RESUMO
Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.
Assuntos
Neoplasias , Uridina Difosfato Ácido Glucurônico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Uridina Difosfato Ácido Glucurônico/biossíntese , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato Xilose/biossíntese , Uridina Difosfato Xilose/metabolismo , Adenocarcinoma de Pulmão , Neoplasias PulmonaresRESUMO
In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.
Assuntos
Sobrevivência Celular , Glândulas Mamárias Animais/citologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Epigênese Genética , Feminino , Impressão Genômica , Masculino , Glândulas Mamárias Animais/fisiologia , Camundongos , Gravidez , Ubiquitina-Proteína Ligases/genética , Inativação do Cromossomo XRESUMO
Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRß/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.
Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Isoformas de Proteínas , Transdução de Sinais/genéticaRESUMO
We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ferroptose , Integrina beta4 , Calinina , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Integrina beta4/metabolismo , Integrina beta4/genética , Calinina/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Transativadores/metabolismo , Transativadores/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.
Assuntos
Neoplasias da Mama/fisiopatologia , Matriz Extracelular/metabolismo , Integrina alfa6beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Laminina/metabolismo , Células-Tronco Neoplásicas/patologia , Feminino , Humanos , Ligantes , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.
Assuntos
Neuropilina-2/genética , Rad51 Recombinase/genética , Neoplasias de Mama Triplo Negativas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína BRCA1/genética , Linhagem Celular Tumoral , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Neuropilinas/genética , Platina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteínas de Sinalização YAPRESUMO
Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6ß4 integrin (ß4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that ß4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.
Assuntos
Integrina beta4/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Células-Tronco/metabolismo , Animais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco/citologiaRESUMO
The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the ß4 subunit of the α6ß4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin ß4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin ß4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin ß4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin ß4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin ß4 expression in cells proximal to the wound edge was evident, and a population of integrin ß4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin ß4 dynamics during invasion and metastasis. Moreover, these integrin ß4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer.This article has an associated First Person interview with the first author of the paper.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Integrina beta4/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina beta4/genética , Microscopia de Vídeo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
Ferroptosis is an iron-dependent form of programmed cell death characterized by the accumulation of lipid-targeting reactive oxygen species that kill cells by damaging their plasma membrane. The lipid repair enzyme GSH peroxidase 4 (GPX4) protects against this oxidative damage and enables cells to resist ferroptosis. Recent work has revealed that matrix-detached carcinoma cells can be susceptible to ferroptosis and that they can evade this fate through the signaling properties of the α6ß4 integrin, which sustains GPX4 expression. Although these findings on ferroptosis are provocative, they differ from those in previous studies indicating that matrix-detached cells are prone to apoptosis via a process referred to as anoikis. In an effort to reconcile these discrepant findings, here we observed that matrix-detached epithelial and carcinoma cells cluster spontaneously via a mechanism that involves the cell adhesion protein PVRL4 (also known as Nectin-4). We found that this clustering process allows these cells to survive by stimulating a PVRL4/α6ß4/Src signaling axis that sustains GPX4 expression and buffers against lipid peroxidation. In the absence of α6ß4, PVRL4-mediated clustering induced an increase in lipid peroxidation that was sufficient for triggering ferroptosis. When the clustering was inhibited, single cells did not exhibit a significant increase in lipid peroxidation in the absence of α6ß4, and they were more susceptible to apoptosis than to ferroptosis. These results indicate that ferroptosis induction depends on cell clustering in matrix-detached cells that lack α6ß4 and imply that the fate of matrix-detached cells can be determined by the state of their cell-cell interactions.
Assuntos
Apoptose , Neoplasias da Mama/patologia , Mama/patologia , Moléculas de Adesão Celular/metabolismo , Adesão Celular , Matriz Extracelular/metabolismo , Integrina alfa6beta4/metabolismo , Ferro/metabolismo , Mama/metabolismo , Neoplasias da Mama/metabolismo , Agregação Celular , Células Cultivadas , Feminino , Humanos , Peroxidação de Lipídeos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies.
Assuntos
Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neuropilinas/metabolismo , Transdução de Sinais , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Terapia de Alvo Molecular , Neoplasias/etiologia , Neoplasias/patologia , Neoplasias/terapia , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neuropilinas/genética , Ligação Proteica , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Cancer stem cells (CSCs) contribute to multidrug resistance, tumor recurrence and metastasis, making them prime therapeutic targets. Their ability to differentiate and lose stem cell properties makes them challenging to study. Currently, there is no simple assay that can quickly capture and trace the dynamic phenotypic changes on the CSC surface. Here, we report rapid discrimination of breast CSCs from non-CSCs using a nanoparticle-fluorescent-protein based sensor. This nanosensor was employed to discriminate CSCs from non-CSCs, as well as CSCs that had differentiated in vitro in two breast cancer models. Importantly, the sensor platform could also discriminate CSCs from the bulk population of cells in patient-derived xenografts of human breast cancer. Taken together, the results obtained demonstrate the feasibility of using the nanosensor to phenotype CSCs and monitor their fate. Furthermore, this approach provides a novel area for therapeutic interventions against these challenging targets.
