RESUMO
Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.
Assuntos
Memória Imunológica , Células Matadoras Naturais , Proteínas Recombinantes de Fusão , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Animais , Proteínas Recombinantes de Fusão/genética , Camundongos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia Adotiva/métodos , Interleucina-15/metabolismoRESUMO
Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-ß (TGF-ß) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.
Assuntos
Antígeno B7-H1 , Células Matadoras Naturais , Animais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Senescência Celular , Docetaxel/metabolismo , Docetaxel/farmacologia , Imunoterapia/métodos , Células Matadoras Naturais/metabolismo , Camundongos , Microambiente TumoralRESUMO
Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.
Assuntos
Interleucina-12/farmacologia , Interleucina-15/farmacologia , Interleucina-18/farmacologia , Células Matadoras Naturais/imunologia , Leucemia/terapia , Animais , Linhagem Celular Tumoral , Humanos , Memória Imunológica/efeitos dos fármacos , Leucemia/imunologia , Camundongos , Receptores de Células Matadoras Naturais/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Indução de Remissão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. However, novel immunotherapeutics with these dual functions are not frequently reported. Here we describe the creation of a heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology. This complex comprises extracellular domains of the human transforming growth factor-ß (TGF-ß) receptor II and a human interleukin-15 (IL-15)/IL-15 receptor α complex. HCW9218 can be readily expressed in CHO cells and purified using antibody-based affinity chromatography in a large-scale manufacturing setting. HCW9218 potently activates mouse natural killer (NK) cells and CD8+ T cells in vitro and in vivo to enhance cell proliferation, metabolism, and antitumor cytotoxic activities. Similarly, human immune cells become activated with increased cytotoxicity following incubation with HCW9218. This fusion complex also exhibits TGF-ß neutralizing activity in vitro and sequesters plasma TGF-ß in vivo. In a syngeneic B16F10 melanoma model, HCW9218 displayed strong antitumor activity mediated by NK cells and CD8+ T cells and increased their infiltration into tumors. Repeat-dose subcutaneous administration of HCW9218 was well tolerated by mice, with a half-life sufficient to provide long-lasting biological activity. Thus, HCW9218 may serve as a novel therapeutic to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Interleucina-15/genética , Células Matadoras Naturais/metabolismo , Melanoma Experimental/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo II/química , Receptores de Interleucina-15/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Injeções Subcutâneas , Interleucina-15/metabolismo , Melanoma Experimental/imunologia , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptores de Interleucina-15/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Neovascularização Patológica/patologia , Microambiente Tumoral/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Camundongos , Camundongos SCID , Neovascularização Patológica/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Resistance to chemotherapy represents a major limitation in the treatment of colorectal cancer. Novel strategies to circumvent resistance are critical to prolonging patient survival. Rac1b, a constitutively activated isoform of the small GTPase Rac1, is upregulated with disease progression and promotes cell proliferation and inhibits apoptosis by activation of NF-κB signaling. Here, we show that Rac1b overexpression correlates with cancer stage and confirmed Rac1b expression is associated with increased growth through enhancing NF-κB activity. Rac1b knockdown reduced cellular proliferation and reduced NF-κB activity. Surprisingly, Rac1b expression and NF-κB activity were upregulated in cells treated with chemotherapeutics, suggesting that Rac1b facilitates chemo-resistance through activation of NF-κB signaling. Knockdown of Rac1b or Rac inhibition increases the sensitivity of the cells to oxaliplatin. When used in combination, inhibition of Rac prevents the increase in NF-κB activity associated with chemotherapy treatment and increases the sensitivity of the cells to oxaliplatin. Although Rac inhibition or oxaliplatin treatment alone reduces the growth of colorectal cancer in vivo, combination therapy results in improved outcomes compared with single agents alone. We provide the first evidence that Rac1b expression confers resistance to chemotherapy in colorectal cancer. Additionally, we show that the use of a Rac inhibitor prevents chemoresistance by blocking activation of chemotherapy induced NF-κB signaling, providing a novel strategy to overcome resistance to chemotherapy in colorectal cancer.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Tratamento Farmacológico , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Camundongos , NF-kappa B/genética , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.
Assuntos
Cisplatino/administração & dosagem , Células Epiteliais/metabolismo , Neoplasias/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidores , Tamoxifeno/administração & dosagem , Animais , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Sinergismo Farmacológico , Tratamento Farmacológico , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Tamoxifeno/farmacologiaRESUMO
Vascular endothelial barrier dysfunction underlies diseases such as acute respiratory distress syndrome (ARDS), characterized by edema and inflammatory cell infiltration. The transcription factor HIF2α is highly expressed in vascular endothelial cells (ECs) and may regulate endothelial barrier function. Here, we analyzed promoter sequences of genes encoding proteins that regulate adherens junction (AJ) integrity and determined that vascular endothelial protein tyrosine phosphatase (VE-PTP) is a HIF2α target. HIF2α-induced VE-PTP expression enhanced dephosphorylation of VE-cadherin, which reduced VE-cadherin endocytosis and thereby augmented AJ integrity and endothelial barrier function. Mice harboring an EC-specific deletion of Hif2a exhibited decreased VE-PTP expression and increased VE-cadherin phosphorylation, resulting in defective AJs. Mice lacking HIF2α in ECs had increased lung vascular permeability and water content, both of which were further exacerbated by endotoxin-mediated injury. Treatment of these mice with Fg4497, a prolyl hydroxylase domain 2 (PHD2) inhibitor, activated HIF2α-mediated transcription in a hypoxia-independent manner. HIF2α activation increased VE-PTP expression, decreased VE-cadherin phosphorylation, promoted AJ integrity, and prevented the loss of endothelial barrier function. These findings demonstrate that HIF2α enhances endothelial barrier integrity, in part through VE-PTP expression and the resultant VE-cadherin dephosphorylation-mediated assembly of AJs. Moreover, activation of HIF2α/VE-PTP signaling via PHD2 inhibition has the potential to prevent the formation of leaky vessels and edema in inflammatory diseases such as ARDS.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Junções Aderentes/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Transdução de Sinais , Lesão Pulmonar Aguda/patologia , Junções Aderentes/genética , Junções Aderentes/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/biossíntese , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/patologiaRESUMO
Triple negative breast cancers (TNBCs) are defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 expression, and are treated with cytotoxic chemotherapy such as paclitaxel or gemcitabine, with a durable response rate of less than 20%. TNBCs are enriched for the basal subtype gene expression profile and the presence of breast cancer stem cells, which are endowed with self-renewing and tumor-initiating properties and resistance to chemotherapy. Hypoxia-inducible factors (HIFs) and their target gene products are highly active in TNBCs. Here, we demonstrate that HIF expression and transcriptional activity are induced by treatment of MDA-MB-231, SUM-149, and SUM-159, which are human TNBC cell lines, as well as MCF-7, which is an ER(+)/PR(+) breast cancer line, with paclitaxel or gemcitabine. Chemotherapy-induced HIF activity enriched the breast cancer stem cell population through interleukin-6 and interleukin-8 signaling and increased expression of multidrug resistance 1. Coadministration of HIF inhibitors overcame the resistance of breast cancer stem cells to paclitaxel or gemcitabine, both in vitro and in vivo, leading to tumor eradication. Increased expression of HIF-1α or HIF target genes in breast cancer biopsies was associated with decreased overall survival, particularly in patients with basal subtype tumors and those treated with chemotherapy alone. Based on these results, clinical trials are warranted to test whether treatment of patients with TNBC with a combination of cytotoxic chemotherapy and HIF inhibitors will improve patient survival.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Análise de Variância , Linhagem Celular Tumoral , Primers do DNA/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Processamento de Imagem Assistida por Computador , Immunoblotting , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Estimativa de Kaplan-Meier , Luciferases , Microscopia , Paclitaxel/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , GencitabinaRESUMO
UNLABELLED: Cystathionine ß-synthase (CBS) catalyzes metabolic reactions that convert homocysteine to cystathionine. To assess the role of CBS in human glioma, cells were stably transfected with lentiviral vectors encoding shRNA targeting CBS or a nontargeting control shRNA, and subclones were injected into immunodeficient mice. Interestingly, decreased CBS expression did not affect proliferation in vitro but decreased the latency period before rapid tumor xenograft growth after subcutaneous injection and increased tumor incidence and volume following orthotopic implantation into the caudate-putamen. In soft-agar colony formation assays, CBS knockdown subclones displayed increased anchorage-independent growth. Molecular analysis revealed that CBS knockdown subclones expressed higher basal levels of the transcriptional activator hypoxia-inducible factor 2α (HIF2α/EPAS1). HIF2α knockdown counteracted the effect of CBS knockdown on anchorage-independent growth. Bioinformatic analysis of mRNA expression data from human glioma specimens revealed a significant association between low expression of CBS mRNA and high expression of angiopoietin-like 4 (ANGPTL4) and VEGF transcripts, which are HIF2 target gene products that were also increased in CBS knockdown subclones. These results suggest that decreased CBS expression in glioma increases HIF2α protein levels and HIF2 target gene expression, which promotes glioma tumor formation. IMPLICATIONS: CBS loss-of-function promotes glioma growth.
Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Carcinogênese/patologia , Cistationina beta-Sintase/metabolismo , Glioma/enzimologia , Glioma/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Cistationina beta-Sintase/deficiência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Injeções Subcutâneas , Masculino , Camundongos SCID , Carga Tumoral , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular vesicles such as exosomes and microvesicles (MVs) are shed by cancer cells, are detected in the plasma of cancer patients, and promote cancer progression, but the molecular mechanisms regulating their production are not well understood. Intratumoral hypoxia is common in advanced breast cancers and is associated with an increased risk of metastasis and patient mortality that is mediated in part by the activation of hypoxia-inducible factors (HIFs). In this paper, we report that exposure of human breast cancer cells to hypoxia augments MV shedding that is mediated by the HIF-dependent expression of the small GTPase RAB22A, which colocalizes with budding MVs at the cell surface. Incubation of naïve breast cancer cells with MVs shed by hypoxic breast cancer cells promotes focal adhesion formation, invasion, and metastasis. In breast cancer patients, RAB22A mRNA overexpression in the primary tumor is associated with decreased overall and metastasis-free survival and, in an orthotopic mouse model, RAB22A knockdown impairs breast cancer metastasis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Exossomos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Intratumoral hypoxia induces the recruitment of stromal cells, such as macrophages and mesenchymal stem cells (MSCs), which stimulate invasion and metastasis by breast cancer cells (BCCs). Production of macrophage colony-stimulating factor 1 (CSF1) by BCCs is required for macrophage recruitment, but the mechanisms underlying CSF1 expression have not been delineated. Triple-negative breast cancers have increased expression of genes regulated by hypoxia-inducible factors (HIFs). In this study, we delineate two feed-forward signaling loops between human MDA-MB-231 triple-negative BCCs and human MSCs that drive stromal cell recruitment to primary breast tumors. The first loop, in which BCCs secrete chemokine (C-X-C motif) ligand 16 (CXCL16) that binds to C-X-C chemokine receptor type 6 (CXCR6) on MSCs and MSCs secrete chemokine CXCL10 that binds to receptor CXCR3 on BCCs, drives recruitment of MSCs. The second loop, in which MSCs secrete chemokine (C-C motif) ligand 5 that binds to C-C chemokine receptor type 5 on BCCs and BCCs secrete cytokine CSF1 that binds to the CSF1 receptor on MSCs, drives recruitment of tumor-associated macrophages and myeloid-derived suppressor cells. These two signaling loops operate independent of each other, but both are dependent on the transcriptional activity of HIFs, with hypoxia serving as a pathophysiological signal that synergizes with chemokine signals from MSCs to trigger CSF1 gene transcription in triple-negative BCCs.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Células da Medula Óssea/citologia , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CXCL10/metabolismo , Quimiocina CXCL16 , Quimiocinas CXC/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Hipóxia , Neoplasias Pulmonares/secundário , Metástase Linfática , Macrófagos/citologia , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Transplante de Neoplasias , Receptores CCR5/metabolismo , Receptores Depuradores/metabolismoRESUMO
Overexpression of Rho kinase 1 (ROCK1) and the G protein RhoA is implicated in breast cancer progression, but oncogenic mutations are rare, and the molecular mechanisms that underlie increased ROCK1 and RhoA expression have not been determined. RhoA-bound ROCK1 phosphorylates myosin light chain (MLC), which is required for actin-myosin contractility. RhoA also activates focal adhesion kinase (FAK) signaling. Together, these pathways are critical determinants of the motile and invasive phenotype of cancer cells. We report that hypoxia-inducible factors coordinately activate RhoA and ROCK1 expression and signaling in breast cancer cells, leading to cell and matrix contraction, focal adhesion formation, and motility through phosphorylation of MLC and FAK. Thus, intratumoral hypoxia acts as an oncogenic stimulus by triggering hypoxia-inducible factor â RhoA â ROCK1 â MLC â FAK signaling in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Colágeno/química , Citoesqueleto/metabolismo , Feminino , Células HEK293 , Humanos , Microscopia Confocal , Mutação , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Fosforilação , Ratos , Transdução de Sinais , Microambiente TumoralRESUMO
UNLABELLED: Targeted therapy against triple-negative breast cancers, which lack expression of the estrogen, progesterone, and HER2 receptors, is not available and the overall response to cytotoxic chemotherapy is poor. One of the molecular hallmarks of triple-negative breast cancers is increased expression of genes that are transcriptionally activated by hypoxia-inducible factors (HIFs), which are implicated in many critical aspects of cancer progression including metabolism, angiogenesis, invasion, metastasis, and stem cell maintenance. Ganetespib is a second-generation inhibitor of heat shock protein 90 (HSP90), a molecular chaperone that is essential for the stability and function of multiple client proteins in cancer cells including HIF-1α. In this study, human MDA-MB-231 and MDA-MB-435 triple-negative breast cancer cells were injected into the mammary fat pad of immunodeficient mice that received weekly intravenous injections of ganetespib or vehicle following the development of palpable tumors. Ganetespib treatment markedly impaired primary tumor growth and vascularization, and eliminated local tissue invasion and distant metastasis to regional lymph nodes and lungs. Ganetespib treatment also significantly reduced the number of Aldefluor-positive cancer stem cells in the primary tumor. Primary tumors of ganetespib-treated mice had significantly reduced levels of HIF-1α (but not HIF-2α) protein and of HIF-1 target gene mRNAs encoding proteins that play key roles in angiogenesis, metabolism, invasion, and metastasis, thereby providing a molecular basis for observed effects of the drug on the growth and metastasis of triple-negative breast cancer. KEY MESSAGES: Triple-negative breast cancers (TNBCs) respond poorly to available chemotherapy. TNBCs overexpress genes regulated by hypoxia-inducible factors (HIFs). Ganetespib induces degradation of HSP90 client proteins, including HIF-1α. Ganetespib inhibited TNBC orthotopic tumor growth, invasion, and metastasis. Ganetespib inhibited expression of HIF-1 target genes involved in TNBC progression.
Assuntos
Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/prevenção & controle , Molécula L1 de Adesão de Célula Nervosa/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The presence of hypoxia and fibrosis within the primary tumor are two major risk factors for metastasis of human breast cancer. In this study, we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Finally, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and show that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hidroxibenzoatos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Extracellular matrix (ECM) composition, organization, and compliance provide both architectural and chemical cues that modulate tissue structure and function. ECM produced by stromal fibroblasts plays a key role in breast cancer invasion and metastasis, which are also stimulated by intratumoral hypoxia. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of ECM remodeling by fibroblasts under hypoxic conditions. HIF-1 activates expression of genes encoding collagen prolyl (P4HA1 and P4HA2) and lysyl (PLOD2) hydroxylases. P4HA1 and P4HA2 are required for collagen deposition, whereas PLOD2 is required for ECM stiffening and collagen fiber alignment. Together P4HA1, P4HA2, and PLOD2 mediate remodeling of ECM composition, alignment, and mechanical properties in response to hypoxia. HIF-1-dependent ECM remodeling by hypoxic fibroblasts induces changes in breast cancer cell morphology, adhesion, and motility that promote invasion and metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Matriz Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/biossíntese , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/biossíntese , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Colágeno/biossíntese , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Feminino , Fibroblastos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/genéticaRESUMO
Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hipóxia Celular/fisiologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Pró-Colágeno/metabolismo , Animais , Neoplasias da Mama/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Pró-Colágeno/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genéticaRESUMO
Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) to promote metastasis. In a mouse orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to LNs and lungs in a HIF-dependent manner. Coculture of MSCs with BCCs augmented HIF activity in BCCs. Additionally, coculture induced expression of the chemokine CXCL10 in MSCs and the cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. CXCR3 expression was blocked in cocultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs cocultured with BCCs that did not express CXCR3 or HIFs. MSC coculture did not enhance the metastasis of HIF-deficient BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGFR1, respectively, in a HIF-dependent manner, and CXCL10 expression by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis.