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1.
Curr Opin Endocrinol Diabetes Obes ; 27(3): 170-176, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32304391

RESUMO

PURPOSE OF REVIEW: The aim of this review is to summarize recent advances on development of in vivo preclinical models of adrenocortical carcinoma (ACC). RECENT FINDINGS: Significant progress has been achieved in the underlying molecular mechanisms of adrenocortical tumorigenesis over the last decade, and recent comprehensive profiling analysis of ACC tumors identified several genetic and molecular drivers of this disease. Therapeutic breakthroughs, however, have been limited because of the lack of preclinical models recapitulating the molecular features and heterogeneity of the tumors. Recent publications on genetically engineered mouse models and development of patient-derived ACC xenografts in both nude mice and humanized mice now provide researchers with novel tools to explore therapeutic targets in the context of heterogeneity and tumor microenvironment in human ACC. SUMMARY: We review current in-vivo models of ACC and discuss potential therapeutic opportunities that have emerged from these studies.

2.
Cell Death Dis ; 11(3): 192, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184394

RESUMO

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10-8, 8.1 × 10-7 and 2.1 × 10-8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

3.
J Clin Endocrinol Metab ; 105(1)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31513709

RESUMO

CONTEXT: Although the development of immune checkpoint inhibitors has transformed treatment strategies of several human malignancies, research models to study immunotherapy in adrenocortical carcinoma (ACC) are lacking. OBJECTIVE: To explore the effect of anti-PD1 immunotherapy on the alteration of the immune milieu in ACC in a newly generated preclinical model and correlate with the response of the matched patient. DESIGN, SETTING, AND INTERVENTION: To characterize the CU-ACC2-M2B patient-derived xenograft in a humanized mouse model, evaluate the effect of a PD-1 inhibitor therapy, and compare it with the CU-ACC2 patient with metastatic disease. RESULTS: Characterization of the CU-ACC2-humanized cord blood-BALB/c-Rag2nullIl2rγnullSirpaNOD model confirmed ACC origin and match with the original human tumor. Treatment of the mice with pembrolizumab demonstrated significant tumor growth inhibition (60%) compared with controls, which correlated with increased tumor infiltrating lymphocyte activity, with an increase of human CD8+ T cells (P < 0.05), HLA-DR+ T cells (P < 0.05) as well as Granzyme B+ CD8+ T cells (<0.001). In parallel, treatment of the CU-ACC2 patient, who had progressive disease, demonstrated a partial response with 79% to 100% reduction in the size of target lesions, and no new sites of metastasis. Pretreatment analysis of the patient's metastatic liver lesion demonstrated abundant intratumoral CD8+ T cells by immunohistochemistry. CONCLUSIONS: Our study reports the first humanized ACC patient-derived xenograft mouse model, which may be useful to define mechanisms and biomarkers of response and resistance to immune-based therapies, to ultimately provide more personalized care for patients with ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Anticorpos Monoclonais Humanizados/farmacologia , Modelos Animais de Doenças , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente Tumoral/imunologia , Neoplasias do Córtex Suprarrenal/imunologia , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/imunologia , Carcinoma Adrenocortical/patologia , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Proliferação de Células , Feminino , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor de Morte Celular Programada 1/imunologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Endocr Soc ; 3(12): 2295-2304, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31745526

RESUMO

Adrenocortical carcinoma (ACC) is a rare orphan disease with a dismal prognosis. Surgery remains the first-line treatment, but most patients eventually develop metastatic disease. Mitotane is often used with chemotherapy with modest success. Little information is available concerning the efficacy of immunotherapy in combination with mitotane. We conducted a retrospective review of our initial six patients with metastatic ACC, for whom mitotane alone or with chemotherapy failed, and who were subsequently treated with a combination of pembrolizumab and mitotane, between July 2016 and March 2019. Imaging was analyzed per Response Evaluation Criteria in Solid Tumours 1.1 criteria. Two patients had a partial response and four patients had stable disease (8 to 19 months). One patient had grade 3 hepatitis and pembrolizumab was discontinued after 8 months. She died with disease progression 16 months after initiating pembrolizumab. One patient developed brain metastasis after 19 months of treatment and was transitioned to hospice. One patient had focal pneumonitis after 18 months of treatment, and pembrolizumab was discontinued. Three remaining patients continue pembrolizumab plus mitotane at the time of this writing. The current standard of care for ACC is a combination of etoposide, doxorubicin, cisplatin, and mitotane with an overall survival of 14.8 months. All six patients lived for at least 16 months after starting pembrolizumab added to mitotane therapy. The therapy appeared to be effective in both microsatellite instability-high and microsatellite stable tumors, suggesting some synergistic effect with mitotane. Combined immunotherapy and mitotane should be considered in future clinical trials in patients with ACC.

