RESUMO
PURPOSE: HER2-positive breast cancer has a high chance of achieving pathological complete response when HSD17B4, responsible for peroxisomal ß-oxidation of very long-chain fatty acids (VLCFA) and estradiol, is methylation-silenced. Here, we aimed to identify the underlying molecular mechanism. METHODS: Using a HER2-positive breast cancer cell line, BT-474, control and knock-out (KO) clones were obtained. Metabolic characteristics were analyzed using a Seahorse Flux analyzer. RESULTS: HSD17B4 KO suppressed cellular proliferation, and enhanced sensitivity to lapatinib approximately tenfold. The KO led to accumulation of VLCFA and a decrease of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid. HSD17B4 KO increased Akt phosphorylation, possibly via decreased DHA, and genes involved in oxidative phosphorylation (OxPhos) and electron transport chain (ETC) were upregulated. Increased mitochondrial ATP production in the KO cells was confirmed by extracellular flux analyzer. Increased OxPhos led to severe dependence of the KO cells on pyruvate from glycolysis. Suppression of glycolysis by lapatinib led to severe delayed suppression of OxPhos in KO cells. CONCLUSION: HSD17B4 KO in BT-474 cells caused a decrease of PUFAs, increased Akt phosphorylation, enhanced glucose dependence of OxPhos, and increased sensitivity to inhibition of HER2, upstream of Akt. This mechanism may be applicable to other HER2-positive glucose-dependent breast cancer cells with HSD17B4 silencing.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Lapatinib/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glucose , Linhagem Celular Tumoral , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismoRESUMO
D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.
Assuntos
Perda Auditiva Neurossensorial , Hipoglicemia , Deficiência de Proteína , Éxons , Perda Auditiva Neurossensorial/genética , Humanos , Hipoglicemia/genética , Recém-Nascido , Proteína Multifuncional do Peroxissomo-2/genética , Deficiência de Proteína/genéticaRESUMO
An electromagnetic field (EMF) may affect the functions of uterine tissues. This study hypothesized that EMF changes the estrogenic activity of pig myometrium during the peri-implantation period. Tissue was collected on days 15-16 of the gestation and incubated in the presence of EMF (50 and 120 Hz, 2 and 4 h). The cytochrome P450 aromatase type 3 (CYP19A3) and hydroxysteroid 17ß dehydrogenase type 4 (HSD17B4) mRNA transcript abundance, cytochrome P450arom (aromatase), and 17ß hydroxysteroid dehydrogenase 17ßHSD) protein abundance and estrone (E1) and estradiol-17ß (E2) release were examined using Real-Time PCR, Western blot and radioimmunoassay. Selected myometrial slices were treated with progesterone (P4) to determine whether it functions as a protector against EMF. CYP19A3 mRNA transcript abundance in slices treated with EMF was less at 50 Hz (2 h) and greater at 120 Hz (2 and 4 h). HSD17B4 mRNA transcript was greater in slices treated with EMF at 120 Hz (2 h). Progesterone diminished EMF-related effects on CYP19A3 and HSD17B4. When P4 was added, EMF had suppressive (50 and 120 Hz, 2 h) or enhancing (50 Hz, 4 h) effects on aromatase abundance. The E1 release was lower after 4 h of EMF treatment at 50 Hz and P4 did not protect myometrial E1 release. In conclusion, EMF alters the synthesis and release of E1 and did not affect E2 release in the myometrium during the peri-implantation period.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Implantação do Embrião/efeitos da radiação , Estradiol/metabolismo , Estrona/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Miométrio/efeitos da radiação , Animais , Aromatase/genética , Aromatase/metabolismo , Radiação Eletromagnética , Feminino , Miométrio/metabolismo , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Técnicas de Cultura de TecidosRESUMO
Molecular mechanisms underlying intratumoral androgenesis and aberrant androgen receptor (AR) activation in prostate cancer remain poorly understood. Here we demonstrate that ectopic expression of the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger domain protein (SPOP) stabilizes 17ßHSD4. SPOP bound a functional substrate-binding consensus (SBC) motif 315RATST319 in 17ßHSD4 and promoted nondegradable K27- and K29-linked polyubiquitination of 17ßHSD4. The effect of SPOP was antagonized by serum- and glucocorticoid kinase-3 (SGK3)-mediated phosphorylation of serine 318 (S318) in the SBC and S318 phosphorylation-dependent binding of SKP2 E3 ligase and subsequent K48-linked polyubiquitination and proteasomal degradation of 17ßHSD4. Prostate cancer-associated SPOP mutations impaired the SPOP-17ßHSD4 interaction, caused 17ßHSD4 protein destruction in prostate cancer cells in culture and patient specimens, and increased testosterone production and prostate cancer cell growth in vitro and in mouse models. Thus, we have identified SPOP and SKP2 as two essential E3 ubiquitin ligases that exert opposite effects on 17ßHSD4 protein degradation and intratumoral androgenesis in prostate cancer cells. We further demonstrate that SPOP mutations or SKP2 overexpression contribute to prostate cancer progression by decreasing 17ßHSD4 expression and increasing intratumoral androgen synthesis. SIGNIFICANCE: This study reveals a novel mechanism of aberrant AR activation in SPOP-mutated prostate cancer and uncovers putative biomarkers for effective treatment by AR-targeted therapies.
