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J Immunother Cancer ; 8(1)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32474412


BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy. Tumor-related glucocorticoid excess is present in ~60% of patients and associated with particularly poor prognosis. Results of first clinical trials using immune checkpoint inhibitors were heterogeneous. Here we characterize tumor-infiltrating T lymphocytes (TILs) in ACC in association with glucocorticoids as potential explanation for resistance to immunotherapy. METHODS: We performed immunofluorescence analysis to visualize tumor-infiltrating T cells (CD3+), T helper cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+) and regulatory T cells (Tregs; CD3+CD4+FoxP3+) in 146 ACC tissue specimens (107 primary tumors, 16 local recurrences, 23 metastases). Quantitative data of immune cell infiltration were correlated with clinical data (including glucocorticoid excess). RESULTS: 86.3% of ACC specimens showed tumor infiltrating T cells (7.7 cells/high power field (HPF)), including T helper (74.0%, 6.7 cells/HPF), cytotoxic T cells (84.3%, 5.7 cells/HPF) and Tregs (49.3%, 0.8 cells/HPF). The number of TILs was associated with better overall survival (HR for death: 0.47, 95% CI 0.25 to 0.87), which was true for CD4+- and CD8+ subpopulations as well. In localized, non-metastatic ACC, the favorable impact of TILs on overall and recurrence-free survival was manifested even independently of ENSAT (European Network for the Study of Adrenal Tumors) stage, resection status and Ki67 index. T helper cells were negatively correlated with glucocorticoid excess (Phi=-0.290, p=0.009). Patients with glucocorticoid excess and low TILs had a particularly poor overall survival (27 vs. 121 months in patients with TILs without glucocorticoid excess). CONCLUSION: Glucocorticoid excess is associated with T cell depletion and unfavorable prognosis. To reactivate the immune system in ACC by checkpoint inhibitors, an inhibition of adrenal steroidogenesis might be pivotal and should be tested in prospective studies.

Mol Cell Endocrinol ; 480: 36-41, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30315857


90 kDa heat shock proteins (Hsp90) act as protein chaperones and play a role in modulating endoplasmic reticulum (ER) stress. Hsp90 inhibitors are under clinical investigation as cancer treatment. Mitotane therapy of adrenocortical carcinoma (ACC) has been shown to act through lipid-induced ER-stress. To explore the potential of Hsp90 inhibitors in ACC as a single agent and in combination with mitotane, we analyzed two independent gene expression data sets of adrenal tumors in silico and treated the ACC cell line model NCI-H295 with Hsp90 inhibitors BIIB021 (B) and CCT18159 (C) alone and in combination with mitotane. ER-stress markers were monitored by immunoblotting. Drug synergism was quantified using the median effect model with cell viability as read-out. Cytosolic Hsp90 isoforms AA1 and AB1 were significantly overexpressed in ACC. Viability of H295 cells was impaired by B and C as single agents with an EC50 of 5.7 × 10-6M and 12.1 × 10-6M. B but not C dose-dependently increased XBP1 splicing and CHOP expression indicative of ER-stress activation. ER-stress marker expression was enhanced by co-incubation of B with 10  µM but not 5  µM mitotane. Maximal CHOP expression was induced by 25 µM mitotane alone with no additional effect of B. Combination indices (CI) of B and C with mitotane ranged from 0.64 to 1.38 and 0.68 to 1.30, respectively where CI values < 0.5 support clinically-relevant drug synergism. In conclusion, Hsp90 paralogs are differentially expressed in ACC and B but not C activates ER-stress in ACC cells. No meaningful drug synergism of Hsp90 inhibitors with mitotane was observed.

Carcinoma Adrenocortical/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Mitotano/uso terapêutico , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/patologia , Linhagem Celular , Simulação por Computador , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Humanos , Mitotano/farmacologia , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
J Clin Endocrinol Metab ; 102(9): 3491-3498, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911143


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.

Neoplasias do Córtex Suprarrenal/sangue , Neoplasias do Córtex Suprarrenal/mortalidade , Carcinoma Adrenocortical/sangue , Carcinoma Adrenocortical/mortalidade , Biomarcadores Tumorais/sangue , Proteínas Proto-Oncogênicas c-vav/metabolismo , Neoplasias do Córtex Suprarrenal/terapia , Carcinoma Adrenocortical/terapia , Adulto , Idoso , Análise de Variância , Biópsia por Agulha , Estudos de Coortes , Terapia Combinada , Intervalo Livre de Doença , Europa (Continente) , Feminino , Humanos , Imuno-Histoquímica , Internacionalidade , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Valor Preditivo dos Testes , Prognóstico , Proteínas Proto-Oncogênicas c-vav/sangue , Análise de Sobrevida , Resultado do Tratamento
Sci Signal ; 10(469)2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-28270555


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.

Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-vav/genética , Fator Esteroidogênico 1/genética , Adolescente , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Citoesqueleto/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-vav/metabolismo , Fator Esteroidogênico 1/metabolismo , Análise de Sobrevida , Adulto Jovem
EMBO Rep ; 17(9): 1264-80, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27402544


Several stimuli induce programmed cell death by increasing Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca(2+) uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca(2+) uptake by mitochondria. FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca(2+) uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca(2+)-dependent pro-apoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs.

Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/mortalidade , Antineoplásicos Hormonais/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/ultraestrutura , Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Mitotano/farmacologia , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Esteroides/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética
Endocrinology ; 156(11): 3895-908, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26305886


Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mitotano/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Intervalo Livre de Doença , Estresse do Retículo Endoplasmático/genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Immunoblotting , Imuno-Histoquímica , Lipídeos/análise , Mitotano/uso terapêutico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transcriptoma/efeitos dos fármacos