RESUMO
Antibody-drug conjugates (ADC) have emerged as one of the pillars of clinical disease management in oncology. The biggest hurdle to widespread development and application of ADCs has been a narrow therapeutic index. Advances in antibody technologies and formats as well as novel linker and payload chemistries have begun to facilitate structural improvements to ADCs. However, the interplay of structural characteristics with physiologic and pharmacologic factors determining therapeutic success has garnered less attention. This review elaborates on the pharmacology of ADCs, the pathophysiology of cancerous tissues, and the reciprocal consequences on ADC properties and functions. While most currently approved ADCs utilize either microtubule inhibition or DNA damage as primary mechanisms of action, we present arguments to expand this repertoire and highlight the need for payload mechanisms that exploit disease-specific vulnerabilities. We promote the idea that the choice of antibody format, targeting antigen, linker properties, and payload of an ADC should be deliberately fit for purpose by taking the pathophysiology of disease and the specific pharmacology of the drug entity into account, thus allowing a higher probability of clinical success.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Anticorpos/uso terapêutico , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
PURPOSE: Depatux-m is an antibody drug conjugate (ADC) that targets and inhibits growth of cancer cells overexpressing the epidermal growth factor receptor (EGFR) or the 2-7 deletion mutant (EGFRvIII) in tumor models in vitro and in vivo. Treatment of patients suffering from relapsed/refractory glioblastoma (GBM) with a combination of depatux-m and temozolomide (TMZ) tended to increase overall survival. As a first step to understand the nature of the interaction between the two drugs, we investigated whether the interaction was synergistic, additive or antagonistic. METHODS: The efficacy of ADCs, antibodies, TMZ and radiation was tested in xenograft models of GBM, U-87MG and U-87MG EGFRvIII. Both models express EGFR. U-87MG EGFRvIII was transduced to express EGFRvIII. Changes in tumor volume, biomarkers of cell death and apoptosis after treatment were used to measure efficacy of the various treatments. Synergism of depatux-m and TMZ was verified in three-dimensional cultures of U-87MG and U-87MG EGFRvIII by the method of Chou and Talalay. RESULTS: Combined with TMZ and radiotherapy (RT), depatux-m inhibited xenograft growth of U-87MG and U-87MG EGFRvIII more than either treatment with depatux-m or TMZ + RT. Durability of the response to depatux-m + TMZ + RT or depatux-m + TMZ was more pronounced in U-87MG EGFRvIII than in U-87MG. Efficacy of depatux-m + TMZ was synergistic in U-87MG EGFRvIII and additive in U-87MG. CONCLUSION: Adding depatux-m enhances the efficacy of standard of care therapy in preclinical models of GBM. Durability of response to depatux-m + TMZ in vivo and synergy of the drug-drug interaction correlates with the amount of antigen expressed by the tumor cells.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas , Glioblastoma , Temozolomida/farmacologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody-drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.
Assuntos
Receptores ErbB/metabolismo , Imunoconjugados/uso terapêutico , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos NusRESUMO
Depatuxizumab mafodotin (depatux-m, ABT-414) is a tumor-selective antibody drug conjugate (ADC) comprised of the anti-EGFR antibody ABT-806 and the monomethyl auristatin F (MMAF) warhead. Depatux-m has demonstrated promising clinical activity in glioblastoma multiforme (GBM) patients and is currently being evaluated in clinical trials in first-line and recurrent GBM disease settings. Depatux-m responses have been restricted to patients with amplified EGFR, highlighting the need for therapies with activity against tumors with nonamplified EGFR overexpression. In addition, depatux-m dosing has been limited by corneal side effects common to MMAF conjugates. We hypothesized that a monomethyl auristatin E (MMAE) ADC utilizing an EGFR-targeting antibody with increased affinity may have broader utility against tumors with more modest EGFR overexpression while mitigating the risk of corneal side effects. We describe here preclinical characterization of ABBV-221, an EGFR-targeting ADC comprised of an affinity-matured ABT-806 conjugated to MMAE. ABBV-221 binds to a similar EGFR epitope as depatux-m and retains tumor selectivity with increased binding to EGFR-positive tumor cells and greater in vitro potency. ABBV-221 displays increased tumor uptake and antitumor activity against wild-type EGFR-positive xenografts with a greatly reduced incidence of corneal side effects relative to depatux-m. ABBV-221 has similar activity as depatux-m against an EGFR-amplified GBM patient derived xenograft (PDX) model and is highly effective alone and in combination with standard-of-care temozolomide in an EGFRvIII-positive GBM xenograft model. Based on these results, ABBV-221 has advanced to a phase I clinical trial in patients with advanced solid tumors associated with elevated levels of EGFR. Mol Cancer Ther; 17(4); 795-805. ©2018 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Glioblastoma/tratamento farmacológico , Imunoconjugados/farmacologia , Oligopeptídeos/química , Animais , Anticorpos Monoclonais Humanizados/química , Apoptose , Proliferação de Células , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Imunoconjugados/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development.
