RESUMEN
The combination of endocrine therapy and CDK4/6 inhibitors such as palbociclib is an effective and well-tolerated treatment for estrogen receptor-positive (ER+) breast cancer, yet many patients relapse with therapy-resistant disease. Determining the mechanisms underlying endocrine therapy resistance is limited by the lack of ability to fully recapitulate inter- and intratumor heterogeneity in vitro and of availability of tumor samples from women with disease progression or relapse. In this study, multiple cell line models of resistant disease were used for both two-dimensional (2D)- and three-dimensional (3D)-based inhibitor screening. The screens confirmed the previously reported role of pro-proliferative pathways, such as PI3K-AKT-mTOR, in endocrine therapy resistance and additionally identified the transcription-associated cyclin-dependent kinase CDK9 as a common hit in ER+ cell lines and patient-derived organoids modeling endocrine therapy-resistant disease in both the palbociclib-sensitive and palbociclib-resistant settings. The CDK9 inhibitor, AZD4573, currently in clinical trials for hematologic malignancies, acted synergistically with palbociclib in these ER+in vitro 2D and 3D models. In addition, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell-cycle regulators differentially downregulated only in combination-treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease. SIGNIFICANCE: Targeting transcription-associated CDK9 synergizes with CDK4/6 inhibitor to drive tumor regression in multiple models of endocrine- and palbociclib-resistant ER+ breast cancer, which could address the challenge of overcoming resistance in patients.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Fosfatidilinositol 3-Quinasas , Resistencia a Antineoplásicos/genética , Receptores de Estrógenos/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Recurrencia , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quinasa 9 Dependiente de la Ciclina/genéticaRESUMEN
Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients' tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA/AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.
Asunto(s)
Neoplasias de la Mama , Animales , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Fosforilación Oxidativa , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Receptores de Estrógenos/metabolismo , Modelos Animales de EnfermedadRESUMEN
The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Cisplatino/farmacología , Cisplatino/uso terapéutico , Platino (Metal)/uso terapéutico , Proteína BRCA1/genética , Recombinación Homóloga , Mutación , Secuenciación Completa del Genoma , Proteína BRCA2/genéticaRESUMEN
Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are associated with higher recurrence and distant metastasis rate. Standard of care for early stage TNBC is anthracyclines combined with cyclophosphamide (AC) followed by taxanes, in the neo-adjuvant or adjuvant setting. This work aimed to identify predictive biomarkers of AC response in patient-derived xenograft (PDX) models of TNBC and to validate them in the clinical setting. By gene and protein expression analysis of 39 PDX with different responses to AC, we found that high expression of HORMAD1 was associated with better response to AC. Both gene and protein expression were associated with promoter hypomethylation. In a cohort of 526 breast cancer patients, HORMAD1 was overexpressed in 71% of TNBC. In a second cohort of 186 TNBC patients treated with AC, HORMAD1 expression was associated with longer metastasis-free survival (MFS). In summary, HORMAD1 overexpression was predictive of an improved response to AC in PDX and is an independent prognostic factor in TNBC patients treated with AC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Antraciclinas/farmacología , Antraciclinas/uso terapéutico , Supervivencia sin Enfermedad , Antibióticos Antineoplásicos/uso terapéutico , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Proteínas de Ciclo CelularRESUMEN
Triple-negative breast cancer (TNBC) is a highly heterogeneous cancer lacking actionable targets. Using a phenotypic screen of TNBC cells, we discovered a novel multiple kinase inhibitor tinengotinib (TT-00420) that strongly inhibited Aurora A/B, FGFR1/2/3, VEGFRs, JAK1/2, and CSF1R in biochemical assays. Exposure to tinengotinib specifically inhibited proliferation across all subtypes of TNBC in vitro and in vivo, while leaving luminal breast cancer cells intact. Incubation of HCC1806 with tinengotinib led to dose-dependent downregulation of genes essential for TNBC cell growth and proliferation. Studies revealed that the potential mechanism of action of tinengotinib involved, predominantly, inhibition of Aurora A or B kinase activity, while inhibition of other pathways contributed to suppression of potency and activity. In vitro treatment of TNBC cell lines or in vivo administration in a syngeneic model with tinengotinib resulted in up-regulation of CXCL10 and 11 or diminished tumor-associated macrophage (TAM) infiltration. Tinengotinib represents a novel combinatorial inhibitory mechanism to treat TNBC. The phase I trial of tinengotinib was completed (ClinicalTrials.gov identifier: NCT03654547).