Assuntos
Técnicas Biossensoriais , Proliferação de Células , Nanopartículas/química , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Apoptose , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The integrin α6ß4 (referred to as ß4) is expressed in epithelial cells where it functions as a laminin receptor. Although in vitro studies have implicated ß4 in the biology of mammary epithelial cells, its contribution to mammary gland development has not been settled. To address this problem, we generated and analyzed itgb4(flox/flox)MMTV-Cre(-) and itgb4(flox/flox)MMTV-Cre(+) mice. The salient features of embryonic mammary tissue from itgb4(flox/flox)MMTV-Cre(+) mice were significantly smaller mammary buds and increased apoptosis in the surrounding mesenchyme. Also, compared to control glands, the itgb4-deleted mammary buds lacked expression of the progenitor cell marker CK14 and they were unable to generate mammary glands upon transplantation into cleared fat pads of recipient mice. Analysis of mammary glands at puberty and during pregnancy revealed that itgb4-diminished mammary tissue was unable to elongate and undergo branching morphogenesis. Micro-dissection of epithelial cells in the mammary bud and of the surrounding mesenchyme revealed that loss of ß4 resulted in a significant decrease in the expression of parathyroid hormone related protein (PTHrP) in epithelial cells and of target genes of the PTHrP receptor in mesenchymal cells. Given that the phenotype of the itgb4-deleted mammary tissue mimicked that of the PTHrP knockout, we hypothesized that ß4 contributes to mammary gland development by sustaining PTHrP expression and enabling PTHrP signaling. Indeed, the inability of itgb4-deleted mammary buds to elongate was rescued by exogenous PTHrP. These data implicate a critical role for the ß4 integrin in mammary gland development and provide a mechanism for this role.
Assuntos
Integrina beta4/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Animais , Feminino , Deleção de Genes , Camundongos Endogâmicos C57BL , Tamanho do ÓrgãoRESUMO
Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin ß4 â PI3K â Akt â FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin ß4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin ß4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing.
Assuntos
Integrina beta4/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Cicatrização/fisiologia , Animais , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina beta4/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Neovascularização Fisiológica , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Transdução de Sinais , Pele/lesões , Pele/metabolismo , Pele/patologia , Regulação para Cima , Cicatrização/genéticaRESUMO
Estrogen receptor ß (ERß) promotes the degradation of hypoxia inducible factor 1α (HIF-1α), which contributes to the ability of this hormone receptor to sustain the differentiation of epithelial and carcinoma cells. Although the loss of ERß and consequent HIF-1 activation occur in prostate cancer with profound consequences, the mechanism by which ERß promotes the degradation of HIF-1α is unknown. We report that ERß regulates the ligand (3ß-adiol)-dependent transcription of prolyl hydroxylase 2 (PHD2) also known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-dependent dioxygenase that hydroxylates HIF-1α and targets it for recognition by the von Hippel-Lindau tumor suppressor and consequent degradation. ERß promotes PHD2 transcription by interacting with a unique estrogen response element in the 5' UTR of the PHD2 gene that functions as an enhancer. PHD2 itself is critical for maintaining epithelial differentiation. Loss of PHD2 expression or inhibition of its function results in dedifferentiation with characteristics of an epithelial-mesenchymal transition, and exogenous PHD2 expression in dedifferentiated cells can restore an epithelial phenotype. Moreover, expression of HIF-1α in cells that express PHD2 does not induce dedifferentiation but expression of HIF-1α containing mutations in the proline residues that are hydroxylated by PHD2 induces dedifferentiation. These data describe a unique mechanism for the regulation of HIF-1α stability that involves ERß-mediated transcriptional regulation of PHD2 and they highlight an unexpected role for PHD2 in maintaining epithelial differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Linhagem Celular Tumoral , Células Epiteliais/citologia , Receptor beta de Estrogênio/genética , Humanos , Hidroxilação/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Masculino , Mutação , Pró-Colágeno-Prolina Dioxigenase/genética , Estabilidade Proteica , ProteóliseRESUMO
IMP3, a member of a family of insulin-like growth factor II (IGF-II) mRNA-binding proteins (IMPs), is expressed preferentially in triple-negative breast cancers, which are resistant to many chemotherapeutics. However, the mechanisms by which it impacts breast cancer have not been elucidated. We hypothesized a role for IMP3 in chemoresistance based on these observations. Depletion of IMP3 expression in triple-negative breast cancer cells increased their sensitivity to doxorubicin and mitoxantrone significantly but not to taxol. Given that doxorubicin and mitoxantrone are effluxed by breast cancer resistance protein (BCRP), we assessed whether IMP3 regulates BCRP. The data obtained demonstrate that IMP3 binds to BCRP mRNA and regulates BCRP expression. These findings are significant because they provide insight into the mechanism by which IMP3 contributes to aggressive cancers, and they highlight the potential for targeting this mRNA-binding protein for the clinical management of cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Proteínas de Ligação a RNA/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Células HEK293 , Humanos , Mitoxantrona/farmacologia , Proteínas de Neoplasias/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6ß1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6ß1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6ß1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6ß1 integrin with the cytoskeleton. NRP2 also facilitates α6ß1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6ß1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6ß1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Adesões Focais , Integrina alfa6beta1/metabolismo , Neuropilina-2/metabolismo , Transdução de Sinais , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Humanos , Laminina/metabolismo , Neuropilina-2/fisiologia , Proteína Quinase C/metabolismoRESUMO
