RESUMO
The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via hypoxia-inducible factor 1 (HIF-1α) and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation, and interaction with von Hippel-Lindau protein (pVHL), resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of endoplasmic reticulum (ER) stress and cancer cell apoptosis mediated through C/EBP homologous protein (CHOP). HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects. Human breast cancers with high levels of HIF-1α contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.
Assuntos
Neoplasias da Mama/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclo do Ácido Cítrico , Estresse do Retículo Endoplasmático , Feminino , Glicólise , Glicosilação , Humanos , Hidroxilação , Estimativa de Kaplan-Meier , Camundongos , Camundongos Nus , N-Acetilglucosaminiltransferases/metabolismo , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Fator de Transcrição CHOP/metabolismoRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. METHODS: In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. RESULTS: We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells. CONCLUSION: We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Células Neoplásicas Circulantes , Animais , Biomarcadores Tumorais , Fibroblastos Associados a Câncer/patologia , Contagem de Células , Análise por Conglomerados , Feminino , Humanos , Camundongos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Microambiente TumoralRESUMO
Interfering with microtubule dynamics is a well-established strategy in cancer treatment; however, many microtubule-targeting agents are associated with drug resistance and adverse effects. Substantial evidence points to ATP-binding cassette (ABC) transporters as critical players in the development of resistance. Herein, we demonstrate the efficacy of DJ95 (2-(1H-indol-6-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine), a novel tubulin inhibitor, in a variety of cancer cell lines, including malignant melanomas, drug-selected resistant cell lines, specific ABC transporter-overexpressing cell lines, and the National Cancer Institute 60 cell line panel. DJ95 treatment inhibited cancer cell migration, caused morphologic changes to the microtubule network foundation, and severely disrupted mitotic spindle formation of mitotic cells. The high-resolution crystal structure of DJ95 in complex with tubulin protein and the detailed molecular interactions confirmed its direct binding to the colchicine site. In vitro pharmacological screening of DJ95 using SafetyScreen44 (Eurofins Cerep-Panlabs) revealed no significant off-target interactions, and pharmacokinetic analysis showed that DJ95 was maintained at therapeutically relevant plasma concentrations for up to 24 hours in mice. In an A375 xenograft model in nude mice, DJ95 inhibited tumor growth and disrupted tumor vasculature in xenograft tumors. These results demonstrate that DJ95 is potent against a variety of cell lines, demonstrated greater potency to ABC transporter-overexpressing cell lines than existing tubulin inhibitors, directly targets the colchicine binding domain, exhibits significant antitumor efficacy, and demonstrates vascular-disrupting properties. Collectively, these data suggest that DJ95 has great potential as a cancer therapeutic, particularly for multidrug resistance phenotypes, and warrants further development. SIGNIFICANCE STATEMENT: Paclitaxel is a widely used tubulin inhibitor for cancer therapy, but its clinical efficacy is often limited by the development of multidrug resistance. In this study, we reported the preclinical characterization of a new tubulin inhibitor DJ95, and demonstrated its abilities to overcome paclitaxel resistance, disrupt tumor vasculature, and exhibit significant antitumor efficacy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Tubulina (Proteína)/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colchicina/metabolismo , Cristalografia por Raios X , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/administração & dosagem , Imidazóis/química , Imidazóis/farmacologia , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Nus , Piridinas/administração & dosagem , Piridinas/química , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Metastasis is responsible for a significant number of breast cancer-related deaths. Hypoxia, a primary driving force of cancer metastasis, induces the expression of BHLHE40, a transcription regulator. This study aimed to elucidate the function of BHLHE40 in the metastatic process of breast cancer cells. METHODS: To define the role of BHLHE40 in breast cancer, BHLHE40 expression was knocked down by a lentiviral construct expressing a short hairpin RNA against BHLHE40 or knocked out by the CRISPR/Cas9 editing system. Orthotopic xenograft and experimental metastasis (tail vein injection) mouse models were used to analyze the role of BHLHE40 in lung metastasis of breast cancer. Global gene expression analysis and public database mining were performed to identify signaling pathways regulated by BHLHE40 in breast cancer. The action mechanism of BHLHE40 was examined by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (CoIP), exosome analysis, and cell-based assays for metastatic potential. RESULTS: BHLHE40 knockdown significantly reduced primary tumor growth and lung metastasis in orthotopic xenograft and experimental metastasis models of breast cancer. Gene expression analysis implicated a role of BHLHE40 in transcriptional activation of heparin-binding epidermal growth factor (HBEGF). ChIP and CoIP assays revealed that BHLHE40 induces HBEGF transcription by blocking DNA binding of histone deacetylases (HDAC)1 and HDAC2. Cell-based assays showed that HBEGF is secreted through exosomes and acts to promote cell survival and migration. Public databases provided evidence linking high expression of BHLHE40 and HBEGF to poor prognosis of triple-negative breast cancer. CONCLUSION: This study reveals a novel role of BHLHE40 in promoting tumor cell survival and migration by regulating HBEGF secretion.