Assuntos
Regulação Neoplásica da Expressão Gênica , Transcriptoma , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Esferoides CelularesRESUMO
ABT-700 is a therapeutic antibody against the hepatocyte growth factor receptor (MET). At doses or regimens that lead to exposures exceeding optimum in vivo, the efficacy of ABT-700 is unexpectedly reduced. We hypothesized that this reduction in efficacy was due to a "prozone-like" effect in vivo. A prozone-like effect, which is a reduction in efficacy beyond optimum exposure, is caused due a mechanism similar to the generation of false negative flocculation tests by excessive antibody titres. In vitro, we demonstrate that at higher ABT-700 concentrations, this "prozone-like" effect is mediated by a progressive conversion from bivalent to ineffective monovalent binding of the antibody. In vivo, the efficacy of ABT-700 is dependent on an optimum range of exposure as well. Our data suggest that the "prozone-like" effect is operative and independent of target expression. ABT-700 dose, regimen, exposure, and tumor burden are interdependent variables influencing the "prozone-like" effect and mediating and in vivo efficacy. By optimization of dosage and regimen we demonstrate that the "prozone-like" effect can be alleviated and ABT-700 efficacy at varying tumor loads can be further extended in combination with cisplatin. Our results suggest that optimization of exposure taking tumor burden into account may alleviate "prozone-like" effects without compromising efficacy.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Linhagem Celular , Cisplatino/administração & dosagem , Humanos , Camundongos , Camundongos Nus , Camundongos SCIDRESUMO
Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic.Experimental Design: ADC ABBV-399 was generated with the c-Met-targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity.Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399-mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy.Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met-overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met-expressing non-small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992-1000. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas c-met/genética , Animais , Anticorpos Monoclonais/efeitos adversos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting tumor-overexpressed EGFR with an antibody-drug conjugate (ADC) is an attractive therapeutic strategy; however, normal tissue expression represents a significant toxicity risk. The anti-EGFR antibody ABT-806 targets a unique tumor-specific epitope and exhibits minimal reactivity to EGFR in normal tissue, suggesting its suitability for the development of an ADC. We describe the binding properties and preclinical activity of ABT-414, an ABT-806 monomethyl auristatin F conjugate. In vitro, ABT-414 selectively kills tumor cells overexpressing wild-type or mutant forms of EGFR. ABT-414 inhibits the growth of xenograft tumors with high EGFR expression and causes complete regressions and cures in the most sensitive models. Tumor growth inhibition is also observed in tumor models with EGFR mutations, including activating mutations and those with the exon 2-7 deletion [EGFR variant III (EGFRvIII)], commonly found in glioblastoma multiforme. ABT-414 exhibits potent cytotoxicity against glioblastoma multiforme patient-derived xenograft models expressing either wild-type EGFR or EGFRvIII, with sustained regressions and cures observed at clinically relevant doses. ABT-414 also combines with standard-of-care treatment of radiation and temozolomide, providing significant therapeutic benefit in a glioblastoma multiforme xenograft model. On the basis of these results, ABT-414 has advanced to phase I/II clinical trials, and objective responses have been observed in patients with both amplified wild-type and EGFRvIII-expressing tumors. Mol Cancer Ther; 15(4); 661-9. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Epitopos , Receptores ErbB/antagonistas & inibidores , Imunoconjugados/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Afinidade de Anticorpos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Modelos Animais de Doenças , Epitopos/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Terapia de Alvo Molecular , Mutação , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: c-Met is the receptor tyrosine kinase for hepatocyte growth factor (HGF) encoded by the MET proto-oncogene. Aberrant activation of c-Met resulting from MET amplification and c-Met overexpression is associated with poor clinical outcome in multiple malignancies underscoring the importance of c-Met signaling in cancer progression. Several c-Met inhibitors have advanced to the clinic; however, the development of inhibitory c-Met-directed therapeutic antibodies has been hampered by inherent agonistic activity. METHOD: We generated and tested a bivalent anti-c-Met monoclonal antibody ABT-700 in vitro for binding potency and antagonistic activity and in vivo for antitumor efficacy in human tumor xenografts. Human cancer cell lines and gastric cancer tissue microarrays were examined for MET amplification by fluorescence in situ hybridization (FISH). RESULTS: ABT-700 exhibits a distinctive ability to block both HGF-independent constitutive c-Met signaling and HGF-dependent activation of c-Met. Cancer cells addicted to the constitutively activated c-Met signaling driven by MET amplification undergo apoptosis upon exposure to ABT-700. ABT-700 induces tumor regression and tumor growth delay in preclinical tumor models of gastric and lung cancers harboring amplified MET. ABT-700 in combination with chemotherapeutics also shows additive antitumor effect. Amplification of MET in human cancer tissues can be identified by FISH. CONCLUSIONS: The preclinical attributes of ABT-700 in blocking c-Met signaling, inducing apoptosis and suppressing tumor growth in cancers with amplified MET provide rationale for examining its potential clinical utility for the treatment of cancers harboring MET amplification.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proteínas Proto-Oncogênicas c-met/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Amplificação de Genes , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Administração Oral , Compostos de Anilina/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzotiazóis/química , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Docetaxel , Perfilação da Expressão Gênica , Granulócitos/metabolismo , Humanos , Isoquinolinas/química , Cinética , Camundongos , Transplante de Neoplasias , Neoplasias/metabolismo , Neutropenia/induzido quimicamente , Neutrófilos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Taxoides/efeitos adversos , Trombocitopenia/induzido quimicamente , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/metabolismoRESUMO
Cancer cells tend to utilize aerobic glycolysis even under normoxic conditions, commonly called the "Warburg effect." Aerobic glycolysis often directly correlates with malignancy, but its purpose, if any, in metastasis remains unclear. When wild-type KISS1 metastasis suppressor is expressed, aerobic glycolysis decreases and oxidative phosphorylation predominates. However, when KISS1 is missing the secretion signal peptide (ΔSS), invasion and metastasis are no longer suppressed and cells continue to metabolize using aerobic glycolysis. KISS1-expressing cells have 30% to 50% more mitochondrial mass than ΔSS-expressing cells, which are accompanied by correspondingly increased mitochondrial gene expression and higher expression of PGC1α, a master coactivator that regulates mitochondrial mass and metabolism. PGC1α-mediated downstream pathways (i.e., fatty acid synthesis and ß-oxidation) are differentially regulated by KISS1, apparently reliant upon direct KISS1 interaction with NRF1, a major transcription factor involved in mitochondrial biogenesis. Since the downstream effects could be reversed using short hairpin RNA to KISS1 or PGC1α, these data appear to directly connect changes in mitochondria mass, cellular glucose metabolism, and metastasis.
