RESUMO
Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in cancer progression is not completely understood. Using stable isotope-resolved metabolomics analysis, we showed that the epithelial mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate levels. The treatment with cell-permeable succinate induced mesenchymal phenotypes in mammary epithelial cells and enhanced cancer cell stemness. Chromatin immunoprecipitation and sequence analysis showed that elevated cytoplasmic succinate levels were sufficient to reduce global 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional repression of EMT-related genes. We showed that expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated with elevation of cytoplasmic succinate during the EMT process. Silencing of PLOD2 expression in breast cancer cells reduced succinate levels and inhibited cancer cell mesenchymal phenotypes and stemness, which was accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced cells, suggesting that PLOD2 promotes cancer progression at least partially through succinate. These results reveal the previously unidentified function of succinate in enhancing cancer cell plasticity and stemness.
Assuntos
Neoplasias , Ácido Succínico , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Succinatos , HumanosRESUMO
Retinoid orphan nuclear receptor alpha (RORα) is a member of the orphan nuclear factor family and regulates gene expression by binding to ROR response elements (ROREs). RORα has been identified as a potential tumor suppressor; however, how downregulation of RORα promotes cancer progression is not fully understood. Here, we showed that protein levels of RORα were downregulated during the Snail-, Twist-, or transforming growth factor-ß-induced epithelial-mesenchymal transition (EMT). We found that silencing of RORα induced expression of mesenchymal markers in MCF10A cells, accompanied by enhanced cell invasion, migration, and mammosphere formation. Furthermore, ectopic expression of RORα suppressed transforming growth factor-ß-induced EMT processes in MCF10A and HMLE cells. These results indicate that downregulation of RORα is crucial for the induction of EMT in mammary epithelial cells. By analyzing gene expression profiles in control and RORα-expressing cells, we also identified Snail, a key regulator of EMT, as a potential target of RORα. We show that RORα expression significantly inhibits Snail transcription in breast cancer cells. Chromatin immunoprecipitation analysis demonstrated that RORα bound to the ROREs in promoter region of SNAI1 gene, and using the luciferase reporter assay, we showed that binding to the ROREs was critical for RORα to repress Snail transcription. Finally, rescue experiments substantiated that Snail mediates RORα function in suppressing EMT and mammosphere formation. These results reveal a novel function of RORα in suppressing EMT and identify Snail as a direct target of RORα in mammary epithelial cells.
Assuntos
Transição Epitelial-Mesenquimal , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptores Nucleares Órfãos , Fatores de Transcrição da Família Snail , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Elementos de Resposta , Receptor alfa de Ácido Retinoico/genética , Retinoides , Fatores de Transcrição da Família Snail/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.
Assuntos
Plaquetas/metabolismo , Neoplasias da Mama/metabolismo , Colágeno/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Transição Epitelial-Mesenquimal , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Amplificação de Genes , Proteínas de Choque Térmico HSP47/genética , Humanos , Camundongos SCID , Metástase NeoplásicaRESUMO
Breast cancer development is associated with macrophage infiltration and differentiation in the tumor microenvironment. Our previous study highlights the crucial function of reactive oxygen species (ROS) in enhancing macrophage infiltration during the disruption of mammary tissue polarity. However, the regulation of ROS and ROS-associated macrophage infiltration in breast cancer has not been fully determined. Previous studies identified retinoid orphan nuclear receptor alpha (RORα) as a potential tumor suppressor in human breast cancer. In the present study, we showed that retinoid orphan nuclear receptor alpha (RORα) significantly decreased ROS levels and inhibited ROS-mediated cytokine expression in breast cancer cells. RORα expression in mammary epithelial cells inhibited macrophage infiltration by repressing ROS generation in the co-culture assay. Using gene co-expression and chromatin immunoprecipitation (ChIP) analyses, we identified complex I subunits NDUFS6 and NDUFA11 as RORα targets that mediated its function in suppressing superoxide generation in mitochondria. Notably, the expression of RORα in 4T1 cells significantly inhibited cancer metastasis, reduced macrophage accumulation, and enhanced M1-like macrophage differentiation in tumor tissue. In addition, reduced RORα expression in breast cancer tissue was associated with an increased incidence of cancer metastasis. These results provide additional insights into cancer-associated inflammation, and identify RORα as a potential target to suppress ROS-induced mammary tumor progression.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Neoplasias/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores , Respiração Celular , Citocinas/metabolismo , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.
