RESUMO
The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Camundongos , Organogênese , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Mutations in genes associated with homologous recombination (HR) increase an individual's risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR. METHODS: Initial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s' role in RAD51 recruitment was determined by immunoblotting. RESULTS: Here, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding. CONCLUSIONS: Together, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Replicação do DNA , Rad51 Recombinase/metabolismo , Estresse Fisiológico , Animais , Linhagem Celular Tumoral , Cromossomos/genética , Cromossomos/metabolismo , Dano ao DNA , Reparo do DNA , Células Epiteliais/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Origem de ReplicaçãoRESUMO
BACKGROUND: Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2. METHODS: For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis. RESULTS: Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in re-expression of SIM2. CONCLUSION: Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/genética , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação , Ligação Proteica , Transdução de SinaisRESUMO
The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor-induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.
Assuntos
Benzo(a)pireno/metabolismo , Mama/citologia , Ritmo Circadiano , Adutos de DNA/metabolismo , Glândulas Mamárias Animais/citologia , Animais , Biomarcadores/metabolismo , Mama/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Ritmo Circadiano/genética , Citocromo P-450 CYP1A1/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos BiológicosRESUMO
We have previously demonstrated that the bHLH/PAS transcription factor, singleminded 2s (Sim2s), is required for proper mammary ductal morphogenesis and luminal epithelial differentiation. Furthermore, loss of Sim2s in breast cancer cells resulted in downregulation of epithelial markers and acquisition of a basal-like phenotype. The objective of this study was to further define the role of Sim2s in mammary differentiation. We found that Sim2s is developmentally regulated throughout mammary gland development with highest expression during lactation. Mammary glands from nulliparous mice expressing Sim2s driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter were morphologically indistinguishable from wild-type mice but displayed hallmarks of precocious lactogenic differentiation. These included elevated expression of the milk protein genes Wap and Csn2, and apical localization of the lactation marker Npt2b. Consistent with the in vivo results, Sim2s enhanced prolactin-mediated Csn2 expression in HC11 and CIT3 mouse mammary epithelial cells, and downregulation of Sim2s by shRNA in HC11 cells inhibited Csn2 expression. Chromatin immunoprecipitation (ChIP) analyses of the Csn2 gene found that Sim2s associates with the Csn2 promoter and re-ChIP experiments showed that Sim2s interacted with the RNA II polymerase (RNAPII) complex. Together, these data demonstrate, for the first time, that Sim2s is required for establishing and maintaining mammary gland differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular/genética , Lactação/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Lactação/fisiologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
The functionally differentiated mammary gland adapts to extreme levels of stress from increased demand for energy by activating specific protective mechanisms to support neonatal health. Here, we identify the breast tumor suppressor gene, single-minded 2 s (SIM2s) as a novel regulator of mitophagy, a key component of this stress response. Using tissue-specific mouse models, we found that loss of Sim2 reduced lactation performance, whereas gain (overexpression) of Sim2s enhanced and extended lactation performance and survival of mammary epithelial cells (MECs). Using an in vitro model of MEC differentiation, we observed SIM2s is required for Parkin-mediated mitophagy, which we have previously shown as necessary for functional differentiation. Mechanistically, SIM2s localizes to mitochondria to directly mediate Parkin mitochondrial loading. Together, our data suggest that SIM2s regulates the rapid recycling of mitochondria via mitophagy, enhancing the function and survival of differentiated MECs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Mitofagia , Camundongos , Feminino , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Células Epiteliais , Modelos Animais de Doenças , Ubiquitina-Proteína Ligases/genéticaRESUMO
Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.
Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Mitocôndrias/metabolismo , Fosforilação OxidativaRESUMO
Dysregulation of cellular metabolism is a hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. Here, we show that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the mitochondrial respiratory chain (MRC) to facilitate functional supercomplex (SC) formation. Loss of SIM2s expression disrupts SC formation through destabilization of MRC Complex III, leading to inhibition of electron transport, although Complex I (CI) activity is retained. A metabolomic analysis showed that knockout of SIM2s leads to a compensatory increase in ATP production through glycolysis and accelerated glutamine-driven TCA cycle production of NADH, creating a favorable environment for high cell proliferation. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC on metabolic adaptation and breast cancer progression.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transporte de Elétrons , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismoRESUMO
Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.Abbreviations: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Animais , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Approximately 70% of all breast cancers are estrogen receptor-positive (ER+ breast cancer), and endocrine therapy has improved survival for patients with ER+ breast cancer. However, up to half of these tumors recur within 20 years. Recurrent ER+ breast cancers develop resistance to endocrine therapy; thus, novel targets are needed to treat recurrent ER+ breast cancer. Here we report that semaphorin 7A (SEMA7A) confers significantly decreased patient survival rates in ER+ breast cancer. SEMA7A was hormonally regulated in ER+ breast cancer, but its expression did not uniformly decrease with antiestrogen treatments. Additionally, overexpression of SEMA7A in ER+ cell lines drove increased in vitro growth in the presence of estrogen deprivation, tamoxifen, and fulvestrant. In vivo, SEMA7A conferred primary tumor resistance to fulvestrant and induced lung metastases. Prosurvival signaling was identified as a therapeutic vulnerability of ER+SEMA7A+ tumors. We therefore propose that targeting this pathway with inhibitors of survival signaling such as venetoclax may prove efficacious for treating SEMA7A+ tumors. SIGNIFICANCE: SEMA7A predicts for and likely contributes to poor response to standard-of-care therapies, suggesting that patients with SEMA7A+ER+ tumors may benefit from alternative therapeutic strategies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/1/187/F1.large.jpg.
Assuntos
Antígenos CD/metabolismo , Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores de Estrogênio/metabolismo , Semaforinas/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Estrogênios/farmacologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , Semaforinas/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: An important component of time course microarray studies is the identification of genes that demonstrate significant time-dependent variation in their expression levels. Until recently, available methods for performing such significance tests required replicates of individual time points. This paper describes a replicate-free method that was developed as part of a study of the estrous cycle in the rat mammary gland in which no replicate data was collected. RESULTS: A temporal test statistic is proposed that is based on the degree to which data are smoothed when fit by a spline function. An algorithm is presented that uses this test statistic together with a false discovery rate method to identify genes whose expression profiles exhibit significant temporal variation. The algorithm is tested on simulated data, and is compared with another recently published replicate-free method. The simulated data consists both of genes with known temporal dependencies, and genes from a null distribution. The proposed algorithm identifies a larger percentage of the time-dependent genes for a given false discovery rate. Use of the algorithm in a study of the estrous cycle in the rat mammary gland resulted in the identification of genes exhibiting distinct circadian variation. These results were confirmed in follow-up laboratory experiments. CONCLUSION: The proposed algorithm provides a new approach for identifying expression profiles with significant temporal variation without relying on replicates. When compared with a recently published algorithm on simulated data, the proposed algorithm appears to identify a larger percentage of time-dependent genes for a given false discovery rate. The development of the algorithm was instrumental in revealing the presence of circadian variation in the virgin rat mammary gland during the estrous cycle.
