is the third most frequent type of cancer worldwide. In the past few years, studies have revealed a protective effect of metformin (MET-an anti-hyperglycemic drug, used to treat type 2 diabetes), against CRC. The protective effect of MET has been associated with AMPK activation (and mTOR inhibition), resulting in suppressed protein synthesis, and reduced cell proliferation in malignant transformed cells. To elucidate new mechanisms for the protective effect of metformin, we evaluated the oxidative stress and inflammatory process modulation, since these processes are strictly involved in colorectal carcinogenesis. The present study evaluated the protective effect of MET in a CRC model induced by 1,2-dimethylhydrazine (DMH) in Balb/c female mice. The simultaneous/continuous treatment (administration of MET and DMH simultaneously), revealed protective activity of MET, preventing the formation of aberrant crypt foci (ACF) in 71.4% at distal colon sections, and was able to restore basal labeling of apoptosis. Treatment with MET also reduced the inflammatory process induced by DMH, resulting in of the reduction of oxidative stress and nitric oxide related parameters. © 2016 Wiley Periodicals, Inc.

Kinin B1 receptor modulates glucose homeostasis and physical exercise capacity by altering adrenal catecholamine synthesis and secretion.

Our group has shown in several papers that kinin B1 receptor (B1R) is involved in metabolic adaptations, mediating glucose homeostasis and interfering in leptin and insulin signaling. Since catecholamines are involved with metabolism management, we sought to evaluate B1R role in catecholamine synthesis/secretion. Using B1R global knockout mice, we observed increased basal epinephrine content, accompanied by decreased hepatic glycogen content and increased glucosuria. When these mice were challenged with maximal intensity exercise, they showed decreased epinephrine and norepinephrine response, accompanied by disturbed glycemic responses to effort and poor performance. This phenotype was related to alterations in adrenal catecholamine synthesis: increased basal epinephrine concentration and reduced norepinephrine content in response to exercise, as well decreased gene expression and protein content of tyrosine hydroxylase and decreased gene expression of dopamine beta hydroxylase and kinin B2 receptor. We conclude that the global absence of B1R impairs catecholamine synthesis, interfering with glucose metabolism at rest and during maximal exercise.

Upregulation of TCF21 inhibits migration of adrenocortical carcinoma cells.

BACKGROUND: Adrenocortical carcinomas (ACC) are rare and aggressive cancer. Our previous study has revealed that the transcription factor 21, TCF21, is downregulated in ACC and regulates steroidogenic factor 1 (SF-1) binding to the SF-1 E-box promoter. In addition, it could be found that TCF21 is a predictor of overall survival (OS) in adult carcinomas. METHODS: In this study, it was investigated the correlation between TCF21 expression and the promoter methylation status in adrenocortical tumor cells, carcinomas and adenoma. The biological function and potential molecular mechanism of TCF21 restoration in migration and invasion of ACC cells was examined. RESULTS: We could be demonstrated a negative correlation between the level of TCF21 expression and methylation of its promoter in adenoma and carcinoma cells indicating the epigenetic control of TCF21 expression. It was also demonstrated that the expression of TCF21 inhibits migration and invasion in the ACC cell line, H295R cells, using plasmid transfection to express TCF21. Furthermore, it could be investigated the TCF21 function as tumor suppressor probably through Kisspeptin 1 (KISS-1) expression and epithelial-mesenchymal transition (EMT) reversion, as well as the modulation of several metalloproteinases in ACC cells. CONCLUSIONS: Our results suggest that enhancement of TCF21 expression levels may be a potential strategy to revert invasive abilities in adrenocortical carcinomas.

ARMC5 mutations are associated with high levels of proliferating cell nuclear antigen and the presence of the serotonin receptor 5HT4R in PMAH nodules.