Assuntos
Androgênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas Nucleares/metabolismo , Proteína Multifuncional do Peroxissomo-2/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos SCID , Proteínas Nucleares/genética , Proteína Multifuncional do Peroxissomo-2/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteólise , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HER2-positive breast cancers that achieve pathological complete response (pCR) after HER2-directed therapy consistently have good survival. We previously identified HSD17B4 methylation as a marker for pCR by methylation screening. Here, we aimed to identify a new marker by conducting a multi-omics analysis of materials prepared by laser capture microdissection, and adding 71 new samples. In the screening set (n = 36), mutations, methylation, and expression were analyzed by targeted sequencing, Infinium 450 K, and expression microarray, respectively, and 15 genes were identified as differentially expressed and eight genomic regions as differentially methylated between cancer samples with and without pCR. In a validation set (n = 47), one gene showed differential expression, and one region had differential methylation. Further, in the re-validation set (n = 55), all new samples, only HSD17B4 methylation was significantly different. The HSD17B4 methylation was at the transcriptional start site of its major variant, and was associated with its silencing. HSD17B4 was highly expressed in the vast majority of human cancers, and its methylation was present only in breast cancers and one lymphoblastic leukemia cell line. A combination of estrogen receptor-negative status and HSD17B4 methylation showed a positive predictive value of 80.0%. During HER2-directed neoadjuvant therapy, HSD17B4 methylation was the most reliable marker to monitor response to the therapy. These results showed that HSD17B4 methylation is a candidate predictive and response marker of HER2-positive breast cancer to HER2-directed therapy.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/genética , Proteína Multifuncional do Peroxissomo-2/genética , Receptor ErbB-2/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/antagonistas & inibidores , Transcriptoma , Resultado do TratamentoRESUMO
Steroidogenic enzymes are crucial in prostate cancer (PCa) progression. 17ß-Hydroxysteroid dehydrogenase type 4 (HSD17B4), encoded by HSD17B4, lacks catalytic capacity in androgen metabolism. Now the detailed role and molecular mechanism of PCa development are largely unknown. Here we showed that the expression of HSD17B4 was increased in PCa tissues compared to paired paratumor tissues. HSD17B4 knockdown in PCa cells significantly suppressed its proliferation, migration and invasion, while overexpressing HSD17B4 had opposite effects. Mechanistically, we found that the protein level of HSD17B4 was regulated by its acetylation at lysine 669(K669). Dihydroxytestosterone (DHT) treatment increased HSD17B4 acetylation and then promoted its degradation via chaperone-mediated autophagy (CMA). SIRT3 directly interacted with HSD17B4 to inhibit its acetylation and enhance its stability. In addition, we identified CREBBP as a regulator of the K669 acetylation and degradation of HSD17B4, affecting PC cell proliferation, migration and invasion. Notably, in PCa tissues and paired paratumor tissues, the level of HSD17B4 was negatively correlated with its K669 acetylation. Taken together, this study identified a novel role of HSD17B4 in PCa progression and suggested that HSD17B4 and its upstream regulators may be potential therapeutic targets for PCa intervention.