Asunto(s)
Antineoplásicos , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proliferación Celular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.
Asunto(s)
Resistencia a Antineoplásicos , Histonas , Neoplasias de la Mama Triple Negativas , Resistencia a Antineoplásicos/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Recurrencia Local de Neoplasia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: Endocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo. METHODS: The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analyzed their anti-proliferative effects on 6 ERα+ and 3 ERα- PDX models. Genomic and non-genomic estrogen signaling were assessed by measuring ERα/PI3K interaction by PLA and the expression of estrogen target genes by RT-QPCR, respectively. RESULTS: We confirmed that ERα/Src and ERα/PI3K interactions were associated with a trend to poorer survival, the latter displaying the most significant effects. In ERα+ tumors, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status, or ERα/PI3K targeting. Remarkably, resistance to fulvestrant was associated with non-genomic ERα signaling, since genomic degradation of ERα was unaltered in these tumors, whereas the treatment did not diminish the level of ERα/PI3K interaction. Interestingly, in 2 ERα- models, fulvestrant alone impacted tumor growth, and this was associated with a decrease in ERα/PI3K interaction. CONCLUSIONS: Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant in ERα+ tumors was associated with a lack of impairment of ERα/PI3K interaction in the cytoplasm. In addition, an efficient targeting of ERα/PI3K in ERα- tumors could constitute a promising therapeutic option.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Fulvestrant/uso terapéutico , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Receptores de Estrógenos/metabolismo , Tiazoles/uso terapéutico , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Genómica , Humanos , Ratones , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Secuenciación del Exoma/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Ciclina D1/genética , Variaciones en el Número de Copia de ADN/genética , Resistencia a Antineoplásicos/genética , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Desnudos , Piperazinas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pteridinas/uso terapéutico , Piridinas/uso terapéutico , Quinasa Tipo Polo 1RESUMEN
Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.
Asunto(s)
Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptores de Estrógenos/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Fulvestrant/administración & dosificación , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Tamoxifeno/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.
Asunto(s)
Inhibidores de Topoisomerasa I , Neoplasias de la Mama Triple Negativas , Humanos , Irinotecán/farmacología , Irinotecán/uso terapéutico , Proteínas Nucleares/metabolismo , Proteínas de Unión a Retinoblastoma , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa I/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Ubiquitina-Proteína LigasasRESUMEN
Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.
Asunto(s)
Dosificación de Gen , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias de la Mama Triple Negativas/genética , Animales , Fosfatidilinositol 3-Quinasa Clase I/genética , Femenino , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Proteínas de la Membrana/genética , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , Trasplante de Neoplasias , Medicina de Precisión , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mutations in SPG11 account for the most common form of autosomal recessive hereditary spastic paraplegia (HSP), characterized by a gait disorder associated with various brain alterations. Mutations in the same gene are also responsible for rare forms of Charcot-Marie-Tooth (CMT) disease and progressive juvenile-onset amyotrophic lateral sclerosis (ALS). To elucidate the physiopathological mechanisms underlying these human pathologies, we disrupted the Spg11 gene in mice by inserting stop codons in exon 32, mimicking the most frequent mutations found in patients. The Spg11 knockout mouse developed early-onset motor impairment and cognitive deficits. These behavioral deficits were associated with progressive brain atrophy with the loss of neurons in the primary motor cortex, cerebellum and hippocampus, as well as with accumulation of dystrophic axons in the corticospinal tract. Spinal motor neurons also degenerated and this was accompanied by fragmentation of neuromuscular junctions and muscle atrophy. This new Spg11 knockout mouse therefore recapitulates the full range of symptoms associated with SPG11 mutations observed in HSP, ALS and CMT patients. Examination of the cellular alterations observed in this model suggests that the loss of spatacsin leads to the accumulation of lipids in lysosomes by perturbing their clearance from these organelles. Altogether, our results link lysosomal dysfunction and lipid metabolism to neurodegeneration and pinpoint a critical role of spatacsin in lipid turnover.
Asunto(s)
Metabolismo de los Lípidos/fisiología , Lisosomas/metabolismo , Enfermedad de la Neurona Motora/metabolismo , Proteínas/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Lisosomas/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/fisiología , Enfermedad de la Neurona Motora/patología , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Proteínas/genética , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.