RESUMO
BACKGROUND: ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease. METHODS: Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, ß1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels. RESULTS: We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a ß1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression. CONCLUSIONS: We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.
Assuntos
Proteínas ADAM/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Invasividade Neoplásica/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas ADAM/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.
RESUMO
Drug resistance is a serious challenge in cancer treatment and can be acquired through multiple mechanisms. These molecular changes may introduce varied extents of resistance to different therapies and need to be characterized for optimal therapy choice. A recently discovered small molecule, ethyl-2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate) (CXL017), reveals selective cytotoxicity toward drug-resistant leukemia. A drug-resistant acute myeloid leukemia cell line, HL60/MX2, also failed to acquire resistance to CXL017 upon chronic exposure and regained sensitivity toward standard therapies. In this study, we investigated the mechanisms responsible for HL60/MX2 cells' drug resistance and the molecular basis for its resensitization. Results show that the HL60/MX2 cell line has an elevated level of Mcl-1 protein relative to the parental cell line, HL60, and its resensitized cell line, HL60/MX2/CXL017, whereas it has a reduced level of topoisomerase IIß. Mcl-1 overexpression in HL60/MX2 cells is mainly regulated through phospho-extracellular signal-regulated protein kinases 1 and 2-mediated Mcl-1 stabilization, whereas the reduction of topoisomerase IIß in HL60/MX2 cells is controlled through genetic downregulation. Upregulating Mcl-1 introduces multidrug resistance to standard therapies, whereas its downregulation results in significant cell death. Downregulating topoisomerase IIß confers resistance specifically to mitoxantrone, not to other topoisomerase II inhibitors. Overall, these data suggest that Mcl-1 overexpression is a critical determinant for cross-resistance to standard therapies, whereas topoisomerase IIß downregulation is specific to mitoxantrone resistance.
Assuntos
DNA Topoisomerases Tipo II/biossíntese , Proteínas de Ligação a DNA/biossíntese , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Mitoxantrona/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Benzopiranos/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
A new and general method for asymmetric synthesis of either enantiomer of 2-substituted pyrrolidines from a single starting material is described. Reductive cyclization of (S(S))-gamma-chloro-N-tert-butanesulfinyl ketimines with LiBHEt(3) in THF at -78 to 23 degrees C afforded (S(S),R)-N-tert-butanesulfinyl-2-substituted pyrrolidines in excellent yields (88-98%) and with high diastereoselectivity (99:1). The diastereoselectivity is controlled effectively by the choice of reducing agent. Thus, the corresponding epimers of (S(S),S)-2-substituted pyrrolidines were synthesized in good yields (87-98%) and with high diastereoslectivity (1:99) by simply switching the reducing agent from LiBHEt(3) to DIBAL-H/LiHMDS. Deprotection of N-tert-butanesulfinyl-2-substituted pyrrolidines using 4 N HCl in dioxane and MeOH gave the corresponding enantiomers of 2-substituted pyrrolidines in quantative yield. This method was found to be effective for a variety of substrates including aromatic, heteroaromatic, and aliphatic substituents. Extension of this methodology to the formation of 2-substituted piperidines is also illustrated. Reductive cyclization of (S(S))-delta-chloro-N-tert-butanesulfinyl ketimine with LiBHEt(3) in THF at -78 to 23 degrees C or DIBAL-H/LiHMDS in toluene at -78 to 0 degrees C afforded the (S(S),R)-N-tert-butanesulfinyl-2-substituted piperidines in excellent yield (98%) and with high diastereoselectivity (99:1) or (S(S),S)-N-tert-butanesulfinyl-2-substituted piperidines in good yield (98%) and with high diastereoselectivity (1:99), respectively.