5.
J Steroid Biochem Mol Biol ; 193: 105422, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31265901

RESUMO

The adrenal cortex governs fundamental metabolic processes though synthesis of glucocorticoid, mineralocorticoids and androgens. Studies in rodents have demonstrated that the cortex undergoes a self-renewal process and that capsular/subcapsular stem/progenitor cell pools differentiate towards functional steroidogenic cells supporting the dynamic centripetal streaming of adrenocortical cells throughout life. We previously demonstrated that the Notch atypical ligand Delta-like homologue 1 (DLK1)/preadipocyte factor 1 (PREF1) is expressed in subcapsular Sf1 and Shh-positive, CYP11B1-negative and CYP11B2-partially positive cortical progenitor cells in rat adrenals, and that secreted DLK1 can modulate GLI1 expression in H295R cells. Here we show that the human adrenal cortex remodels with age to generate clusters of relatively undifferentiated cells expressing DLK1. These clusters (named DLK1-expressing cell clusters or DCCs) increased with age in size and were found to be different entities to aldosterone-producing cell clusters, another well-characterized and age-dependent cluster structure. DLK1 was markedly overexpressed in adrenocortical carcinomas but not in aldosterone-producing adenomas. Thus, this data identifies a novel cell population in the human adrenal cortex and might suggest a yet-to be identified role of DLK1 in the pathogenesis of adrenocortical carcinoma in humans.


Assuntos
Córtex Suprarrenal/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos
6.
Endocr Relat Cancer ; 26(10): 765-778, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325906

RESUMO

Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.

7.
Pituitary ; 22(1): 37-45, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30456434

RESUMO

PURPOSE: In view of mounting attention related to possible brain retention of gadolinium-based contrast agents (GBCAs) in patients with normal renal function, our purpose was to detail results from a survey of pituitary experts to assess: 1) the timing interval and frequency of pituitary magnetic resonance imaging (MRI) following surgical and/or medical and/or radiation therapy of pituitary tumors, 2) awareness of the types of GBCAs used and their possible safety issues. METHODS: The Pituitary Society Education Committee composed a survey with 12 multiple choice questions, 8 of which specifically addressed the time interval and frequency of MRI in the longitudinal management of pituitary tumors. The survey was distributed at two meetings; the International Pituitary Neurosurgeons Society conference in San Diego, CA, on February 18th, 2018, and the Pituitary Society Membership and Career Development Forum, Chicago, IL on March 18th, 2018. RESULTS: There is consensus among pituitary endocrinologists and neurosurgeons that long-term repeated imaging is recommended in most pituitary tumors, although the precise strategy of timing varied depending on the specialist group and the specific clinical context of the adenoma. The data also suggest that International Pituitary Neurosurgeons Society neurosurgeons, as well as Pituitary Society neuroendocrinologists, are sometimes unaware of which contrast agents are used by their institution, and many are also unaware that evidence of long-term brain retention has been reported with the use of GBCAs in patients with normal function. CONCLUSIONS: International pituitary endocrinologists and pituitary neurosurgeons experts suggest ongoing MRIs for the management of pituitary tumors; strategies vary based on clinical context, but also on individual experience and practice.


Assuntos
Adenoma/diagnóstico por imagem , Meios de Contraste/análise , Gadolínio/análise , Imagem por Ressonância Magnética/métodos , Neoplasias Hipofisárias/diagnóstico por imagem , Humanos , Inquéritos e Questionários
8.
Endocrinology ; 159(7): 2532-2544, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29790920

RESUMO

Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.