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Proteínas de Homeodomínio/genética , Neoplasias Pulmonares/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo , Interferência de RNA , Terapêutica com RNAi/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF-7 breast cancer cells and a patient-derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 µg per day) led to increased tumor size of MCF-7 xenografts and ER+ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF-7 cell proliferation or induction of canonical estrogen-responsive genes (pS2 and progesterone receptor), in contrast to 17ß-estradiol (E2) treatment. MCF-7 and PDX-derived mammospheres exhibited increased size and up-regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA-MB-231 (ER- ) mammospheres. As tumor-initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti-estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non-classic estrogen response or an ER-independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/genética , Carcinógenos/toxicidade , Disruptores Endócrinos/toxicidade , Células-Tronco Neoplásicas/efeitos dos fármacos , Parabenos/toxicidade , Receptores de Estrogênio/genética , Animais , Carcinógenos/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Ovariectomia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: While aberrant activation of the chromatin-remodeling SWI/SNF complexes has been associated with cancer development and progression, the role of each subunit in tumor cells is poorly defined. This study is aimed to characterize the role of SMARCE1/BAF57 in regulating metastasis of breast cancer cells. METHODS: Genetic approaches and chemical inhibitors were used to manipulate the activities of SMARCE1 and its downstream targets in multiple breast cancer cell lines. Xenograft mouse models were used to analyze the role of SMARCE1 in lung metastasis in vivo. Nonadherent culture conditions were used to elucidate the role of SMARCE1 in regulating anoikis. Chromatin immunoprecipitation (ChIP), immunoprecipitation, and immunoblotting assays were designed to dissect the mechanism of action of SMARCE1. Public databases were used to investigate the relationship between SMARCE1 deregulation and breast cancer prognosis. RESULTS: SMARCE1 knockdown reduced lung metastasis of breast cancer cells and sensitized tumor cells to anoikis. In response to loss of attachment, SMARCE1 interacted with and potentiated transcriptional activity of HIF1A, resulting in rapid PTK2 activation. Both HIF1A and PTK2 were indispensable for SMARCE1-mediated protection against anoikis by promoting activation of ERK and AKT pathways while suppressing the expression of pro-apoptotic BIM protein. Expression data analysis of a large cohort of human breast tumors revealed that high expression of SMARCE1 or PTK2 is associated with poor prognosis and tumor relapse, and PTK2 expression is positively correlated with SMARCE1 expression in basal-like and luminal B subtypes of breast tumors. CONCLUSIONS: SMARCE1 plays an essential role in breast cancer metastasis by protecting cells against anoikis through the HIF1A/PTK2 pathway. SMARCE1-mediated PTK2 activation likely plays a key role in promoting metastasis of basal-like and luminal B subtype of breast tumors.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Anoikis , Sequência de Bases , Sítios de Ligação , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Feminino , Quinase 1 de Adesão Focal/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Regiões Promotoras Genéticas , Transdução de Sinais , Ativação TranscricionalRESUMO
BACKGROUND: Hypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion. METHODS: We used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f (+) cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49f(neg or low) cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients. RESULTS: We report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes. CONCLUSIONS: Our data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f (+) cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Integrina alfa6/genética , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Regulação para Baixo/genética , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Integrina alfa6/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Transcrição GênicaRESUMO
Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Receptor ErbB-4/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Endocitose , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lactação/genética , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/metabolismo , Gravidez , Transdução de SinaisRESUMO
ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.