Although the neuropilins were characterized as semaphorin receptors that regulate axon guidance, they also function as vascular endothelial growth factor (VEGF) receptors and contribute to the development of other tissues. Here, we assessed the role of NRP2 in mouse mammary gland development based on our observation that NRP2 is expressed preferentially in the terminal end buds of developing glands. A floxed NRP2 mouse was bred with an MMTV-Cre strain to generate a mammary gland-specific knockout of NRP2. MMTV-Cre;NRP2(loxP/loxP) mice exhibited significant defects in branching morphogenesis and ductal outgrowth compared with either littermate MMTV-Cre;NRP2(+/loxP) or MMTV-Cre mice. Mechanistic insight into this morphological defect was obtained from a mouse mammary cell line in which we observed that VEGF(165), an NRP2 ligand, induces branching morphogenesis in 3D cultures and that branching is dependent upon NRP2 as shown using shRNAs and a function-blocking antibody. Epithelial cells in the mouse mammary gland express VEGF, supporting the hypothesis that this NRP2 ligand contributes to mammary gland morphogenesis. Importantly, we demonstrate that VEGF and NRP2 activate focal adhesion kinase (FAK) and promote FAK-dependent branching morphogenesis in vitro. The significance of this mechanism is substantiated by our finding that FAK activation is diminished significantly in developing MMTV-Cre;NRP2(loxP/loxP) mammary glands compared with control glands. Together, our data reveal a VEGF/NRP2/FAK signaling axis that is important for branching morphogenesis and mammary gland development. In a broader context, our data support an emerging hypothesis that directional outgrowth and branching morphogenesis in a variety of tissues are influenced by signals that were identified initially for their role in axon guidance.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glândulas Mamárias Animais/embriologia , Morfogênese/fisiologia , Neuropilina-2/metabolismo , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neuropilina-2/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Patients with castration-resistant prostate cancer (CRPC) are generally unresponsive to tumor targeted and immunotherapies. Whether genetic alterations acquired during the evolution of CRPC impact immune and immunotherapy responses is largely unknown. Using our innovative electroporation-based mouse models, we generated distinct genetic subtypes of CRPC found in patients and uncovered unique immune microenvironments. Specifically, mouse and human prostate tumors with MYC amplification and p53 disruption had weak cytotoxic lymphocyte infiltration and an overall dismal prognosis. MYC and p53 cooperated to induce tumor intrinsic secretion of VEGF, which by signaling through VEGFR2 expressed on CD8+ T cells, could directly inhibit T cell activity. Targeting VEGF-VEGFR2 signaling in vivo led to CD8+ T cell-mediated tumor and metastasis growth suppression and significantly increased overall survival in MYC and p53 altered CPRC. VEGFR2 blockade also led to induction of PD-L1, and in combination with PD-L1 immune checkpoint blockade produced anti-tumor efficacy in multiple preclinical CRPC mouse models. Thus, our results identify a genetic mechanism of immune suppression through VEGF signaling in prostate cancer that can be targeted to reactivate immune and immunotherapy responses in an aggressive subtype of CRPC. Significance: Though immune checkpoint blockade (ICB) therapies can achieve curative responses in many treatment-refractory cancers, they have limited efficacy in CRPC. Here we identify a genetic mechanism by which VEGF contributes to T cell suppression, and demonstrate that VEGFR2 blockade can potentiate the effects of PD-L1 ICB to immunologically treat CRPC.
RESUMO
The α6ß4 integrin (referred to as "ß4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in ß4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of ß4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates ß4-mediated invasion. Expression of ß4 in ß4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous ß4, miR-29a expression is low and ß4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that ß4 can regulate SPARC expression and that SPARC is an effector of ß4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.
Assuntos
Regulação Neoplásica da Expressão Gênica , Integrina beta4/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Osteonectina/biossíntese , Biossíntese de Proteínas , Linhagem Celular Tumoral , Humanos , Integrina beta4/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Osteonectina/genéticaRESUMO
Understanding the cell biological mechanisms that enable tumor cells to persist after therapy is necessary to improve the treatment of recurrent disease. Here, we demonstrate that transient receptor potential channel 6 (TRPC6), a channel that mediates calcium entry, contributes to the properties of breast cancer stem cells, including resistance to chemotherapy, and that tumor cells that persist after therapy are dependent on TRPC6. The mechanism involves the ability of TRPC6 to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated calcium entry represses the epithelial splicing factor ESRP1 (epithelial splicing regulatory protein 1), which enables expression of the integrin α6B splice variant. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic inhibition of TRPC6 sensitizes triple-negative breast cancer (TNBC) cells and tumors to chemotherapy by targeting the splicing of α6 integrin mRNA and inducing Myc. These data reveal a Ca2+-dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing.