RESUMEN
Objectives: Therapeutic monoclonal antibody biosimilars are expected to help reduce the sizeable economic burden of targeted treatments. Trastuzumab (Herceptin®), a recombinant humanized monoclonal antibody that binds to the extracellular domain of HER2, is approved for use in HER2-overexpressing breast cancer (in both the adjuvant and metastatic settings) and HER2-positive gastric cancer. CT-P6 (Herzuma®) is a biosimilar of trastuzumab, designed to bind with high affinity and specificity to the same HER2 epitope as the reference product. We investigated whether CT-P6 exerts its effects through the same mechanism of action as trastuzumab. Methods: The mechanism of action of CT-P6 and trastuzumab, both as monotherapy and in combination with paclitaxel or pertuzumab, was compared in HER2-overexpressing breast cancer and gastric cancer cell models. Results: We confirmed that CT-P6 functions in a manner similar to trastuzumab by binding to the HER2 receptor, which is central to the effects of trastuzumab in all indications. Conclusions: Collectively, the results of this study show that the mechanisms of action of CT-P6 and trastuzumab are similar in HER2-positive breast cancer and gastric cancer models and, therefore, CT-P6 can be expected to perform similarly in the clinical setting.
Asunto(s)
Biosimilares Farmacéuticos/metabolismo , Trastuzumab/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Antineoplásicos Fitogénicos/farmacología , Biosimilares Farmacéuticos/química , Biosimilares Farmacéuticos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Paclitaxel/farmacología , Fagocitosis/efectos de los fármacos , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Trastuzumab/química , Trastuzumab/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.
Asunto(s)
Ciclo Celular , Estructuras del Núcleo Celular/enzimología , Factores de Empalme Serina-Arginina/metabolismo , Telomerasa/metabolismo , Telómero/enzimología , Ciclo Celular/genética , Línea Celular Tumoral , Estructuras del Núcleo Celular/genética , Células HeLa , Humanos , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismo , Factores de Empalme Serina-Arginina/química , Telomerasa/química , Homeostasis del Telómero , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Telomeres are essential for chromosome integrity and protection, and their maintenance requires the ribonucleoprotein enzyme telomerase. Previously, we have shown that human telomerase reverse transcriptase (hTERT) contains a bipartite nuclear localization signal (NLS; residues 222-240) that is responsible for nuclear import, and that Akt-mediated phosphorylation of residue S227 is important for efficient nuclear import of hTERT. Here, we show that hTERT binds to importin-α proteins through the bipartite NLS and that this heterodimer then forms a complex with importin-ß proteins to interact with the nuclear pore complex. Depletion of individual importin-α proteins results in a failure of hTERT nuclear import, and the resulting cytoplasmic hTERT is degraded by ubiquitin-dependent proteolysis. Crystallographic analysis reveals that the bipartite NLS interacts with both the major and minor sites of importin-α proteins. We also show that Akt-mediated phosphorylation of S227 increases the binding affinity for importin-α proteins and promotes nuclear import of hTERT, thereby resulting in increased telomerase activity. These data provide details of a binding mechanism that enables hTERT to interact with the nuclear import receptors and of the control of the dynamic nuclear transport of hTERT through phosphorylation.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/genética , Proteínas Mutantes/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Telomerasa/metabolismo , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Técnica del Anticuerpo Fluorescente , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Señales de Localización Nuclear , Fosforilación , Fosfoserina/química , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Telomerasa/química , Telomerasa/genética , Células Tumorales Cultivadas , alfa Carioferinas/genéticaRESUMEN
The maintenance of human telomeres requires the ribonucleoprotein enzyme telomerase, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component, and several additional proteins for assembly and activity. Telomere elongation by telomerase in human cancer cells involves multiple steps including telomerase RNA biogenesis, holoenzyme assembly, intranuclear trafficking, and telomerase recruitment to telomeres. Although telomerase has been shown to accumulate in Cajal bodies for association with telomeric chromatin, it is unclear where and how the assembly and trafficking of catalytically active telomerase is regulated in the context of nuclear architecture. Here, we show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus during S phase. The telomerase RNP is retained in nucleoli through the interaction of hTERT with nucleolin, a major nucleolar phosphoprotein. Upon association with TCAB1 in S phase, the telomerase RNP is transported from nucleoli to Cajal bodies, suggesting that TCAB1 acts as an S-phase-specific holoenzyme component. Furthermore, depletion of TCAB1 caused an increase in the amount of telomerase RNP associated with nucleolin. These results suggest that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli. Thus, we propose that the dense fibrillar component is the provider of active telomerase RNP for supporting the continued proliferation of cancer and stem cells.