Assuntos
Kisspeptinas/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Feminino , Expressão Gênica , Glucose/metabolismo , Glicólise , Humanos , Concentração de Íons de Hidrogênio , Kisspeptinas/metabolismo , Ácido Láctico/metabolismo , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gap junctional intercellular communication (GJIC) regulates cellular homeostasis by propagating signaling molecules, exchanging cellular metabolites, and coupling electrical signals. In cancer, cells exhibit altered rates of GJIC which may play a role in neoplastic progression. K(ATP) channels help maintain membrane polarity and linkages between K(ATP) channel activity and rates of GJIC have been established. The mechanistic relationship has not been fully elucidated. We report the effects of treatment with multiple K(ATP) antagonist compounds on GJIC in metastatic cell lines demonstrating an increase in communication rates following treatment with compounds possessing specificities towards the SUR2 subunit of K(ATP). These effects remained consistent using cell lines with different expression levels of SUR1 and SUR2, suggesting possible off target effects on GJIC by these compounds.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Canais KATP/antagonistas & inibidores , Complexo Mediador/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral/patologia , Junções Comunicantes/metabolismo , Humanos , Metástase Neoplásica , Receptores de SulfonilureiasRESUMO
Gli1 is an established oncogene and its expression in Estrogen Receptor (ER) α negative and triple negative breast cancers is predictive of a poor prognosis; however, the biological functions regulated by Gli1 in breast cancer have not been extensively evaluated. Herein, Gli1 was over-expressed or down-regulated (by RNA interference and by expression of the repressor form of Gli3) in the ERα negative, human breast cancer cell lines MDA-MB-231 and SUM1315. Reduced expression of Gli1 in these two cell lines resulted in a decrease in migration and invasion. Gli1 over-expression increased the migration and invasion of MDA-MB-231 cells with a corresponding increase in expression of MMP-11. Silencing MMP-11 in MDA-MB-231 cells that over-expressed Gli1 abrogated the Gli1-induced enhancement of migration and invasion. Sustained suppression of Gli1 expression decreased growth of MDA-MB-231 in vitro by increasing apoptosis and decreasing proliferation. In addition, silencing of Gli1 reduced the numbers and sizes of pulmonary metastases of MDA-MB-231 in an in vivo experimental metastasis assay. In summary, Gli1 promotes the growth, survival, migration, invasion and metastasis of ERα negative breast cancer. Additionally, MMP-11 is up-regulated by Gli1 and mediates the migration and invasion induced by Gli1 in MDA-MB-231.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Receptor alfa de Estrogênio/deficiência , Metaloproteinase 11 da Matriz/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima , Feminino , Humanos , Metaloproteinase 11 da Matriz/genética , Invasividade Neoplásica , Metástase Neoplásica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteína GLI1 em Dedos de ZincoRESUMO
Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosphorylation events play a key role in their trafficking and degradation. The metastasis suppressor breast cancer metastasis suppressor 1 (BRMS1) upregulates GJIC and decreases phosphoinositide-3-kinase (PI3K) signaling. On the basis of these observations, we set out to determine whether there was a link between PI3K and GJIC in tumorigenic and metastatic cell lines. Treatment of cells with the well-known PI3K inhibitor LY294002, and its structural analogue LY303511, which does not inhibit PI3K, increased homotypic GJIC; however, we found the effect to be independent of PI3K/AKT inhibition. We show in multiple cancer cell lines of varying metastatic capability that GJIC can be restored without enforced expression of a connexin gene. In addition, while levels of connexin 43 remained unchanged, its relocalization from the cytosol to the plasma membrane was observed. Both LY294002 and LY303511 increased the activity of protein kinase A (PKA). Moreover, PKA blockade by the small molecule inhibitor H89 decreased the LY294002/LY303511-mediated increase in GJIC. Collectively, our findings show a connection between PKA activity and GJIC mediated by PI3K-independent mechanisms of LY294002 and LY303511. Manipulation of these signaling pathways could prove useful for antimetastatic therapy.