Objective To analyze the morphological and functional characteristics of primary macronodular adrenal hyperplasia (PMAH) nodules carrying or not carrying ARMC5 mutations and the consequences of the presence of mutations in terms of the pattern of macronodule composition and functional state. Subjects and methods The analyses were performed by hematoxylin-eosin staining, immunohistochemistry, microdissection of spongiocyte tissue and RT-qPCR of histological sections from 16 patients diagnosed with PMAH with germline (5) or germline/somatic mutations (5) and without mutations (6) in the ARMC5 gene. Results Hyperplastic nodules were predominantly composed of spongiocytes in mutated and nonmutated sections. ARMC5 mRNA expression in spongiocytes was higher in ARMC5-mutated nodules than in ARMC5-nonmutated nodules, and homogenous ARMC5 protein distribution was observed. The presence of arginine-vasopressin receptor (AVP1AR) and ectopic ACTH production were observed in both cell populations regardless of ARMC5 mutations; the numbers of serotonin receptor (5HT4R)- and proliferating cell nuclear antigen (PCNA)-positive cells were higher in macronodules carrying ARMC5 mutations than in those without mutations. Conclusions Our results suggest that the presence of ARMC5 mutations does not interfere with the pattern of distribution of spongiocytes and compact cells or with the presence of AVP1AR, gastric-inhibitory polypeptide receptor (GIPR) and ectopic ACTH. Nevertheless, the higher numbers of PCNA-positive cells in mutated nodules than in nonmutated nodules suggest that mutated ARMC5 can be related to higher proliferation rates in these cells. In conclusion, our results provide more information about the crosstalk among abnormal GPCRs, ectopic ACTH in steroidogenesis and the ARMC5 gene, which may be relevant in understanding the pathogenesis and diagnosis of patients with PMAH.

Citral prevents UVB-induced skin carcinogenesis in hairless mice.

The incidence of skin cancers has increased worldwide, requiring more prevention of this type of cancer. The use of sunscreen and the control of the time of exposure to sunlight are the recognized forms of prevention. However, new substances have been researched in order to develop formulations with more efficient protective activity. Citral is a natural compound with lemon scent that is used in food and cosmetic industries. The present work evaluated the chemoprotective effect of citral during UVB-induced skin carcinogenesis. Male hairless mice HRS/J, 8-12 weeks old, were exposed to UVB irradiation for 24 weeks, with a cumulative radiation dose of 13.875 J/cm2. Citral (0.1, 0.5 and 1%) was applied to the skin at a dosage of 0.1 g/animal, 5 min after UVB exposure. At the end of the experiment, the number of lesion/animal, and size of lesions were measured. The histological sections of the skin were evaluated for the presence and intensity of actinic keratosis and squamous cell carcinoma. TUNEL assay was performed for apoptosis evaluation. Skin samples were used for the measurement of oxidative stress parameters (total radical-trapping antioxidant parameter of skin, glutathione, catalase activity and malondialdehyde), and cytokines levels (IL-1ß, IL-4, IL-10, IL-23, TNF-α, and IFNγ). Citral 1% completely inhibited UVB-induced skin carcinogenesis by reducing levels of oxidative stress and pro-inflammatory cytokines, increasing apoptotic rate in the skin.

The human adrenal cortex: growth control and disorders.

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.

TCF21/POD-1, a Transcritional Regulator of SF-1/NR5A1, as a Potential Prognosis Marker in Adult and Pediatric Adrenocortical Tumors.

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

The human adrenal cortex: growth control and disorders

This review summarizes key knowledge regarding the development, growth, and growth disorders of the adrenal cortex from a molecular perspective. The adrenal gland consists of two distinct regions: the cortex and the medulla. During embryological development and transition to the adult adrenal gland, the adrenal cortex acquires three different structural and functional zones. Significant progress has been made in understanding the signaling and molecules involved during adrenal cortex zonation. Equally significant is the knowledge obtained regarding the action of peptide factors involved in the maintenance of zonation of the adrenal cortex, such as peptides derived from proopiomelanocortin processing, adrenocorticotropin and N-terminal proopiomelanocortin. Findings regarding the development, maintenance and growth of the adrenal cortex and the molecular factors involved has improved the scientific understanding of disorders that affect adrenal cortex growth. Hypoplasia, hyperplasia and adrenocortical tumors, including adult and pediatric adrenocortical adenomas and carcinomas, are described together with findings regarding molecular and pathway alterations. Comprehensive genomic analyses of adrenocortical tumors have shown gene expression profiles associated with malignancy as well as methylation alterations and the involvement of miRNAs. These findings provide a new perspective on the diagnosis, therapeutic possibilities and prognosis of adrenocortical disorders.