Assuntos
Carcinoma Adrenocortical/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma Adrenocortical/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Naftiridinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
9.
Endocr Relat Cancer ; 25(4): 437-451, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29371329

RESUMO

Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in CTNNB1 and secreted cortisol but not aldosterone. CU-ACC2 cells had a TP53 mutation and loss of MSH2 consistent with the patient's known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets.


Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Aldosterona/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Mutação em Linhagem Germinativa , Humanos
10.
J Neurosurg ; 129(5): 1260-1267, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29219752

RESUMO

OBJECTIVEThe authors report their single-institution experience with the pathological findings, rates of remission, and complications in patients with presumed Cushing's disease (CD) who underwent a two-thirds pituitary gland resection when no adenoma was identified at the time of transsphenoidal surgery (TSS). The authors also review the literature on patients with CD, negative surgical exploration, and histological findings.METHODSThis study is a retrospective analysis of cases found in neurosurgery and pathology department databases between 1989 and 2011. In all cases, patients had been operated on by the same neurosurgeon (K.O.L.). Twenty-two (13.6%) of 161 patients who underwent TSS for CD had no adenoma identified intraoperatively after systematic exploration of the entire gland; these patients all underwent a two-thirds pituitary gland resection. A chart review was performed to assess treatment data points as well as clinical and biochemical remission status.RESULTSOf the 22 patients who underwent two-thirds gland resection, 6 (27.3%) ultimately had lesions found on final pathology. All 6 patients were found to have a distinct adrenocorticotropic hormone (ACTH) cell adenoma. Sixteen (72.7%) of the patients had no tumor identified, with 3 of these patients suspected of having ACTH cell hyperplasia. The follow-up duration for the entire group was between 14 and 315 months (mean 98.9 months, median 77 months). Remission rates were 100% (6/6 patients) for the ACTH cell adenoma group and 75% (12/16) for the group without adenoma. Overall, 18 (81.8%) of the 22 patients had no evidence of hypercortisolism at last follow-up, and 4 patients (18%) had persistent hypercortisolism, defined as a postoperative cortisol level > 5 µg/dl. Of these 4 patients, 1 was suspected of harboring a cavernous sinus adenoma, 2 were found to have lung tumors secreting ACTH, and 1 remained with an undiagnosed etiology. Rates of postoperative complications were low.CONCLUSIONSThe diagnosis and treatment of CD can be challenging for neurosurgeons, endocrinologists, and pathologists alike. Failure to find a discrete adenoma at the time of surgery occurs in at least 10%-15% of cases, even in experienced centers. The current literature provides little guidance regarding rational intraoperative approaches in such cases. The authors' experience with 161 patients with CD, when no intraoperative tumor was localized, demonstrates the utility of a two-thirds pituitary gland resection with a novel and effective surgical strategy, as suggested by a high initial remission rate and a low operative morbidity.


Assuntos
Adenoma/cirurgia , Hipersecreção Hipofisária de ACTH/cirurgia , Hipófise/cirurgia , Neoplasias Hipofisárias/cirurgia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Neurocirúrgicos , Período Pós-Operatório , Indução de Remissão , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
11.
Endocrinology ; 158(5): 1450-1460, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28323918

RESUMO

Densely granulated and sparsely granulated (SG) growth hormone (GH) pituitary adenomas differ in biological behavior, which may be correlated with their known differences in cytoplasmic keratin distribution and E-cadherin expression. We wanted to explore candidate genes that might further explain this behavior. Exon expression microarray was performed on 21 GH tumors (10 SG and 11 densely granulated) and 20 normal control pituitaries from autopsy. Bioinformatic analyses confirmed a differential molecular signature between normal pituitary and GH tumors as well as between the GH tumor subtypes. There was a consistent downregulation of transcripts involved in the structure and function of the desmosome, including desmoplakin (eightfold), desmoglein 2 (sixfold), plakophilin 2 (sevenfold), and p53 apoptosis effector related to PMP-22 (PERP; sixfold) in SG tumors compared with normal pituitary. PERP is lost in more aggressive SG human GH pituitary tumors. PERP re-expression in GH3 rat GH tumor cells resulted in decreased colony formation compared with vector transfectants, confirming the role of PERP as a tumor suppressor with no effects on proliferation. Increased PERP expression was associated with loss of a survival advantage in a hypoxic environment, as assessed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (P < 0.05) and cleaved caspase-3 (P < 0.05). Downregulation of desmosomal formation transcripts including PERP may contribute to the aggressive phenotype seen in SG GH pituitary tumors and their behavior in response to surgery and medical therapy.