Assuntos
Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Ritmo Circadiano/fisiologia , Glândulas Mamárias Animais/metabolismo , RatosRESUMO
The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 (Per1(ldc) and Per1(ldc)/Per2(ldc)). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/efeitos dos fármacos , Carcinógenos/toxicidade , Proteínas de Ciclo Celular/metabolismo , Fígado/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Indução Enzimática , Feminino , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Circadianas Period , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/genéticaRESUMO
17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/fisiologia , Fatores de Transcrição Sp/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição Sp/genética , Fatores de Transcrição Sp/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição Sp3/fisiologia , TransfecçãoRESUMO
There is increasing evidence that genomic instability is a prerequisite for cancer progression. Here we show that SIM2s, a member of the bHLH/PAS family of transcription factors, regulates DNA damage repair through enhancement of homologous recombination (HR), and prevents epithelial-mesenchymal transitions (EMT) in an Ataxia-telangiectasia mutated (ATM)-dependent manner. Mechanistically, we found that SIM2s interacts with ATM and is stabilized through ATM-dependent phosphorylation in response to IR. Once stabilized, SIM2s interacts with BRCA1 and supports RAD51 recruitment to the site of DNA damage. Loss of SIM2s through the introduction of shSIM2 or the mutation of SIM2s at one of the predicted ATM phosphorylation sites (S115) reduces HR efficiency through disruption of RAD51 recruitment, resulting in genomic instability and induction of EMT. The EMT induced by the mutation of S115 is characterized by a decrease in E-cadherin and an induction of the basal marker, K14, resulting in increased invasion and metastasis. Together, these results identify a novel player in the DNA damage repair pathway and provides a link in ductal carcinoma in situ progression to invasive ductal carcinoma through loss of SIM2s, increased genomic instability, EMT, and metastasis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transição Epitelial-Mesenquimal/genética , Recombinação Homóloga/genética , Animais , Proteína BRCA1/genética , Caderinas/genética , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Instabilidade Genômica/genética , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Fosforilação/genética , Rad51 Recombinase/genéticaRESUMO
Apparent full-length cDNA sequences coding for manganese superoxide dismutase (HvMnSOD) and extracellular superoxide dismutase (HvEC-SOD) were isolated from Hydra vulgaris in order to understand their expression and 3D structures; and explore their possibility of being used as for biomarkers for environmental stress and toxicity. The deduced HvMnSOD protein consists of 219 amino acids of which first 21 amino acids constitute a presumed mitochondria-targeting signal peptide whereas HvEC-SOD protein consists of 189 amino acids of which first 19 amino acids constitute a presumed signal peptide. Molecular model generated for HvMnSOD displayed the N-terminal long alpha antiparallel hairpin and the C-terminal mixed alpha/beta fold characteristic of MnSODs and that for HvEC-SOD displayed the characteristic CuZnSOD â-barrel fold. Hydrae subjected to thermal, starvation, metal and oxidative stress responded by regulating MnSOD and EC-SOD mRNA transcription. These results indicated that these genes are involved in the cellular stress response and (anti)oxidative processes triggered by stressor and contaminant exposure. Hence the expression of these SODs in hydra may have potential as molecular biomarkers for assessing stress, toxicity and pro-oxidant quality of chemicals and aquatic environmental quality.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Hydra/enzimologia , Superóxido Dismutase/genética , Animais , DNA Complementar/análise , Hydra/genética , Modelos Moleculares , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/química , Superóxido Dismutase/metabolismoRESUMO
Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Neoplasias da Mama/metabolismo , Proteínas Oncogênicas/deficiência , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica , Feminino , Xenoenxertos , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Metástase Neoplásica , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação , Podossomos/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Sp/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Estradiol/metabolismo , Humanos , Dados de Sequência Molecular , Neovascularização Patológica , Regiões Promotoras GenéticasRESUMO
Apparent full-length cDNA sequences coding respectively for mitochondrial (HvGPx41) and nuclear (HvGPx42) phospholipid hydroperoxide glutathione peroxidase were isolated from Hydra vulgaris. The cDNA sequences share total identity in their 3'-end and differ in their 5'-end. The protein-coding regions of the HvGPx41 and HvGPx42 cDNA encode polypeptides of 190 and 168 amino acids, including a TGA-encoded selenocysteine, respectively. Phylogenetic analysis showed that the HvGPx41 and HvGPx42 are clustered together along with other phospholipid hydroperoxide glutathione peroxidases (PHGPx) from several organisms. A tertiary structure model generated for the H. vulgaris PHGPx displayed the thioredoxin fold. Hydrae exposed to starvation, metal and oxidative stress responded by regulating their PHGPx mRNA transcription. These results indicated that the PHGPx gene is affected by the cellular stress response and (anti)oxidative processes triggered by stressor and contaminant exposure. Hence the expression of PHGPx mRNA in hydra may have potential use as molecular biomarkers for assessing stress, toxicity and pro-oxidant quality of chemicals and aquatic environmental quality.