Assuntos
Pirrolidinas/síntese química , Reagentes de Ligações Cruzadas , Ciclização , Modelos Moleculares , Estrutura Molecular , Pirrolidinas/química , EstereoisomerismoRESUMO
HA 14-1, a small-molecule antagonist against anti-apoptotic Bcl-2 proteins, was demonstrated to induce selective cytotoxicity toward malignant cells and to overcome drug resistance. Due to its poor stability and the reactive oxygen species (ROS) generated by its decomposition, chemical modification of HA 14-1 is needed for its future development. We have synthesized a stabilized analog of HA 14-1--sHA 14-1, which did not induce the formation of ROS. As expected from a putative antagonist against anti-apoptotic Bcl-2 proteins like HA 14-1, sHA 14-1 disrupted the binding interaction of a Bak BH3 peptide with Bcl-2 or Bcl-X(L) protein, inhibited the growth of tumor cells through the induction of apoptosis, and circumvented the drug resistance induced by the over-expression of anti-apoptotic Bcl-2 and Bcl-X(L) proteins. Interestingly, the impairment of extrinsic apoptotic pathway induced moderate resistance to sHA 14-1. The moderate resistance suggested that sHA 14-1 generated part of its apoptotic stress through the intrinsic pathway, possibly through its antagonism against anti-apoptotic Bcl-2 proteins. The resistance indicated that sHA 14-1 generated apoptotic stress through the extrinsic apoptotic pathway as well. The ability of sHA 14-1 to induce apoptotic stress through both pathways was further supported by the synergism of sHA 14-1 towards the cytotoxicities of Fas ligand and dexamethasone in Jurkat cells. Taken together, these findings suggest that sHA 14-1 may represent a promising candidate for the treatment of drug-resistant cancers either as a monotherapy or in combination with current cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia/tratamento farmacológico , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzopiranos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Sinergismo Farmacológico , Proteína Ligante Fas/metabolismo , Humanos , Concentração Inibidora 50 , Células Jurkat , Leucemia/metabolismo , Leucemia/patologia , Nitrilas/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Multidrug resistance (MDR) is a major hurdle in the treatment of cancer, and there is a pressing need for new therapies. We have recently developed ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017), derived from a dual inhibitor of Bcl-2 and SERCA proteins, sHA 14-1, with selective cytotoxicity toward MDR cancer cell lines in vitro. In this study, we present new evidence for its therapeutic potential in treatment of MDR cancers and offer mechanistic insights toward its preferential targeting of drug-resistant cancer. CXL017 selectively suppressed the growth of tumors derived from the MDR cancer cell line, HL60/MX2, in vivo. In addition, even after chronic exposure to CXL017, HL60/MX2 failed to develop stable resistance to CXL017, whereas it acquired >2000-fold resistance to cytarabine (Ara-C), the major first-line chemotherapy for the treatment of acute myeloid leukemia (AML). Remarkably, instead of acquiring further cross-resistance, HL60/MX2 cells exposed to CXL017 were resensitized to standard therapies (10- to 100-fold). Western blotting analyses revealed that CXL017 exposure significantly down-regulated Mcl-1 and Bax and up-regulated Noxa, Bim, Bcl-X(L), SERCA2, and SERCA3 proteins, along with a reduction in endoplasmic reticulum (ER) calcium content. Given the well-established functions of Bcl-2 family proteins and ER calcium in drug resistance, our results suggest that the down-regulation of Mcl-1 and the up-regulation of Noxa and Bim along with the decrease in ER calcium content are likely responsible for CXL017-induced resensitization of MDR cancer cells. These data also demonstrate the unique potential of CXL017 to overcome MDR in cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzopiranos/farmacologia , Citarabina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzopiranos/química , Sobrevivência Celular/efeitos dos fármacos , Citarabina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an average IC(50) of 1.04 µM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database based on COMPARE analysis. Further structure-activity relationship study led to the development of a more potent analogue, compound 7d, with an IC(50) of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer cells with the potential for the treatment of drug-resistant cancers.
Assuntos
Benzopiranos/química , Benzopiranos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/farmacologia , Sinergismo Farmacológico , Células HCT116 , Células HL-60 , Células HT29 , Humanos , Concentração Inibidora 50 , Células K562 , Mitoxantrona/farmacologia , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Paclitaxel/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Vincristina/farmacologiaRESUMO
Rapid development of multiple drug resistance against current therapies is a major barrier in the treatment of cancer. Therefore, anticancer agents that can overcome acquired drug resistance in cancer cells are of great importance. Previously, we have demonstrated that ethyl 2-amino-4-(2-ethoxy-2-oxoethyl)-6-phenyl-4H-chromene-3-carboxylate (5a, sHA 14-1), a stable analogue of ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (6, HA 14-1), mitigates drug resistance and synergizes with a variety of cancer therapies in leukemia cells. Structure-activity relationship (SAR) studies of 5a guided the development of ethyl 2-amino-6-(3',5'-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (5q, CXL017), a compound with low micromolar cytotoxicity against a wide-range of hematologic and solid tumor cells. More excitingly, our studies of 5q in camptothecin (CCRF-CEM/C2) and mitoxantrone (HL-60/MX2) resistant cancer cells highlight its ability to selectively kill drug-resistant cells over parent cancer cells. 5q inhibits tumor cell growth through the induction of apoptosis, with detailed mechanism of its selectivity toward drug-resistant cancer cells under investigation. These results suggest that 5q is a promising candidate for treatment of cancers with multiple drug resistance.