Carbonic anhydrases III and IX are new players in the crosstalk between adrenocortical carcinoma and its altered adipose microenvironment.

PURPOSE: Adrenocortical carcinoma (ACC), a rare malignancy of the adrenocortex, is characterized by a crosstalk between the adipose microenvironment and tumor. Here, we assessed the involvement of carbonic anhydrase (CA) enzymes III and IX (CAIII and CAIX), in the metabolic alterations of the adipose tissue characterizing obesity and in the local crosstalk between the tumor adipose microenvironment and ACC. RESULTS/METHODS: CAIII and CAIX expression is altered in visceral adipose tissue (VAT) in obesity and in ACC. A significant CAIX upregulation was present in ACC at advanced stages (n = 14) (fold increase FI = 7.4 ± 0.1, P < 0.05) associated with lower CAIII levels (FI = 0.25 ± 0.06, P < 0.001), compared with lower stages (n = 9). In vitro coculture between visceral adipose stem cells (ASCs) and ACC cell lines, H295R and MUC-1, mimicking the interaction occurring between VAT and advanced ACC, showed a significant CAIX upregulation in H295R but not in MUC-1 cells, and a decreased expression of CAIII. The effect on adipose cells was different when cocultured with H295R or MUC-1 cells. Coculture did not modulate CAIII expression in ASCs, which, however, was significantly downregulated with H295R (FI = 0.34 ± 0.11, P < 0.05) and upregulated by MUC-1 when cocultured ASCs were induced to differentiate toward adipocytes, with an expression profile similar to what found in VAT of obese subjects. CAIX expression was markedly increased in ASCs cocultured with H295R and to a less extent following adipogenesis induction (FI = 150.9 ± 46.5 and FI = 4.6 ± 1.1, P < 0.01, respectively). CONCLUSION: Our findings highlight a modulation of CAIII and CAIX in the metabolic crosstalk between ACC and its local adipose microenvironment, suggesting that CAs might represent a potential target for novel anticancer therapies.

Evaluation of a standardized protocol for processing adrenal tumor samples: preparation for a European adrenal tumor bank.

Storage and tissue handling of surgical tumor specimen have been recognized as critical steps that can potentially affect reproducibility and comparability of molecular endpoints between laboratories. In the preparation of adrenal tumor tissue banking, three different protocols that simulate warm ischemia upon tumor removal (protocol I), thawing and refreezing cycles (protocol II), as well as storage of vital tumor samples (protocol III) were applied. For the first two protocols, samples were subdivided and either snap frozen or treated with a RNA preserving agent (RPA) while in protocol III different storage media were compared. Following these procedures, recovery and integrity of DNA, RNA, and protein by means of pulsed field electrophoresis, long-range PCR, real-time PCR, immunoblot, and immunohistochemistry (protocol I and II) as well as cell viability and steroidogenic capacity (protocol III) were investigated. While DNA integrity was not influenced by different treatment modalities, expression levels of adrenal marker genes were more affected in samples after snap freezing in comparison to RPA pretreatment. Moreover, storage at room temperature before and after freezing could be demonstrated to decrease the relative amount of protein phosphorylation (ERK) and enzymatic activity (succinate cytochrome c reductase) while overall protein levels were not significantly affected. Similarly, morphological or immunohistochemical evaluation was comparable between groups. For primary cell cultures generated after storage of tumor samples similar rates of viability were observable while steroid output varied between the groups. Overall, on the basis of the presented endpoints standardized operational procedures can be defined for a proposed European adrenal tumor biobank.

Genetic characterization of a mouse line with primary aldosteronism.

In an attempt to define novel genetic loci involved in the pathophysiology of primary aldosteronism, a mutagenesis screen after treatment with the alkylating agent N-ethyl-N-nitrosourea was established for the parameter aldosterone. One of the generated mouse lines with hyperaldosteronism was phenotypically and genetically characterized. This mouse line had high aldosterone levels but normal creatinine and urea values. The steroidogenic enzyme expression levels in the adrenal gland did not differ significantly among phenotypically affected and unaffected mice. Upon exome sequencing, point mutations were identified in seven candidate genes (Sspo, Dguok, Hoxaas2, Clstn3, Atm, Tipin and Mapk6). Subsequently, animals were stratified into wild-type and mutated groups according to their genotype for each of these candidate genes. A correlation of their genotypes with the respective aldosterone, aldosterone-to-renin ratio (ARR), urea and creatinine values as well as steroidogenic enzyme expression levels was performed. Aldosterone values were significantly higher in animals carrying mutations in four different genes (Sspo, Dguok, Hoxaas2 and Clstn3) and associated statistically significant adrenal Cyp11b2 overexpression as well as increased ARR was present only in mice with Sspo mutation. In contrast, mutations of the remaining candidate genes (Atm, Tipin and Mapk6) were associated with lower aldosterone values and lower Hsd3b6 expression levels. In summary, these data demonstrate association between the genes Sspo, Dguok, Hoxaas2 and Clstn3 and hyperaldosteronism. Final proofs for the causative nature of the mutations have to come from knock-out and knock-in experiments.

Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of ß-catenin in adrenocortical carcinoma.

Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/ß-catenin signaling pathway. However, the adrenal-specific targets of oncogenic ß-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous ß-catenin activating mutation was done to identify the Wnt/ß-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of ß-catenin in ACC. The Wnt response element site located at nucleotide position -1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/ß-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/ß-catenin pathway involved in ACC, acting on transcription and RNA splicing.