Assuntos
Glutationa Peroxidase/metabolismo , Hydra/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa Peroxidase/genética , Dados de Sequência Molecular , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
INTRODUCTION: Voluntary physical activity levels are regulated by sex hormones. The purpose of this study was to determine the effect of the endocrine disruptor benzyl butyl phthalate (BBP) on the regulation of physical activity in mice. METHODS: Mouse dams were treated with 500 mg·kg·d of BBP or vehicle on gestation days 9-16. Pups were weaned and analyzed for voluntary physical activity levels, puberty development, sex hormone levels, and body composition during the 20-wk period. RESULTS: Seventy-three offspring from BBP-treated dams were studied (n = 43 males and n = 30 females). Endocrine disruption was indicated by decreased anogenital distances in BBP-treated male offspring at 10 (P = 0.001) and 20 wk (P = 0.038) and delayed vaginal openings in BBP-treated female offspring (P = 0.001). Further, there was a significant decrease in serum testosterone concentration in male mice between control and BBP at 10 wk (P = 0.039) and at 20 wk (P = 0.022). In female mice, there was a significant increase in serum testosterone concentration in BBP mice at 20 wk (P = 0.002) and a significant increase in estrogen (estradiol) concentrations at 20 wk in the control female mice (P = 0.015). Overall, BBP mice ran significantly less distance (males, P = 0.008; females, P = 0.042) than controls. Other than a significant increase in BBP-treated males in fat mass at 20 wk (P = 0.040), there was no significant decrease in weight, lean mass, or fat mass in either female or male mice, regardless of treatment. CONCLUSION: Maternal endocrine disruption altered hormone response, but not body composition in either sex of offspring, with a corresponding decreased activity throughout early adulthood in all offspring. These results suggest that exposure to common environmental endocrine disruptors in utero can reduce and alter physical activity levels in offspring.
Assuntos
Disruptores Endócrinos/efeitos adversos , Exposição Materna/efeitos adversos , Atividade Motora , Ácidos Ftálicos/efeitos adversos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testosterona/sangueRESUMO
1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF(3)) and several p-substituted phenyl analogues have been investigated as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Structure-activity studies in PPARgamma-dependent transactivation assays in MCF-7 breast cancer cells show that 5-20 micro M concentrations of compounds containing p-trifluoromethyl, t-butyl, cyano, dimethylamino, and phenyl groups were active, whereas p-methyl, hydrogen, methoxy, hydroxyl, or halogen groups were inactive as PPARgamma agonists. Induction of PPARgamma-dependent transactivation by 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2) and DIM-C-pPhCF(3) was inhibited in MCF-7 cells cotreated with the PPARgamma-specific antagonist N-(4'-aminopyridyl)-2-chloro-5-nitrobenzamide. In mammalian two-hybrid assays, DIM-C-pPhCF(3) and PGJ2 (5-20 micro M) induced interactions of PPARgamma with steroid receptor coactivator (SRC) 1, SRC2 (TIFII), and thyroid hormone receptor-associated protein 220 but not with SRC3 (AIB1). In contrast, DIM-C-pPhCF(3), but not PGJ2, induced interactions of PPARgamma with PPARgamma coactivator-1. C-substituted diindolylmethanes inhibit carcinogen-induced rat mammary tumor growth, induce differentiation in 3T3-L1 preadipocytes, inhibit MCF-7 cell growth and G(0)/G(1)-S phase progression, induce apoptosis, and down-regulate cyclin D1 protein and estrogen receptor alpha in breast cancer cells. These compounds are a novel class of synthetic PPARgamma agonists that induce responses in MCF-7 cells similar to those observed for PGJ2.