Assuntos
Adenoma/genética , Desmossomos/metabolismo , Genes Supressores de Tumor/fisiologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Proteínas de Membrana/fisiologia , Hipófise/metabolismo , Adenoma/patologia , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Análise em Microsséries , Transcriptoma , Células Tumorais Cultivadas
12.
J Appl Physiol (1985) ; 120(11): 1355-63, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27032901

RESUMO

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.


Assuntos
Exercício Físico/fisiologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Adulto , Atletas , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Teste de Tolerância a Glucose/métodos , Humanos , Masculino , Consumo de Oxigênio/fisiologia
13.
Mol Cancer Ther ; 15(3): 412-20, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26721946

RESUMO

Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. No medical treatments are currently available. Using a combined genetic and genomic screen of individual human gonadotrope pituitary tumor samples, we recently identified the mammalian sterile-20 like kinase 4 (MST4) as a protumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. To identify novel inhibitors of the MST4 kinase for potential future clinical use, computational-based virtual library screening was used to dock the SelleckChem kinase inhibitor library into the ATP-binding site of the MST4 crystal structure. Several inhibitor candidates were identified with the potential to bind with high affinity. Using a TR-FRET in vitro recombinant kinase assay, hesperadin, initially described as an Aurora kinase inhibitor, exhibited potent inhibition of the MST4 kinase at nanomolar concentrations. The LßT2 gonadotrope pituitary cell hypoxic model was used to test the ability of this inhibitor to antagonize MST4 actions. Under short-term severe hypoxia (1% O2), MST4 protection from hypoxia-induced apoptosis was abrogated in the presence of hesperadin. Similarly, under chronic hypoxia (5%), hesperadin blocked the proliferative and colony-forming actions of MST4 as well as the ability to activate specific downstream signaling and hypoxia-inducible factor-1 effectors. Together, these data identify hesperadin as the first potent, selective inhibitor of the MST4 kinase with the capacity to block pituitary tumor cell growth in a hypoxic microenvironment.


Assuntos
Antineoplásicos/química , Modelos Moleculares , Neoplasias Hipofisárias/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hipóxia/metabolismo , Imuno-Histoquímica , Indóis/química , Indóis/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Estresse Fisiológico , Sulfonamidas/química , Sulfonamidas/farmacologia , Microambiente Tumoral/efeitos dos fármacos
14.
Mol Cell Endocrinol ; 417: 73-83, 2015 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-26391562

RESUMO

PURPOSE: The aim of this study was to examine whether differential expression of somatostatin receptors (SSTR) 1-5 and downstream effectors are different in densely (DG) and sparsely (SG) granulated histological growth hormone (GH) pituitary tumor subtypes. METHODS: The study included 33 acromegalic patients with 23 DG and 10 SG tumors. SSTR1-5 were measured by qPCR and immunoblotting. Signaling candidates downstream of SSTR2 were also assessed. RESULTS: SSTR2 mRNA and protein levels were significantly higher in DG compared to SG tumors. Downstream of SSTR2, p27(kip1) was decreased (2.6-fold) in SG compared to DG tumors, suggesting a potential mechanism of SSA resistance in SG tumors with intact SSTR2 expression. Re-expression of E-cadherin in GH pituitary cell increased p27(kip1) levels. CONCLUSIONS: Histological subtyping correlated with SSTR2, E cadherin and p27(kip) protein levels and these may serve as useful biomarkers in GH tumors to predict behavior and response to therapy with SSA.


Assuntos
Caderinas/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/metabolismo , Estudos Retrospectivos , Transdução de Sinais
15.
Mol Endocrinol ; 29(3): 460-72, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25650755

RESUMO

The genetic and molecular mechanisms that initiate and maintain pituitary tumorigenesis are poorly understood. Nonfunctioning tumors of the gonadotrope lineage represent 35% of all tumors; are usually macroadenomas, often resulting in hypopituitarism; and have no medical treatments. Using expression microarrays combined with whole-genome copy number screens on individual human tumors, we identified the mammalian sterile-20-like kinase (MST4) transcript, which was amplified within chromosome Xq26.2 in one tumor and up-regulated in all gonadotrope tumor samples. MST4 mRNA and protein were consistently overexpressed in human tumors compared with normal pituitaries. To mimic the pituitary tumor microenvironment, a hypoxia model using LßT2 murine gonadotrope cells was created to examine the functional role of the kinase. During long-term hypoxia, MST4 expression increased colony formation in a soft agar assay and rates of cell proliferation by activating p38 MAPK and AKT. Under short-term severe hypoxic stress, MST4 decreased the rates of apoptosis via p38 MAPK, AKT, hypoxia-inducible factor-1, and its cell-specific downstream targets. Analysis of MST4 mutants confirmed the importance of the kinase sequence but not the regulatory C terminus for its functional effects. Together these data identify the MST4 kinase as a novel candidate to mediate human pituitary tumorigenesis in a hypoxic environment and position it as a potential therapeutic target.


Assuntos
Hipófise/enzimologia , Hipófise/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose , Hipóxia Celular , Proliferação de Células , Sobrevivência Celular , Citoproteção , Variações do Número de Cópias de DNA/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Gonadotrofos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Estresse Fisiológico , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Endocrine ; 49(1): 231-41, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25129651

RESUMO

Growth hormone (GH) pituitary tumors are associated with significant morbidity and mortality. Current treatments, including surgery and medical therapy with somatostatin analogs (SSA), dopamine agonists and/or a GH receptor antagonist, result in disease remission in approximately half of patients. Predictors of GH tumor response to different therapies have been incompletely defined based on histologic subtype, particularly densely (DG) versus sparsely (SG) granulated adenomas. The aim of this study was to examine our own institutional experience with GH adenomas and correlate how subtype related to clinical parameters as well as response to surgery and medical therapies. A retrospective chart review of 101 acromegalic patients operated by a single neurosurgeon was performed. Clinical data were correlated with histologic subtype and disease control, as defined by IGF-1 levels, and random growth hormone levels in response to surgery and/or medical therapies. SG tumors, compared to DG, occurred in younger patients (p = 0.0010), were 3-fold larger (p = 0.0030) but showed no differences in tumor-invasion characteristics (p = 0.12). DG tumors had a higher rate of remission in response to surgery compared to SG, 65.7 vs. 14.3 % (p < 0.0001), as well as to medical therapy with SSAs (68.8 % for DG vs. 28.6 % for SG tumors; p = 0.028). SG tumors not controlled with SSAs consistently responded to a switch to, or addition of, a GH receptor antagonist. Histological GH tumor subtyping implicates a different clinical phenotype and biologic behavior, and provides prognostic significance for surgical success and response to medical therapies.


Assuntos
Acromegalia , Adenoma , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Hormônio do Crescimento Humano/análogos & derivados , Procedimentos Neurocirúrgicos/métodos , Avaliação de Resultados em Cuidados de Saúde , Acromegalia/classificação , Acromegalia/tratamento farmacológico , Acromegalia/patologia , Acromegalia/cirurgia , Adenoma/classificação , Adenoma/tratamento farmacológico , Adenoma/patologia , Adenoma/cirurgia , Adulto , Fatores Etários , Idoso , Feminino , Adenoma Hipofisário Secretor de Hormônio do Crescimento/classificação , Adenoma Hipofisário Secretor de Hormônio do Crescimento/tratamento farmacológico , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/cirurgia , Hormônio do Crescimento Humano/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
17.
Endocrinology ; 153(7): 2963-73, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22562171

RESUMO

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G(2)/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LßT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G(2)/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LßT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/fisiologia , Neoplasias Hipofisárias/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ilhas de CpG , Citoplasma/metabolismo , Fase G2 , Genes Supressores de Tumor , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Hipófise/metabolismo , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas
18.
Cancer ; 118(21): 5302-9, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22488744

RESUMO

BACKGROUND: The objective of this study was to document the differences in testosterone (T) levels between crizotinib-treated and noncrizotinib-treated patients with metastatic nonsmall cell lung cancer (NSCLC). METHODS: Testosterone levels were measured in 19 men with metastatic NSCLC who received crizotinib and in 19 men with metastatic NSCLC who did not receive crizotinib. Clinical characteristics of the patients were compared, and additional hormone assays were performed as appropriate. Two patients who began crizotinib and 4 patients who had dose interruptions or who stopped crizotinib therapy had serial hormone measurements, permitting the documentation of dynamic hormone changes on and off crizotinib treatment. RESULTS: Total T levels were low (<241 ng/dL) in 19 of 19 (100%) crizotinib-treated men and in 6 of 19 men (32%) with metastatic NSCLC who did not receive crizotinib (mean T levels, 131 ng/dL and 311 ng/dL, respectively; P = .0002). Only 1 in 5 patients who had anaplastic lymphoma kinase (ALK) gene rearrangements and had not yet received crizotinib had low T. The initiation of crizotinib in 2 patients who had previously normal T levels was associated with a rapid decreases in T and in luteinizing hormone and follicle stimulating hormone levels within 14 to 21 days. Discontinuation of crizotinib led to increases back to normal T levels. CONCLUSIONS: Crizotinib therapy caused rapid suppression of T levels in men. The current results indicated that the site of action must include a central (hypothalamic or pituitary) effect, but additional direct testicular effects could not be excluded. Further work is required to assess the correlation between low T levels and crizotinib side effects as well as the exact molecular mechanism and site of drug toxicity.


Assuntos
Antineoplásicos/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Hipogonadismo/induzido quimicamente , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , Piridinas/efeitos adversos , Testosterona/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Crizotinibe , Humanos , Hipogonadismo/sangue , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica
19.
Endocrine ; 42(1): 18-28, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22434413

RESUMO

Growth hormone (GH) pituitary tumors are almost always benign adenomas, yet are associated with significant morbidity and mortality. Surgical and medical responses of GH tumors are often incomplete, and therefore predictors of residual or recurrent disease are needed. Clinical features, including patient gender, age or size of adenoma, have proven to be unreliable predictors of recurrence. Differing clinical behavior between the two GH tumor subtypes, sparsely granulated (SG) versus densely granulated (DG), has been reported, but has not been used routinely in clinical management. SG tumors are more common in younger patients (<50 years), and are usually larger tumors. SG tumors have been reported to be less responsive to somatostatin analogs (SSA) than DG tumors. The mechanisms underlying these potential differences in tumor behavior, however, are poorly defined. Subsets (up to 50 %) of DG adenomas harbor a gsp mutation that can activate cAMP that provides a theoretical intracellular target for somatostatin therapy. In contrast, some SG tumors have reduced somatostatin receptor expression and mutations in the extracellular domain of the GH receptor that may contribute to SSA resistance. While DG versus SG growth hormone adenomas are readily distinguished by immunohistochemistry, other less common GH adenoma variants still require electron microscopy (EM) for confident subclassification. Whether these less common variants possess unique clinical features is unknown. Research is needed to identify clinically relevant biomarkers of GH pituitary tumors that predict risk of recurrence and response to medical therapy.


Assuntos
Adenoma/diagnóstico , Adenoma/terapia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/diagnóstico , Adenoma Hipofisário Secretor de Hormônio do Crescimento/terapia , Adenoma/epidemiologia , Adenoma/patologia , Adulto , Animais , Adenoma Hipofisário Secretor de Hormônio do Crescimento/epidemiologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico
20.
Endocrinology ; 152(10): 3603-13, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21810943

RESUMO

Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45ß in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45ß in LßT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45ß showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and ß in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45ß promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45ß is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.


Assuntos
Antígenos de Diferenciação/fisiologia , Gonadotrofos/patologia , Neoplasias Hipofisárias/patologia , Proteínas Supressoras de Tumor/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Diferenciação/genética , Linhagem Celular , Dano ao DNA , Feminino , Genes p53 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos
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