Assuntos
Anoikis , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Mamárias Animais/metabolismo , Receptor ErbB-2/biossíntese , Animais , Adesão Celular/genética , Feminino , Deleção de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , Estresse Fisiológico/genéticaRESUMO
INTRODUCTION: In breast cancer, distinct expression profiles of microRNAs (miRNAs) have been associated with molecular subgroups and clinicopathological characteristics, implicating a diagnostic and prognostic role of miRNAs. However, the biological functions of deregulated miRNAs in tumor progression are not yet completely defined. In this study, we investigated the function of miR-18a in regulating breast cancer metastasis through the hypoxia-inducible factor 1α (HIF1A)-dependent hypoxic response. METHODS: An orthotopic metastatic breast cancer xenograft model (MDA-MB-231 cells) was used to identify miRNAs associated with spontaneous lung metastasis. The function of miR-18a in regulating HIF1A expression, as well as cellular responses to hypoxia and metastasis, were then studied in vitro and in vivo by assessing ectopic miR-18a expression or miR-18a inhibition. miRNA-mRNA interactions (AGO2 immunoprecipitation and 3' untranslated region Luciferase reporter assays), gene expression (quantitative PCR and microarray), cell migration and invasion, and cell growth were assessed under normoxic or hypoxic conditions, complemented by orthotopic xenograft of tumor cells to the mammary fat pad to investigate the effect of modulating miR-18a expression on primary tumor growth and lung metastasis. Last, clinically relevant correlations between miR-18a, HIF1A, hypoxia-responsive gene expression and distant metastasis-free survival (DMFS) were assessed using published expression array breast tumors data sets. RESULTS: miRNAs encoded by the MIR17HG gene were downregulated in lung metastases compared to primary tumors. Ectopic expression of miR-18a, a MIR17HG family member, in a metastatic variant of MDA-MB-231 cells reduced primary tumor growth and lung metastasis, whereas miR-18a inhibition in the parental cells promoted tumor growth and lung metastasis. We identified HIF1A as a direct target of miR-18a. Modulating miR-18a expression significantly affected hypoxic gene expression, cell invasiveness and sensitivity to anoikis and hypoxia in vitro in a HIF1A-dependent manner. Analysis of previously published data revealed that higher expression of HIF1A and a panel of hypoxic genes is associated with shorter DMFS interval in patients with basal-like breast tumors, and that, within this subtype, miR-18a expression is inversely correlated with hypoxic gene expression. Together, these data support a role of miR-18a in repressing distant metastasis through a HIF1A-dependent pathway. CONCLUSIONS: The results of this study reveal a novel role for miR-18a in targeting HIF1A and repressing metastasis of basal-like breast tumors.
Assuntos
Neoplasias da Mama/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/metabolismo , MicroRNAs/fisiologia , Neoplasia de Células Basais/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasia de Células Basais/genética , Neoplasia de Células Basais/secundário , Interferência de RNARESUMO
BACKGROUND: Improving survival for patients diagnosed with metastatic disease and overcoming chemoresistance remain significant clinical challenges in treating breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of therapeutically targetable receptors (ER/PR/HER2). TNBC therapy includes a combination of cytotoxic chemotherapies, including microtubule-targeting agents (MTAs) like paclitaxel (taxane class) or eribulin (vinca class); however, there are currently no FDA-approved MTAs that bind to the colchicine-binding site. Approximately 70 % of patients who initially respond to paclitaxel will develop taxane resistance (TxR). We previously reported that an orally bioavailable colchicine-binding site inhibitor (CBSI), VERU-111, inhibits TNBC tumor growth and treats pre-established metastatic disease. To further improve the potency and metabolic stability of VERU-111, we created next-generation derivatives of its scaffold, including 60c. RESULTS: 60c shows improved in vitro potency compared to VERU-111 for taxane-sensitive and TxR TNBC models, and suppress TxR primary tumor growth without gross toxicity. 60c also suppressed the expansion of axillary lymph node metastases existing prior to treatment. Comparative analysis of excised organs for metastasis between 60c and VERU-111 suggested that 60c has unique anti-metastatic tropism. 60c completely suppressed metastases to the spleen and was more potent to reduce metastatic burden in the leg bones and kidney. In contrast, VERU-111 preferentially inhibited liver metastases and lung metastasis repression was similar. Together, these results position 60c as an additional promising CBSI for TNBC therapy, particularly for patients with TxR disease.
Assuntos
Colchicina , Neoplasias de Mama Triplo Negativas , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Humanos , Feminino , Animais , Colchicina/farmacologia , Linhagem Celular Tumoral , Sítios de Ligação , Moduladores de Tubulina/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Piridinas/farmacologia , Camundongos Nus , Camundongos , Carga Tumoral/efeitos dos fármacosRESUMO
INTRODUCTION: Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression. METHODS: The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme. RESULTS: AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors. CONCLUSIONS: These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.
