RESUMO
Receptor interacting protein kinase 1 (RIPK1) is an enzyme acting downstream of tumor necrosis factor alpha to control cell survival and death. RIPK1 expression has been reported to cause drug resistance in cancer cells, but so far, no published studies have investigated the role of RIPK1 in vitamin D signaling. In the present study, we investigated whether RIPK1 plays any roles in 1,25-dihydroxyvitamin D3 (1,25D3)-induced growth suppression. In our studies, RIPK1 decreased the transcriptional activity of vitamin D receptor (VDR) in luciferase reporter assays independent of its kinase activity, suggesting a negative role of RIPK1 in 1,25D3 action. RIPK1 also formed a complex with VDR, and deletion analyses mapped the RIPK1 binding region to the C-terminal ligand-binding domain of the VDR. Subcellular fractionation analyses indicated that RIPK1 increased VDR retention in the cytoplasm, which may account for its inhibition of VDR transcriptional activity. Consistent with the reporter analyses, 1,25D3-induced growth suppression was more pronounced in RIPK1-null MEFs and RIPK1-knockdown ovarian cancer cells than in control cells. Our studies have defined RIPK1 as a VDR repressor, projecting RIPK1 depletion as a potential strategy to increase the potency of 1,25D3 and its analogs for cancer intervention.
Assuntos
Proliferação de Células , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Calcitriol/metabolismo , Vitamina D/análogos & derivados , Linhagem Celular Tumoral , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Vitamina D/metabolismoRESUMO
Fe65 is a brain-enriched adaptor protein known for its role in the action of the Aß amyloid precursor protein in neuronal cells and Alzheimer's disease, but little is known about its functions in cancer cells. The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion. Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation. The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.
Assuntos
Cortactina/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Células HEK293 , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Imunoprecipitação , Lisina Acetiltransferase 5 , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25D3) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D3 suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D3, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D3 as well as a 1,25D3-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages. This article is part of a Special Issue entitled "Vitamin D Workshop".
Assuntos
Calcitriol/farmacologia , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Omento/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Calcitriol/metabolismo , Vitaminas/farmacologia , Animais , Carcinoma Epitelial do Ovário , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Omento/metabolismo , Omento/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
Obesity is a pandemic and major risk factor for cancers. The reduction of obesity would have been an effective strategy for cancer prevention, but the reality is that worldwide obesity has kept increasing for decades, remaining a major avoidable cancer risk secondary only to smoke. The present studies suggest that vitamin D may be an effective agent to reduce obesity-associated cancer risks in women. Molecular analyses showed that leptin increased human telomerase reverse transcriptase (hTERT) mRNA expression and cell growth through estrogen receptor-α (ERα) activation in ovarian cancer cells, which was suppressed by 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The suppression was compromised when miR-498 induction by the hormone was depleted with microRNA (miRNA) sponges. In mice, high-fat diet (HFD) stimulation of ovarian tumor growth was remarkably suppressed by 1,25(OH)2D3 analogue EB1089, which was also compromised by miR-498 sponges. EB1089 did not alter HFD-induced increase in serum leptin levels but increased miR-498 and decreased the diet-induced hTERT expression in tumors. Quantitative RT-PCR analyses revealed an inverse correlation between hTERT mRNA and miR-498 in response to 1,25(OH)2D3 in estrogen-sensitive ovarian, endometrial, and breast cancers. The studies suggest that miR-498-mediated hTERT downregulation is a key event mediating the anti-leptin activity of 1,25(OH)2D3 in estrogen-sensitive tumors in women.
Assuntos
Neoplasias da Mama/genética , Leptina/biossíntese , MicroRNAs/biossíntese , Neoplasias Ovarianas/genética , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dieta Hiperlipídica , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leptina/metabolismo , Camundongos , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Telomerase/biossíntese , Telomerase/genética , Vitamina D/administração & dosagemRESUMO
Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).
Assuntos
Calcitriol/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Neoplasias/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/biossíntese , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Feminino , Genoma , Humanos , Camundongos , Camundongos Nus , MicroRNAs/fisiologia , Mutagênese , Análise de Sequência com Séries de Oligonucleotídeos , RNA não Traduzido/metabolismoRESUMO
The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.
Assuntos
Baratas/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/agonistas , Animais , Encéfalo/metabolismo , Corpora Allata/metabolismo , Feminino , Gânglios/metabolismo , Trato Gastrointestinal/metabolismo , Masculino , Ovário/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Testículo/metabolismoRESUMO
Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a lambda ZAP-cDNA library prepared from the corpora allata of the D. punctata. As an approach to testing the function of this receptor in vivo, the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.