Assuntos
Antígeno AC133/metabolismo , Sistemas de Liberação de Medicamentos/métodos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Nanopartículas/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células RAW 264.7 , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Loss of epithelial cell polarity promotes cell invasion and cancer dissemination. Therefore, identification of factors that disrupt polarized acinar formation is crucial. Reactive oxygen species (ROS) drive cancer progression and promote inflammation. Here, we show that the non-polarized breast cancer cell line T4-2 generates significantly higher ROS levels than polarized S1 and T4R cells in three-dimensional (3D) culture, accompanied by induction of the nuclear factor κB (NF-κB) pathway and cytokine expression. Minimizing ROS in T4-2 cells with antioxidants reestablished basal polarity and inhibited cell proliferation. Introducing constitutively activated RAC1 disrupted cell polarity and increased ROS levels, indicating that RAC1 is a crucial regulator that links cell polarity and ROS generation. We also linked monocyte infiltration with disruption of polarized acinar structure using a 3D co-culture system. Gain- and loss-of-function experiments demonstrated that increased ROS in non-polarized cells is necessary and sufficient to enhance monocyte recruitment. ROS also induced cytokine expression and NF-κB activity. These results suggest that increased ROS production in mammary epithelial cell leads to disruption of cell polarity and promotes monocyte infiltration.
Assuntos
Técnicas de Cultura de Células/métodos , Polaridade Celular , Células Epiteliais/citologia , Glândulas Mamárias Humanas/citologia , Monócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.
Assuntos
Anoikis , Neoplasias da Mama/metabolismo , Colágeno Tipo VIII/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular , Linhagem Celular Tumoral , Colágeno Tipo VIII/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Proteínas de Membrana/genética , Camundongos SCID , Interferência de RNA , Terapêutica com RNAi/métodos , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
BACKGROUND: Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated. METHODS: Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis. RESULTS: Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models. CONCLUSION: These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.
Assuntos
Neoplasias da Mama/enzimologia , Colágeno/metabolismo , Neoplasias Pulmonares/enzimologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Invasividade Neoplásica , Fenótipo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/genética , Prognóstico , Prolil Hidroxilases/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Retinoic acid receptor-related orphan receptor alpha (RORα), a candidate tumor suppressor, is prevalently downregulated or lost in malignant breast cancer cells. However, the mechanisms of how RORα expression is regulated in breast epithelial cells remain incompletely understood. Protein arginine N-methyltransferase 5 (PRMT5), a type II methyltransferase catalyzing the symmetric methylation of the amino acid arginine in target proteins, was reported to regulate protein stability. To study whether and how PRMT5 regulates RORα, we examined the direct interaction between RORα and PRMT5 by immunoprecipitation and GST pull-down assays. The results showed that PRMT5 directly bound to RORα, and PRMT5 mainly symmetrically dimethylated the DNA-binding domain (DBD) but not the ligand-binding domain (LBD) of RORα. To investigate whether RORα protein stability is regulated by PRMT5, we transfected HEK293FT cells with RORα and PRMT5-expressing or PRMT5-silencing (shPRMT5) vectors and then examined RORα protein stability by a cycloheximide chase assay. The results showed that PRMT5 increased RORα protein stability, while silencing PRMT5 accelerated RORα protein degradation. In PRMT5-silenced mammary epithelial cells, RORα protein expression was decreased, accompanied by an enhanced epithelial-mesenchymal transition morphology and cell invasion and migration abilities. In PRMT5-overexpressed mammary epithelial cells, RORα protein was accumulated, and cell invasion was suppressed. These findings revealed a novel mechanism by which PRMT5 regulates RORα protein stability.
RESUMO
Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.
Assuntos
Implantação do Embrião/genética , Glicoproteínas/fisiologia , Prenhez , Proteínas Secretadas pela Próstata/fisiologia , Útero/metabolismo , Animais , Decídua/metabolismo , Implantação do Embrião/fisiologia , Implantação Tardia do Embrião/genética , Feminino , Regulação da Expressão Gênica , Idade Gestacional , Glicoproteínas/genética , Glicoproteínas/metabolismo , Camundongos , Comunicação Parácrina/genética , Comunicação Parácrina/fisiologia , Gravidez , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/metabolismo , Pseudogravidez/genética , Pseudogravidez/metabolismo , Pseudogravidez/patologia , Fatores de Tempo , Distribuição Tecidual , Inibidor da Tripsina Pancreática de KazalRESUMO
Tumor cell invasion and metastasis are the most complicated problems of cancer medicine and cancer biology, and cancer becomes a fatal disease because of cancer cell invasion and metastasis. The mechanisms of invasion and metastasis remain elusive, but tumor cell invasion still is the hotspot of research. This review summarizes the development of researching in tumor cell invasion.
Assuntos
Neoplasias/patologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Animais , Proteínas Ativadoras de GTPase , Humanos , Invasividade Neoplásica , Neoplasias/genéticaRESUMO
Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC) and HER2 positive breast cancer. By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent docetaxel and doxorubicin in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Animais , Linhagem Celular , Colágeno/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ácidos Cetoglutáricos/metabolismo , Camundongos , Camundongos SCID , Metástase Neoplásica/prevenção & controle , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The extracellular matrix (ECM) is a determining factor in the tumor microenvironment that restrains or promotes malignant growth. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. A transcription network analysis showed that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or noninvasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Coexpression network analysis also showed that levels of microRNA(miR)-29b and -29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and -29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins.
Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Matriz Extracelular/genética , Redes Reguladoras de Genes/genética , Proteínas de Choque Térmico HSP47/fisiologia , Invasividade Neoplásica/genética , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , MicroRNAs/fisiologia , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; "active" targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.
Assuntos
MicroRNAs/antagonistas & inibidores , Nanopartículas/metabolismo , RNA Interferente Pequeno/administração & dosagem , Neoplasias de Mama Triplo Negativas/terapia , Animais , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Nanotecnologia , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Retinoic acid receptor-related orphan nuclear receptor alpha (RORα) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which RORα binds to E2F1 to inhibit cell cycle progression. We showed that RORα bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of RORα inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by RORα. Importantly, RORα levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing RORα in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between RORα and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis.
Assuntos
Fator de Transcrição E2F1/metabolismo , Células Epiteliais/metabolismo , Histona Desacetilase 1/genética , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Acetilação , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Regulação da Expressão Gênica , Células HEK293 , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , Morfogênese , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ligação Proteica , Transcrição GênicaRESUMO
Extracellular matrix (ECM), a major component of the cellular microenvironment, plays critical roles in normal tissue morphogenesis and disease progression. Binding of ECM to membrane receptor proteins, such as integrin, discoidin domain receptors, and dystroglycan, elicits biochemical and biomechanical signals that control cellular architecture and gene expression. These ECM signals cooperate with growth factors and hormones to regulate cell migration, differentiation, and transformation. ECM signaling is tightly regulated during normal mammary gland development. Deposition and alignment of fibrillar collagens direct migration and invasion of mammary epithelial cells during branching morphogenesis. Basement membrane proteins are required for polarized acinar morphogenesis and milk protein expression. Deregulation of ECM proteins in the long run is sufficient to promote breast cancer development and progression. Recent studies demonstrate that the integrated biophysical and biochemical signals from ECM and soluble factors are crucial for normal mammary gland development as well as breast cancer progression.
Assuntos
Neoplasias da Mama/metabolismo , Matriz Extracelular/metabolismo , Glândulas Mamárias Humanas/embriologia , Glândulas Mamárias Humanas/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Matriz Extracelular/patologia , Feminino , Humanos , Transdução de Sinais/fisiologiaRESUMO
The microenvironment of cells controls their phenotype, and thereby the architecture of the emerging multicellular structure or tissue. We have reported more than a dozen microenvironmental factors whose signaling must be integrated in order to effect an organized, functional tissue morphology. However, the factors that prevent integration of signaling pathways that merge form and function are still largely unknown. We have identified nuclear factor kappa B (NFkB) as a transcriptional regulator that disrupts important microenvironmental cues necessary for tissue organization. We compared the gene expression of organized and disorganized epithelial cells of the HMT-3522 breast cancer progression series: the non-malignant S1 cells that form polarized spheres ('acini'), the malignant T4-2 cells that form large tumor-like clusters, and the 'phenotypically reverted' T4-2 cells that polarize as a result of correction of the microenvironmental signaling. We identified 180 genes that display an increased expression in disorganized compared to polarized structures. Network, GSEA and transcription factor binding site analyses suggested that NFkB is a common activator for the 180 genes. NFkB was found to be activated in disorganized breast cancer cells, and inhibition of microenvironmental signaling via EGFR, beta1 integrin, MMPs, or their downstream signals suppressed its activation. The postulated role of NFkB was experimentally verified: Blocking the NFkB pathway with a specific chemical inhibitor or shRNA induced polarization and inhibited invasion of breast cancer cells in 3D cultures. These results may explain why NFkB holds promise as a target for therapeutic intervention: Its inhibition can reverse the oncogenic signaling involved in breast cancer progression and integrate the essential microenvironmental control of tissue architecture.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , NF-kappa B/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Imageamento Tridimensional/métodos , Análise em Microsséries , NF-kappa B/genética , Fenótipo , Transdução de Sinais , Ativação Transcricional , Microambiente TumoralRESUMO
Inactivation of tumor suppressors and inhibitory microenvironmental factors is necessary for breast cancer invasion; therefore, identifying those suppressors and factors is crucial not only to advancing our knowledge of breast cancer, but also to discovering potential therapeutic targets. By analyzing gene expression profiles of polarized and disorganized human mammary epithelial cells in a physiologically relevant three-dimensional (3D) culture system, we identified retinoid orphan nuclear receptor alpha (RORα) as a transcription regulator of semaphorin 3F (SEMA3F), a suppressive microenvironmental factor. We showed that expression of RORα was downregulated in human breast cancer tissue and cell lines, and that reduced mRNA levels of RORα and SEMA3F correlated with poor prognosis. Restoring RORα expression reprogrammed breast cancer cells to form noninvasiveness structures in 3D culture and inhibited tumor growth in nude mice, accompanied by enhanced SEMA3F expression. Inactivation of RORα in nonmalignant human mammary epithelial cells inhibited SEMA3F transcription and impaired polarized acinar morphogenesis. Using chromatin immunoprecipitation and luciferase reporter assays, we showed that transcription of SEMA3F is directly regulated by RORα. Knockdown of SEMA3F in RORα-expressing cancer cells rescued the aggressive 3D phenotypes and tumor invasion. These findings indicate that RORα is a potential tumor suppressor and inhibits tumor invasion by inducing suppressive cell microenvironment.
Assuntos
Neoplasias da Mama/etiologia , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Polaridade Celular , Microambiente Celular , Feminino , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Proteínas do Tecido Nervoso/fisiologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/análise , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , PrognósticoRESUMO
Previously we have found that DNA vaccine, pCMV4-rZPC' can generate specific antibodies against rabbit ZPC (amino acid 263-415, rZPC'), which binds to ovarian ZP and leads to a significant reduction of fertility in vivo. The purpose of this study was to evaluate the effect of antisera from pCMV4-rZPC(')-immunized mice on sperm-oocyte interaction in vitro. The effect of antisera from DNA vaccine-immunized mice on fertilization and early embryonic development was studied using an in vitro fertilization system. The results showed that the antisera supplemented in fertilization medium (10%, v/v) significantly decreased the rate of fertilization compared to that of control groups (P<0.05); whereas the antisera showed no significant effect on the rate of fertilization when ZP-free eggs were used. Moreover, the antisera pre-neutralized with mouse soluble zona pellucida lost the capacity to inhibit fertilization when compared with that of control groups. In addition, the antisera showed no detrimental effect on early developmental potential of mouse embryos in vitro. Taken together, our study provided herein direct evidence showing that antisera generated by DNA vaccine can block sperm-egg recognition during fertilization via targeting the oocyte ZP proteins.
Assuntos
Autoanticorpos/administração & dosagem , Autoanticorpos/imunologia , Anticoncepção , Proteínas do Ovo/antagonistas & inibidores , Imunização Passiva/métodos , Glicoproteínas de Membrana/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Vacinas Anticoncepcionais/imunologia , Vacinas de DNA/imunologia , Animais , Células Cultivadas , Proteínas do Ovo/imunologia , Feminino , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óvulo/imunologia , Receptores de Superfície Celular/imunologia , Espermatozoides/imunologia , Vacinas Anticoncepcionais/administração & dosagem , Vacinas de DNA/administração & dosagem , Glicoproteínas da Zona PelúcidaRESUMO
Secretory protein proline-rich acid protein 1 (PRAP1) is abundantly expressed in late pregnant uterus. However, regulation and function of PRAP1 in pregnant uterus is still elusive. We firstly reported differential expression of PRAP1 in peri-implantation uteri and its localization in endometrial epithelia. Expression of PRAP1 in uterus was induced by 17ß-estradiol and its expression showed a negative correlation with that of class Ihistone deacetylases (HDACs) in isolated endometrial epithelia. PRAP1 was increased by HDACs inhibitor sodium butyrate treatment, while decreased significantly by estrogen receptor inhibitor ICI182,780 via up-regulating class IHDACs. Number of implanted embryos was decreased in mice immunized with pCR3.1-PRAP1 or injected with rabbit anti-PRAP1 antibody. DNA immunization or antibody injection could affect apoptosis and expression of cytokines (IL-4, IFN-γ). In conclusion, both 17ß-estradiol and class IHDACs are involved in modulating PRAP1 expression in peri-implantation uteri. Preliminary functional research indicates that neutralizing PRAP1 protein causes reduction of implanted embryos.