RESUMO
ABSTRACT: Glucocorticoids are key components of the standard-of-care treatment regimens for B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. ABBV-319 is a CD19-targeting antibody-drug conjugate engineered to reduce glucocorticoid-associated toxicities while possessing 3 distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of a glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced fragment crystallizable (Fc)-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven antitumor activity against multiple malignant B-cell lines in vitro, as well as in cell line-derived xenografts and patient-derived xenografts (PDXs) in vivo. Remarkably, a single dose of ABBV-319 induced sustained tumor regression and enhanced antitumor activity compared with repeated dosing of systemic prednisolone at the maximum tolerated dose in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed antiproliferative activity in a subset of B-cell lymphoma cell lines through the inhibition of phosphoinositide 3-kinase signaling. Moreover, afucosylation of CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity. Notably, ABBV-319 displayed superior efficacy compared with afucosylated CD19 mAb in human CD34+ peripheral blood mononuclear cell-engrafted NSG-Tg(Hu-IL15) transgenic mice, demonstrating enhanced antitumor activity when multiple MOAs are enabled. ABBV-319 also showed durable antitumor activity across multiple B-cell lymphoma PDX models, including nongerminal center B-cell diffuse large B-cell lymphoma and relapsed lymphoma after R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in a phase 1 clinical trial.
Assuntos
Antígenos CD19 , Imunoconjugados , Receptores de Glucocorticoides , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Antígenos CD19/imunologia , Camundongos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Receptores de Glucocorticoides/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Linhagem Celular Tumoral , Camundongos SCID , Feminino , Maitansina/análogos & derivadosRESUMO
Abundant fibrotic stroma is a typical feature of most solid tumors, and stromal activation promotes oncogenesis, therapy resistance, and metastatic dissemination of cancer cells. Therefore, targeting the tumor stroma in combination with standard-of-care therapies has become a promising therapeutic strategy in recent years. The leucine-rich repeat-containing protein 15 (LRRC15) is involved in cell-cell and cell-matrix interactions and came into focus as a promising anticancer target owing to its overexpression in mesenchymal-derived tumors such as sarcoma, glioblastoma, and melanoma and in cancer-associated fibroblasts in the microenvironment of breast, head and neck, lung, and pancreatic tumors. Effective targeting of LRRC15 using specific antibody-drug conjugates (ADC) has the potential to improve the outcome of patients with LRRC15-positive (LRRC15+) cancers of mesenchymal origin or stromal desmoplasia. Moreover, LRRC15 expression may serve as a predictive biomarker that could be utilized in the preclinical assessment of cancer patients to support personalized clinical outcomes. This review focuses on the role of LRRC15 in cancer, including clinical trials involving LRRC15-targeted therapies, such as the ABBV-085 ADC for patients with LRRC15+ tumors. This review spans perceived knowledge gaps and highlights the clinical avenues that need to be explored to provide better therapeutic outcomes in patients.
Assuntos
Fibroblastos Associados a Câncer , Glioblastoma , Imunoconjugados , Sarcoma , Fibroblastos Associados a Câncer/metabolismo , Glioblastoma/metabolismo , Humanos , Imunoconjugados/farmacologia , Proteínas de Membrana/metabolismo , Sarcoma/tratamento farmacológico , Microambiente TumoralRESUMO
Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with ß1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases.Significance: This study identifies that LRRC15 activates ß1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody-drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.
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Imunoconjugados , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Adesão Celular , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Integrinas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologiaRESUMO
PURPOSE: Leucine-rich repeat containing 15 (LRRC15) is expressed on stromal fibroblasts in the tumor microenvironment of multiple solid tumor types and may represent an interesting target for therapy, particularly in patients with sarcomas where LRRC15 is also expressed by malignant cells. ABBV-085 is a monomethyl auristatin-E antibody-drug conjugate that targets LRRC15 and showed antineoplastic efficacy in preclinical experiments. Herein, we report findings of ABBV-085 monotherapy or combination therapy in adult patients with sarcomas and other advanced solid tumors. PATIENTS AND METHODS: This first-in-human phase I study (NCT02565758) assessed ABBV-085 safety, pharmacokinetics/pharmacodynamics, and preliminary antitumor activity. The study consisted of two parts: dose escalation and dose expansion. ABBV-085 was administered by intravenous infusion at 0.3 to 6.0 mg/kg every 14 days. RESULTS: In total, 85 patients were enrolled; 45 patients received the recommended expansion dose of 3.6 mg/kg ABBV-085 monotherapy, including 10 with osteosarcoma and 10 with undifferentiated pleomorphic sarcoma (UPS). Most common treatment-related adverse events were fatigue, nausea, and decreased appetite. The overall response rate for patients with osteosarcoma/UPS treated at 3.6 mg/kg was 20%, including four confirmed partial responses. No monotherapy responses were observed for other advanced cancers treated at 3.6 mg/kg. One patient treated with ABBV-085 plus gemcitabine achieved partial response. CONCLUSIONS: ABBV-085 appeared safe and tolerable at a dose of 3.6 mg/kg every 14 days, with preliminary antitumor activity noted in patients with osteosarcoma and UPS. Given the high unmet need in these orphan malignancies, further investigation into targeting LRRC15 in these sarcomas may be warranted.
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Antineoplásicos , Neoplasias Ósseas , Imunoconjugados , Neoplasias , Sarcoma , Adulto , Antineoplásicos/efeitos adversos , Neoplasias Ósseas/tratamento farmacológico , Humanos , Imunoconjugados/efeitos adversos , Proteínas de Membrana/genética , Neoplasias/patologia , Sarcoma/tratamento farmacológico , Sarcoma/genética , Microambiente TumoralRESUMO
Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.
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Imunoconjugados , Preparações Farmacêuticas , Imunoconjugados/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína) , Raios XRESUMO
Thailanstatin A and spliceostatin D, two naturally occurring molecules endowed with potent antitumor activities by virtue of their ability to bind and inhibit the function of the spliceosome, and their natural siblings and designed analogues, constitute an appealing family of compounds for further evaluation and optimization as potential drug candidates for cancer therapies. In this article, the design, synthesis, and biological investigation of a number of novel thailanstatin A analogues, including some accommodating 1,1-difluorocyclopropyl and tetrahydrooxazine structural motifs within their structures, are described. Important findings from these studies paving the way for further investigations include the identification of several highly potent compounds for advancement as payloads for antibody-drug conjugates (ADCs) as potential targeted cancer therapies and/or small molecule drugs, either alone or in combination with other anticancer agents.
Assuntos
Antineoplásicos , Imunoconjugados , Antineoplásicos/farmacologia , Piranos/farmacologiaRESUMO
BACKGROUND: Osteosarcoma (OS), the most common bone tumor in children and adolescents, has high rates of metastasis leading to poor survival. Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein whose expression is modulated by TGFß, was recently shown to be highly expressed on the surface of OS tumor cells. Here, we evaluate a novel antibody-drug conjugate (ADC) targeting LRRC15 in OS human cell lines and murine xenografts. We compare this new ADC, which is conjugated to the anthracycline derivative PNU-159682 (PNU), to a previously studied LRRC15 ADC that is conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE), since anthracyclines are standard of care in OS. PROCEDURE: We evaluated LRRC15 expression in OS cells using Western blots and flow cytometry, and analyzed the epigenetic landscape of the LRRC15 locus using chromatin immunoprecipitation. Efficacy of ADCs on cell growth was analyzed by IncuCyte live cell imaging. Intramuscular xenograft tumor growth was assessed by bioluminescence imaging and hematoxylin and eosin staining. RESULTS: LRRC15-PNU is more effective at inhibiting growth in vitro and in vivo than an isotype antibody control or the LRRC15-MMAE ADC in two high LRRC15 expressing OS cell lines. Low expressing cell lines are not sensitive to either ADC. Importantly, cells with low LRRC15 expression are amenable to re-expression after TGFß treatment, suggesting a potential to sensitize insensitive OS cells to LRRC15 ADC treatment. In vivo, LRRC15-PNU had cure rates of 40-100% in OS xenograft models. CONCLUSIONS: Overall, LRRC15-directed ADCs are a promising new avenue for OS treatment.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/análogos & derivados , Imunoconjugados/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Osteossarcoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica/tratamento farmacológico , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Doxorrubicina/análogos & derivados , Imunoconjugados/química , Imunoconjugados/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Doxorrubicina/síntese química , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/tratamento farmacológicoRESUMO
Taking advantage of the C2-symmetry of the antitumor naturally occurring disorazole B1 molecule, a symmetrical total synthesis was devised with a monomeric advanced intermediate as the key building block, whose three-step conversion to the natural product allowed for an expeditious entry to this family of compounds. Application of the developed synthetic strategies and methods provided a series of designed analogues of disorazole B1, whose biological evaluation led to the identification of a number of potent antitumor agents and the first structure-activity relationships (SARs) within this class of compounds. Specifically, the substitutions of the epoxide units and lactone moieties with cyclopropyl and lactam structural motifs, respectively, were found to be tolerable for biological activities and beneficial with regard to chemical stability.
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Antineoplásicos/farmacologia , Desenho de Fármacos , Oxazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Oxazóis/síntese química , Oxazóis/química , Relação Estrutura-AtividadeRESUMO
Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFß on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085's unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059-72. ©2018 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Proteínas de Membrana/metabolismo , Neoplasias/tratamento farmacológico , Células Estromais/efeitos dos fármacos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Neoplasias/metabolismo , Oligopeptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Células Estromais/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192), a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab's tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft) activation of both classical (p50, p65) and non-classical (p52, RelB) NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB-regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52) and upstream kinases (IKK1, IKK2) with siRNA and chemical inhibitors consistently blocked enavatuzumab's activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.
RESUMO
BRCA1 mediates resistance to apoptosis in response to DNA-damaging agents, causing BRCA1 wild-type tumours to be significantly more resistant to DNA damage than their mutant counterparts. In this study, we demonstrate that following treatment with the DNA-damaging agents, etoposide or camptothecin, BRCA1 is required for the activation of nuclear factor-κB (NF-κB), and that BRCA1 and NF-κB cooperate to regulate the expression of the NF-κB antiapoptotic targets BCL2 and XIAP. We show that BRCA1 and the NF-κB subunit p65/RelA associate constitutively, whereas the p50 NF-κB subunit associates with BRCA1 only upon DNA damage treatment. Consistent with this BRCA1 and p65 are present constitutively on the promoters of BCL2 and XIAP, whereas p50 is recruited to these promoters only in damage treated cells. Importantly, we demonstrate that the recruitment of p50 onto the promoters of BCL2 and XIAP is dependent upon BRCA1, but independent of its NF-κB partner subunit p65. The functional relevance of NF-κB activation by BRCA1 in response to etoposide and camptothecin is demonstrated by the significantly reduced survival of BRCA1 wild-type cells upon NF-κB inhibition. This study identifies a novel BRCA1-p50 complex, and demonstrates for the first time that NF-κB is required for BRCA1-mediated resistance to DNA damage. It reveals a functional interdependence between BRCA1 and NF-κB, further elucidating the role played by NF-κB in mediating cellular resistance of BRCA1 wild-type tumours to DNA-damaging agents.
Assuntos
Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Dano ao DNA , Etoposídeo/farmacologia , Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Humanos , NF-kappa B/genética , Subunidade p50 de NF-kappa B/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
PURPOSE: Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer. EXPERIMENTAL DESIGN: We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies. RESULTS: In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone. CONCLUSIONS: These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer.
Assuntos
Benzamidas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Cinesinas/antagonistas & inibidores , Quinazolinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Quinazolinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BRCA1 encodes a tumor suppressor gene that is mutated in the germ line of women with a genetic predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and ubiquitination. Using an Affymetrix U95A microarray, IRF-7 was identified as a BRCA1 transcriptional target and was also shown to be synergistically up-regulated by BRCA1 specifically in the presence of IFN-gamma, coincident with the synergistic induction of apoptosis. We show that BRCA1, signal transducer and activator of transcription (STAT)-1, and STAT2 are all required for the induction of IRF-7 following stimulation with IFN-gamma. We also show that the induction of IRF-7 by BRCA1 and IFN-gamma is dependent on the type I IFNs, IFN-alpha and IFN-beta. We show that BRCA1 is required for the up-regulation of STAT1, STAT2, and the type I IFNs in response to IFN-gamma. We show that BRCA1 is localized at the promoters of the molecules involved in type I IFN signaling leading to their up-regulation. Blocking this intermediary type I IFN step using specific antisera shows the requirement for IFN-alpha and IFN-beta in the induction of IRF-7 and apoptosis. Finally, we outline a mechanism for the BRCA1/IFN-gamma regulation of target genes involved in the innate immune response, which is dependent on type I IFN signaling.
Assuntos
Proteína BRCA1/metabolismo , Imunidade Inata/genética , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/metabolismo , Interferon gama/farmacologia , Apoptose , Proteína BRCA1/análise , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/farmacologia , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Regulação para CimaRESUMO
The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor kappaB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-gamma. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-gamma, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (alpha and beta) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-gamma and doxorubicin (combination index = 0.34) at doses lower than IC(50) values for this drug (0.67 micromol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-gamma, and that this combination results in enhanced apoptosis in breast cancer cells.