Assuntos
Neoplasias da Mama/tratamento farmacológico , DNA Catalítico/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Metaloproteinase 9 da Matriz/biossíntese , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Invasividade Neoplásica/genética , Metástase NeoplásicaRESUMO
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RESUMO
Corticosteroids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and are routinely prescribed to breast cancer patients undergoing chemotherapy treatment to alleviate side effects. Triple-negative breast cancers (TNBCs) account for 15% to 20% of diagnoses and lack expression of estrogen and progesterone receptors as well as amplified HER2, but they often express high GR levels. GR is a mediator of TNBC progression to advanced metastatic disease; however, the mechanisms underpinning this transition to more aggressive behavior remain elusive. We previously showed that tissue/cellular stress (hypoxia, chemotherapies) as well as factors in the tumor microenvironment (transforming growth factor ß [TGF-ß], hepatocyte growth factor [HGF]) activate p38 mitogen-activated protein kinase (MAPK), which phosphorylates GR on Ser134. In the absence of ligand, pSer134-GR further upregulates genes important for responses to cellular stress, including key components of the p38 MAPK pathway. Herein, we show that pSer134-GR is required for TNBC metastatic colonization to the lungs of female mice. To understand the mechanisms of pSer134-GR action in the presence of GR agonists, we examined glucocorticoid-driven transcriptomes in CRISPR knock-in models of TNBC cells expressing wild-type or phospho-mutant (S134A) GR. We identified dexamethasone- and pSer134-GR-dependent regulation of specific gene sets controlling TNBC migration (NEDD9, CSF1, RUNX3) and metabolic adaptation (PDK4, PGK1, PFKFB4). TNBC cells harboring S134A-GR displayed metabolic reprogramming that was phenocopied by pyruvate dehydrogenase kinase 4 (PDK4) knockdown. PDK4 knockdown or chemical inhibition also blocked cancer cell migration. Our results reveal a convergence of GR agonists (ie, host stress) with cellular stress signaling whereby pSer134-GR critically regulates TNBC metabolism, an exploitable target for the treatment of this deadly disease.
Assuntos
Receptores de Glucocorticoides , Neoplasias de Mama Triplo Negativas , Animais , Feminino , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Fosfofrutoquinase-2/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Microambiente TumoralRESUMO
INTRODUCTION: Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1α is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1α is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer. METHODS: Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus ß-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis. RESULTS: Efficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1α also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells. CONCLUSION: These results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1α directly regulates breast TIC activity in vivo.
Assuntos
Proliferação de Células , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Caspase 3/metabolismo , Separação Celular , Fator de Crescimento Epidérmico/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Fosfoproteínas/metabolismo , Esferoides Celulares/metabolismo , Transativadores/metabolismo , Carga Tumoral , Células Tumorais CultivadasRESUMO
HER2+ breast cancer accounts for 15% of all breast cancer cases. Current frontline therapy for HER2+ metastatic breast cancer relies on targeted antibodies, trastuzumab and pertuzumab, combined with microtubule inhibitors in the taxane class (paclitaxel or docetaxel). It is well known that the clinical efficacy of taxanes is limited by the development of chemoresistance and hematological and neurotoxicities. The colchicine-binding site inhibitors (CBSIs) are a class of promising alternative agents to taxane therapy. Sabizabulin (formerly known as VERU-111) is a potent CBSI that overcomes P-gp-mediated taxane resistance, is orally bioavailable, and inhibits tumor growth and distant metastasis in triple negative breast cancer (TNBC). Herein, we demonstrate the efficacy of sabizabulin in HER2+ breast cancer. In vitro, sabizabulin inhibits the proliferation of HER2+ breast cancer cell lines with low nanomolar IC50 values, inhibits clonogenicity, and induces apoptosis in a concentration-dependent manner. In vivo, sabizabulin inhibits breast tumor growth in the BT474 (ER+/PR+/HER2+) xenograft model and a HER2+ (ER-/PR-) metastatic patient-derived xenograft (PDX) model, HCI-12. We demonstrate that sabizabulin is a promising alternative agent to target tubulin in HER2+ breast cancer with similar anti-metastatic efficacy to paclitaxel, but with the advantage of oral bioavailability and lower toxicity than taxanes.
RESUMO
Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2-M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.
Assuntos
Neoplasias de Mama Triplo Negativas , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Colchicina/farmacologia , Colchicina/uso terapêutico , Humanos , Paclitaxel/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêuticoRESUMO
Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Deleção de Genes , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Gravidez , Prenhez , RiscoRESUMO
We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Metástase Neoplásica/prevenção & controle , Piridinas/síntese química , Piridinas/metabolismo , Piridinas/farmacocinética , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.