Assuntos
Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Acetilação , Animais , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Lisina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Invasividade Neoplásica/genética , Sirtuína 3/genética , Testosterona/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adult male muskrat (Ondatra zibethicus) has a pair of scent glands which secret musk to attract females during the breeding stage. The goal of the present study was to investigate the genetic mechanisms of musk secretion of muskrats at the whole genome level. Comparative genomics illustrated obvious expansion in 809 gene families, of which nine gene families played pivotal roles in steroid biosynthesis, possibly related to muskrat musk secretion. We identified 1112 positively selected genes (PSGs) in the muskrat, including estrogen receptor 1 (ER1), an important influencing factor to the weight and size of the scented glands of muskrats. HSD17B3, HSD17B4, CYP7B1 and CYP17B1, crucial to steroid hormone biosynthesis, were under strong positive selection in the muskrat, and phylogenetic analysis of HSD and CYP450 classes revealed high gene diversity. Functional enrichment revealed many pathways associated with musk secretion and/or growth and degeneration of scented gland significantly, such as peroxisome, PI3K-Akt signaling pathway, apoptosis, and prostate cancer. Two muskrat-specific missense mutations (Pro237Thr and Ser297Ile) were detected in LIPC, which were reported to be involved cholesterol metabolic process. More importantly, the missense mutations discovered in LIPC were classified as deleterious by PolyPhen-2, possibly affecting the musk secretion of muskrats.
Assuntos
Arvicolinae/genética , Arvicolinae/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Genômica , Luz , 17-Hidroxiesteroide Desidrogenases , Animais , Apoptose , Sistema Enzimático do Citocromo P-450 , Família 7 do Citocromo P450 , Receptor alfa de Estrogênio/metabolismo , Ácidos Graxos Monoinsaturados/química , Feminino , Masculino , Redes e Vias Metabólicas , Modelos Moleculares , Proteína Multifuncional do Peroxissomo-2 , Fosfatidilinositol 3-Quinases/metabolismo , Conformação Proteica , Glândulas Odoríferas/efeitos da radiação , Esteroide HidroxilasesRESUMO
Accumulating evidence has implicated that the activation of signal transducer and activator of transcription 3 (STAT3) contributes to the progression of liver cancer by affecting the expression of proliferationassociated genes. A previous study reported that elevated levels of 17ßhydroxysteroid dehydrogenase 4 (HSD17B4) are observed in patients with liver cancer. The current study investigated how upregulated HSD17B4 expression promoted the expression of proliferationassociated genes in rats with liver cancer. HSD17B4 expression in rats with liver cancer was significantly increased compared with the control group as determined by reverse transcriptionquantitative polymerase chain reaction and western blot assays. Immunohistochemical results revealed that STAT3 activation was positively correlated with increased HSD17B4 expression in tumor tissues from patients with liver cancer. Western blot results further suggested that HSD17B4 overexpression increased STAT3 activation via the protein kinase B and the mitogenactivated protein kinase/extracellularsignalregulated kinase signaling pathways in HepG2 cells. The present study suggested that overexpression may promote HepG2 proliferation by enhancing expression of various downstream targets of STAT3. Targeted inhibition of HSD17B4 may describe a novel approach in the prevention and treatment of liver cancer.
Assuntos
Proliferação de Células/genética , Neoplasias Hepáticas/genética , Proteína Multifuncional do Peroxissomo-2/genética , Fator de Transcrição STAT3/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Wistar , Transdução de Sinais/genética , Ativação Transcricional/genética , Regulação para Cima/genéticaAssuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Proliferação de Células/fisiologia , Neoplasias Hepáticas/metabolismo , Proteína Multifuncional do Peroxissomo-2/biossíntese , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Neoplasias Hepáticas/genética , Proteína Multifuncional do Peroxissomo-2/genética , RatosRESUMO
OBJECTIVE: Pathogenesis and progression of liver cancer are correlated with inflammatory response and estrogen level. 17ß-estradiol dehydrogenase IV (HSD17B4) is highly expressed in human liver cancer tissues. HSD17B4 participates in liver cancer cell proliferation via suppressing estradiol (E2) activity. This study generated a rat liver cancer model, on which the correlations between HSD17B4 and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), proliferating cell nucleus antigen (PCNA) expression were analyzed. MATERIALS AND METHODS: Male Sprague Dawley (SD) rats were randomly assigned into control and model group (N=30). Diethylnitrosamine was used to induce liver cancer in a rat model. HE staining was used to observe liver injury whilst ELISA was used to measure serum TNF-α and IL-6 levels. The level of serum E2 was quantified by radioimmunoassay. Serum liver function indexes were measured by automatic biochemical analyzer. Protein expressions of HSD17B4, p-Akt, p-ERK and PCNA were measured by Western blot. RESULTS: The inflammatory infiltration and necrosis of hepatocytes were shown in model group by HE staining, along with aggravated liver indexes. Significantly high phosphorylation level of Akt and ERK, along with the increase of HSD17B3 and PCNA expressions, was found in model group (p<0.05 compared to control group). Serum E2 level was statistically decreased, whilst TNF-α and IL-6 were up-regulated (p<0.05). HSD17B4 was positively correlated with TNF-α, IL-6 and PCNA expressions (r=0.68, 0.62 and 0.56, p<0.05). CONCLUSIONS: HSD17B4 is over-expressed in rat liver cancer tissues. Its expression was positively correlated with TNF-α, IL-6 and PCNA levels, and probably participates in liver cancer cell proliferation via ERK and Akt signal pathway.
Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Inflamação/complicações , Inflamação/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteína Multifuncional do Peroxissomo-2/fisiologia , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/complicações , Dietilnitrosamina , Estradiol/sangue , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Hepatócitos/metabolismo , Humanos , Inflamação/patologia , Interleucina-6/biossíntese , Interleucina-6/sangue , Testes de Função Hepática , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/complicações , Masculino , Proteína Multifuncional do Peroxissomo-2/biossíntese , Fosforilação , Antígeno Nuclear de Célula em Proliferação/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangue , Regulação para Cima/efeitos dos fármacosRESUMO
Androgen receptor signaling is critical for prostate adenocarcinoma, even after androgen deprivation therapy. Persistence of intratumoral androgens has been found in castration-resistant prostate cancer and attributed to increased in situ synthesis. Recently, Sharifi and colleagues reported an additional mechanism that can enhance local androgenic exposure: downregulation of an androgen-inactivating enzyme.
Assuntos
Androgênios , Receptores Androgênicos/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Proteína Multifuncional do Peroxissomo-2 , Neoplasias da Próstata , Neoplasias de Próstata Resistentes à Castração , Isoformas de ProteínasRESUMO
Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Isoflavonas/farmacologia , Proteína Multifuncional do Peroxissomo-2/antagonistas & inibidores , Alcaloides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Azocinas/química , Azocinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Isoflavonas/síntese química , Isoflavonas/química , Estrutura Molecular , Proteína Multifuncional do Peroxissomo-2/metabolismo , Quinolizinas/química , Quinolizinas/farmacologia , Relação Estrutura-AtividadeRESUMO
An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein-2 (MFP2), the key enzyme in peroxisomal ß-oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti-inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2-/- mice were treated with the selective colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of >95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2-/- mice. Despite microglial diminution in Mfp2-/- brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2-/- mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2-/- phenotype.
Assuntos
Anti-Inflamatórios/uso terapêutico , Encefalite , Gliose/etiologia , Proteína Multifuncional do Peroxissomo-2/deficiência , Estimulação Acústica , Análise de Variância , Animais , Anti-Inflamatórios/farmacologia , Antígenos de Diferenciação/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Encefalite/complicações , Encefalite/genética , Encefalite/patologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Força Muscular/efeitos dos fármacos , Força Muscular/genética , Proteína Multifuncional do Peroxissomo-2/genética , Índice de Gravidade de DoençaRESUMO
Dysregulation of hormone metabolism is implicated in human breast cancer. 17ß-hydroxysteroid dehydrogenase type 4 (HSD17B4) catalyzes the conversion of estradiol (E2) to estrone (E1), and is associated with the pathogenesis and development of various cancers. Here we show that E1 upregulates HSD17B4 acetylation at lysine 669 (K669) and thereby promotes HSD17B4 degradation via chaperone-mediated autophagy (CMA), while a single mutation at K669 reverses the degradation and confers migratory and invasive properties to MCF7 cells upon E1 treatment. CREBBP and SIRT3 dynamically control K669 acetylation level of HSD17B4 in response to E1. More importantly, K669 acetylation is inversely correlated with HSD17B4 in human breast cancer tissues. Our study reveals a crosstalk between acetylation and CMA degradation in HSD17B4 regulation, and a critical role of the regulation in the malignant progression of breast cancer.
Assuntos
Autofagia/efeitos dos fármacos , Estrona/farmacologia , Proteína Multifuncional do Peroxissomo-2/metabolismo , Proteólise/efeitos dos fármacos , Acetilação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Biológicos , Mutação/genética , Invasividade Neoplásica , Proteína Multifuncional do Peroxissomo-2/genética , Sirtuína 3/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation.
Assuntos
Ativação de Macrófagos , Macrófagos/imunologia , Peroxissomos/imunologia , Fenilbutiratos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Multifuncional do Peroxissomo-2/genética , Cultura Primária de Células , Células RAW 264.7 , Proteínas Repressoras/genéticaRESUMO
Human epidermal growth factor (HER) 2-directed therapy is the standard treatment for HER2-positive breast cancer. Patients who achieved a pathological complete response (pCR) to the therapy are associated with excellent disease-free survival. However, few molecular markers are available to predict pCR. Here, we aimed to establish a DNA methylation marker to predict the response to trastuzumab and chemotherapy. A total of 67 patients were divided into screening (n = 21) and validation (n = 46) sets. Genome-wide DNA methylation analysis of the screening set identified eight genomic regions specifically methylated in patients with pCR. Among these, HSD17B4 encoding type 4 17ß-hydroxysteroid dehydrogenase was most significantly differentially methylated. The differential methylation was confirmed by pyrosequencing (P = 0.03), and a cutoff value was determined. This association was successfully validated in the validation set (P < 0.001), and patients with pCR were predicted with a high specificity (79%). Multivariate analysis, including tumor stage and hormone receptor status, showed that HSD17B4 methylation was an independent predictive factor (odds ratio: 10.0, 95% confidence interval 2.54-39.50, P = 0.001). Combination with ER status and HSD17B4 methylation improved the specificity up to 91%. Identification of HER2-positive breast cancer patients who would achieve pCR only by trastuzumab and chemotherapy may lead to surgery-free treatment for this group of breast cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Multifuncional do Peroxissomo-2/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ilhas de CpG/genética , Feminino , Humanos , Regiões Promotoras Genéticas/genética , Receptor ErbB-2/genética , Trastuzumab/uso terapêutico , Resultado do TratamentoRESUMO
SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Quinase 1 de Adesão Focal/antagonistas & inibidores , Glicolipídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antineoplásicos/síntese química , Sítios de Ligação , Carcinoma de Células Pequenas/enzimologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glicolipídeos/síntese química , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteína Multifuncional do Peroxissomo-2/química , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The discrete effects of obesity on infertility in females remain undefined to date. To investigate obesity-induced ovarian dysfunction, we characterized metabolic parameters, steroidogenesis, and folliculogenesis in obese and lean female Ossabaw mini-pigs. Nineteen nulliparous, sexually mature female Ossabaw pigs were fed a high fat/cholesterol/fructose diet (n=10) or a control diet (n=9) for eight months. After a three-month diet-induction period, pigs remained on their respective diets and had ovarian ultrasound and blood collection conducted during a five-month study period after which ovaries were collected for histology, cell culture, and gene transcript level analysis. Blood was assayed for steroid and protein hormones. Obese pigs developed abdominal obesity and metabolic syndrome, including hyperglycemia, hypertension, insulin resistance and dyslipidemia. Obese pigs had elongated estrous cycles and hyperandrogenemia with decreased LH, increased FSH and luteal phase progesterone, and increased numbers of medium, ovulatory, and cystic follicles. Theca cells of obese, compared to control, pigs displayed androstenedione hypersecretion in response to in vitro treatment with LH, and up-regulated 3-beta-hydroxysteroid dehydrogenase 1 and 17-beta-hydroxysteroid dehydrogenase 4 transcript levels in response to in vitro treatment with LH or LH + insulin. Granulosa cells of obese pigs had increased 3-beta-hydroxysteroid dehydrogenase 1 transcript levels. In summary, obese Ossabaw pigs have increased transcript levels and function of ovarian enzymes in the delta 4 steroidogenic pathway. Alterations in LH, FSH, and progesterone, coupled with theca cell dysfunction, contribute to the hyperandrogenemia and disrupted folliculogenesis patterns observed in obese pigs. The obese Ossabaw mini-pig is a useful animal model in which to study the effects of obesity and metabolic syndrome on ovarian function and steroidogenesis. Ultimately, this animal model may be useful toward the development of therapies to improve fertility in obese and/or hyperandrogenemic females or in which to examine the effects of obesity on the maternal-fetal environment and offspring health.
Assuntos
Androstenodiona/sangue , Hormônio Foliculoestimulante/sangue , Infertilidade Feminina/metabolismo , Hormônio Luteinizante/sangue , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Progesterona/sangue , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Ciclo Estral , Feminino , Hormônio Foliculoestimulante/farmacologia , Expressão Gênica , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Humanos , Infertilidade Feminina/etiologia , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Insulina/farmacologia , Hormônio Luteinizante/farmacologia , Síndrome Metabólica/etiologia , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Obesidade/etiologia , Obesidade/genética , Obesidade/patologia , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo , Cultura Primária de Células , RNA Mensageiro/agonistas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Porco Miniatura , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , Células Tecais/patologiaRESUMO
ß-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several traditional medicines, including saw palmetto and devil's claw. Although ß-sitosterol is effective against enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the mechanisms of action are poorly understood. Here, we report the identification of two new binding proteins for ß-sitosterol that may underlie its beneficial effects.
Assuntos
Proteína Multifuncional do Peroxissomo-2/metabolismo , Sitosteroides/metabolismo , Sitosteroides/farmacologia , Sinaptotagmina I/metabolismo , Animais , Sítios de Ligação , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Conformação Molecular , Proteína Multifuncional do Peroxissomo-2/química , Ligação Proteica , Sitosteroides/química , Sinaptotagmina I/químicaRESUMO
Hepatocellular carcinoma (HCC) arises in a setting of chronic inflammation induced by inflammatory cytokines, such as nuclear factor-kappaB (NF-κB). HCC is a male-predominant cancer that can be attenuated by estradiol (E2) in vitro and in vivo. Although 17ß-hydroxysteroid dehydrogenase 4 (HSD17B4) has been implicated as an estradiol-inactivating enzyme, and its promoter sequence contains two putative NF-κB elements: it is currently unknown whether HSD17B4 is the link between inflammation, estradiol and proliferation in hepatoma cells. In this study, HepG2 cells were used to investigate the role of HSD17B4 in the proliferation of liver cancer cells treated with the NF-κB activator, tumor necrosis factor-alpha (TNF-α), with the inhibitor of NF-κB activation, pyrrolidinedithiocarbamate (PDTC), or with a related specific siRNA. We demonstrated that the human HSD17B4 gene is a target for NF-κB activation in inflammation-stimulated HepG2 cells. HSD17B4 is up-regulated via the binding of activated NF-κB to the distal NF-κB-responsive element via TNF-α stimulation, which then promotes cell proliferation by decreasing the levels of E2 and enhancing the expression of interleukin 6 (IL-6), cyclin D1 and proliferating cell nuclear antigen (PCAN). These results from HepG2 cells are consistent with the observation that HSD17B4 is highly expressed and activated NF-κB is highly co-localized with the NF-κB-responsive element of HSD17B4 in liver tumor tissues from HCC patients. Our findings indicate for the first time that HSD17B4 plays an important role in aggravated HCC progression and provides a novel therapeutic target for HCC.