Assuntos
Comunicação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Junções Comunicantes/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cromonas/farmacologia , Conexina 43/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Citosol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fluoresceínas/metabolismo , Imunofluorescência , Junções Comunicantes/efeitos dos fármacos , Humanos , Immunoblotting , Isoquinolinas/farmacologia , Morfolinas/farmacologia , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , WortmaninaRESUMO
Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis-associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. MicroRNA arrays were done comparing small RNAs that were purified from metastatic MDA-MB-231 and MDA-MB-435 and their nonmetastatic BRMS1-transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT-PCR. BRMS1 decreased metastasis-promoting (miR-10b, -373 and -520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR-10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR-146a, -146b and -335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis-associated miRNA and suggests that recruitment of BRMS1-containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas de Neoplasias/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The Breast Cancer Metastasis Suppressor 1 (BRMS1) belongs to an expanding category of proteins called metastasis suppressors that demonstrate in vivo metastasis suppression while still allowing growth of the orthotopic tumor. Since BRMS1 decreases either the expression or function of multiple mediators implicated in resistance to chemotherapy (NF-kappaB, AKT, EGFR), we asked whether breast carcinoma cells expressing BRMS1 could be sensitized upon exposure to commonly used therapeutic agents that inhibit some of these same cellular mediators as BRMS1. In this report, we demonstrate that chemosensitivity of breast cancer cells is preserved in the presence of BRMS1. Further, BRMS1 does not change expression of AKT isoforms or PTEN, implicated in chemoresistance to common drug agents. Overall, our data with two different metastatic breast cancer cell lines indicates that BRMS1 expression status may not interfere with the response to commonly used chemotherapeutic agents in the management of solid tumors such as breast cancer. Since tumor protein expression analysis increasingly guides therapy decisions, our data may be of clinical benefit in disease management including profiling for BRMS1 expression before start of therapy.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Neoplasias/fisiologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Paclitaxel/farmacologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras , Ensaio Tumoral de Célula-Tronco , Vincristina/farmacologiaRESUMO
Metastasis occurs, in part, due to tumor cell responses to chemokine secretion by ectopic organs or tissues. SDF-1 is constitutively expressed in tissues where metastases frequently develop while breast carcinoma cells express the receptor for SDF-1, CXCR4, which is correlated with increased bone metastasis and poor overall survival. We hypothesized that treatment with a CXCR4 antagonist, CTCE-9908, would decrease incidence of bone and lung metastasis. Treatment with CTCE-9908 (25 mg/kg) began the day prior to or the day of intravenous or intracardiac tumor cell inoculation of MDA-MB-231 human breast carcinoma cells expressing enhanced green fluorescent protein (GFP) into athymic mice. After 5 or 8 weeks (i.c. and i.v. injections, respectively), the presence of fluorescent foci at metastatic sites was assessed. Somewhat surprisingly, CTCE-9908 treatment did not decrease incidence of metastasis as hypothesized. However, CTCE-9908 did decrease metastatic burden (i.e., size of metastases) in all organs examined (lungs, bone, heart, liver, kidneys, pancreas and spleen). Based upon this and other studies, the use of CTCE-9908 is promising as an adjuvant therapy for metastatic disease.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Peptídeos/uso terapêutico , Receptores CXCR4/antagonistas & inibidores , Animais , Neoplasias Ósseas/secundário , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Receptores CXCR4/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth in multiple human and murine cancer cells of diverse origins. We hypothesized that miR-146 may be involved in the ability of BRMS1 to supress metastasis because miR-146 expression is altered by BRMS1 and because BRMS1 and miR-146 are both associated with decreased signaling through the nuclear factor-kappaB pathway. BRMS1 significantly up-regulates miR-146a by 6- to 60-fold in metastatic MDA-MB-231 and MDA-MB-435 cells, respectively, and miR-146b by 40-fold in MDA-MB-435 as measured by real-time quantitative reverse transcription-PCR. Transduction of miR-146a or miR-146b into MDA-MB-231 down-regulated expression of epidermal growth factor receptor, inhibited invasion and migration in vitro, and suppressed experimental lung metastasis by 69% and 84%, respectively (mean +/- SE: empty vector = 39 +/- 6, miR-146a = 12 +/- 1, miR-146b = 6 +/- 1). These results further support the recent notion that modulating the levels of miR-146a or miR-146b could have a therapeutic potential to suppress breast cancer metastasis.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Metástase Neoplásica/prevenção & controle , Proteínas de Neoplasias/fisiologia , RNA Neoplásico/genética , Divisão Celular , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , NF-kappa B/fisiologia , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para CimaRESUMO
BRMS1 and SUDS3 are related members of SIN3-HDAC chromatin remodeling complexes. We hypothesized that they might have overlapping functions and that SUDS3 over-expression could compensate for BRMS1 deficiency. SUDS3 expression was ubiquitous in seven breast cell lines, regardless of metastatic potential. SUDS3 over-expression in BRMS1-non-expressing metastatic cells did not suppress metastasis, motility, osteopontin secretion, or EGF receptor expression, phenotypes associated with BRMS1-mediated metastasis suppression. This study demonstrates functional differences for BRMS1 family members and highlights how the composition of SIN3-HDAC (BRMS1/SUDS3) complexes uniquely affects protein expression and biological behaviors.