RESUMEN
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Asunto(s)
Caspasa 8/metabolismo , Núcleo Celular/enzimología , Proliferación Celular , Puntos de Control de la Fase G2 del Ciclo Celular , Neoplasias/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Antineoplásicos/farmacología , Apoptosis , Caspasa 8/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Células PC-3 , Estabilidad Proteica , Transducción de Señal , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
Infantile neuronal ceroid lipofuscinosis (CLN1 Batten Disease) is a devastating pediatric lysosomal storage disease caused by pathogenic variants in the CLN1 gene, which encodes the depalmitoylation enzyme, palmitoyl-protein thioesterase 1 (PPT1). CLN1 patients present with visual deterioration, psychomotor dysfunction, and recurrent seizures until neurodegeneration results in death, typically before fifteen years of age. Histopathological features of CLN1 include aggregation of lysosomal autofluorescent storage material (AFSM), as well as profound gliosis. The current management of CLN1 is relegated to palliative care. Here, we examine the therapeutic potential of a small molecule PPT1 mimetic, N-tert-butyl hydroxylamine (NtBuHA), in a Cln1-/- mouse model. Treatment with NtBuHA reduced AFSM accumulation both in vitro and in vivo. Importantly, NtBuHA treatment in Cln1-/- mice reduced neuroinflammation, mitigated epileptic episodes, and normalized motor function. Live cell imaging of Cln1-/- primary cortical neurons treated with NtBuHA partially rescued aberrant synaptic calcium dynamics, suggesting a potential mechanism contributing to the therapeutic effects of NtBuHA in vivo. Taken together, our findings provide supporting evidence for NtBuHA as a potential treatment for CLN1 Batten Disease.
RESUMEN
We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Histonas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Reparación del ADN por Recombinación , Transposasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Cromatina/química , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Endodesoxirribonucleasas , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Fosforilación , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transposasas/antagonistas & inhibidores , Transposasas/metabolismoRESUMEN
BACKGROUND: The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. METHODS: We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically "cold" 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically "hot" CT26 colon carcinoma model. RESULTS: V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. CONCLUSIONS: Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically "cold" tumors "hot" and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.
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Anticuerpos Biespecíficos , Carcinoma , Melanoma Experimental , Humanos , Ratones , Animales , Sulfatos de Condroitina/farmacología , Sulfatos de Condroitina/metabolismo , Memoria Inmunológica , Inhibidores de Puntos de Control Inmunológico , Melanoma Experimental/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Mamíferos/metabolismoRESUMEN
Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2ß1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.
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Matriz Extracelular/patología , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas , Animales , Antígenos de Superficie/análisis , Biomarcadores de Tumor/análisis , Células Clonales/patología , Humanos , Inmunofenotipificación , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Lectinas/metabolismo , Neoplasias/metabolismo , Transporte de Proteínas , Línea Celular Tumoral , Membrana Celular/metabolismo , Endosomas/metabolismo , Exosomas/metabolismo , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Lineage plasticity of prostate cancer is associated with resistance to androgen receptor (AR) pathway inhibition (ARPI) and supported by a reactive tumor microenvironment. Here we show that changes in chondroitin sulfate (CS), a major glycosaminoglycan component of the tumor cell glycocalyx and extracellular matrix, is AR-regulated and promotes the adaptive progression of castration-resistant prostate cancer (CRPC) after ARPI. AR directly represses transcription of the 4-O-sulfotransferase gene CHST11 under basal androgen conditions, maintaining steady-state CS in prostate adenocarcinomas. When AR signaling is inhibited by ARPI or lost during progression to non-AR-driven CRPC as a consequence of lineage plasticity, CHST11 expression is unleashed, leading to elevated 4-O-sulfated chondroitin levels. Inhibition of the tumor cell CS glycocalyx delays CRPC progression, and impairs growth and motility of prostate cancer after ARPI. Thus, a reactive CS glycocalyx supports adaptive survival and treatment resistance after ARPI, representing a therapeutic opportunity in patients with advanced prostate cancer.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Andrógenos , Sulfatos de Condroitina , Glicocálix/metabolismo , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Transducción de Señal , Microambiente TumoralRESUMEN
Broad-spectrum therapeutics in non-small cell lung cancer (NSCLC) are in demand. Most human solid tumors express proteoglycans modified with distinct oncofetal chondroitin sulfate (CS) chains that can be detected and targeted with recombinant VAR2CSA (rVAR2) proteins and rVAR2-derived therapeutics. Here, we investigated expression and targetability of oncofetal CS expression in human NSCLC. High oncofetal CS expression is associated with shorter disease-free survival and poor overall survival of clinically annotated stage I and II NSCLC patients (n = 493). Oncofetal CS qualifies as an independent prognosticator of NSCLC in males and smokers, and high oncofetal CS levels are more prevalent in EGFR/KRAS wild-type cases, as compared to mutation cases. NSCLC cell lines express oncofetal CS-modified proteoglycans that can be specifically detected and targeted by rVAR2 proteins in a CSA-dependent manner. Importantly, a novel VAR2-drug conjugate (VDC-MMAE) efficiently eliminates NSCLC cells in vitro and in vivo. In summary, oncofetal CS is a prognostic biomarker and an actionable glycosaminoglycan target in NSCLC.
RESUMEN
Solid tumors remain a major challenge for targeted therapeutic intervention strategies such as antibody-drug conjugates and immunotherapy. At a minimum, clear and actionable solid tumor targets have to comply with the key biological requirement of being differentially over-expressed in solid tumors and metastasis, in contrast to healthy organs. Oncofetal chondroitin sulfate is a cancer-specific secondary glycosaminoglycan modification to proteoglycans expressed in a variety of solid tumors and metastasis. Normally, this modification is found to be exclusively expressed in the placenta, where it is thought to facilitate normal placental implantation during pregnancy. Informed by this biology, oncofetal chondroitin sulfate is currently under investigation as a broad and specific target in solid tumors. Here, we discuss oncofetal chondroitin sulfate as a potential therapeutic target in childhood solid tumors in the context of current knowhow obtained over the past five years in adult cancers.
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Sulfatos de Condroitina/metabolismo , Feto/metabolismo , Terapia Molecular Dirigida , Neoplasias/metabolismo , Adulto , Niño , Femenino , Humanos , Placenta/metabolismo , EmbarazoRESUMEN
PURPOSE: Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. RESULTS: We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. CONCLUSIONS: Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Proteínas de Unión al Calcio/metabolismo , Resistencia a Antineoplásicos , Factor 4F Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Taxoides/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Biología Computacional , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Unión Proteica , ARN Interferente Pequeño/genética , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. ß-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.
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Proteínas Hedgehog/genética , Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata/genética , Receptores Androgénicos/metabolismo , Proteína Gli3 con Dedos de Zinc/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The significance of lactate transporters has been recognized in various cancer types, but their role in urothelial carcinoma remains mostly unknown. The aim of this study was to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in preclinical models of urothelial carcinoma and to assess its relevance in patient tumors. The association of MCT4 expression with molecular subtypes and outcome was determined in The Cancer Genome Atlas (TCGA) cohort and two independent cohorts of patients with urothelial carcinoma. Silencing of MCT4 was performed using siRNAs in urothelial carcinoma cell lines. Effects of MCT4 inhibition on cell growth, apoptosis, and production of reactive oxygen species (ROS) were assessed. Moreover, effects on lactate efflux were determined. The in vivo effects of MCT4 silencing were assessed in an orthotopic xenograft model. MCT4 expression was higher in the basal subtype. Decreased MCT4 methylation and increased RNA and protein expression were associated with worse overall survival (OS). Inhibition of MCT4 led to a reduction in cell growth, induction of apoptosis, and an increased synthesis of ROS. MCT4 inhibition resulted in intracellular accumulation of lactate. In vivo, stable knockdown of MCT4 reduced tumor growth. The expression of MCT4 in urothelial carcinoma is associated with features of aggressive tumor biology and portends a poor prognosis. Inhibition of MCT4 results in decreased tumor growth in vitro and in vivo Targeting lactate metabolism via MCT4 therefore provides a promising therapeutic approach for invasive urothelial carcinoma, especially in the basal subtype.
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Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Proteínas Musculares/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/patología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Desnudos , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.
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Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Animales , Proliferación Celular , Humanos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/patología , Semaforinas/antagonistas & inhibidores , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. OBJECTIVE: To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. DESIGN, SETTING, AND PARTICIPANTS: An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients. INTERVENTION: An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Antineoplastic effects of targeting ofCS. RESULTS AND LIMITATIONS: In situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design. CONCLUSIONS: Targeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC. PATIENT SUMMARY: Cisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin.
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Antígenos de Protozoos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Sulfatos de Condroitina/metabolismo , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Oligopéptidos/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Antígenos de Protozoos/metabolismo , Antineoplásicos/efectos adversos , Colombia Británica , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/efectos adversos , Relación Dosis-Respuesta a Droga , Europa (Continente) , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Ratones , Factores de Tiempo , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors.
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Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Proliferación Celular/efectos de los fármacos , Clusterina/metabolismo , Mitosis/efectos de los fármacos , Neoplasias de la Próstata/patología , Taxoides/farmacología , Línea Celular Tumoral , Clusterina/genética , Técnicas de Silenciamiento del Gen , Humanos , MasculinoRESUMEN
Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-ß1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes ß-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. IMPLICATIONS: The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR.
Asunto(s)
Antígenos de Protozoos/genética , Sulfatos de Condroitina/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Antígenos de Protozoos/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Sulfatos de Condroitina/genética , Humanos , Melanoma Experimental/metabolismo , Ratones , Metástasis de la Neoplasia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de SeñalRESUMEN
BACKGROUND: Cabazitaxel, abiraterone acetate (AA), and enzalutamide have been approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) following docetaxel chemotherapy. Whether taxanes and next-generation androgen receptor (AR) axis inhibitors are cross-resistant or not is a subject of debate. OBJECTIVE: To evaluate the antitumour activity of cabazitaxel in mCRPC pretreated with abiraterone or enzalutamide. DESIGN, SETTING, AND PARTICIPANTS: The antitumour activity of cabazitaxel was assessed in patients with mCRPC and progressive disease after treatment with docetaxel and AA. In parallel, cabazitaxel antitumour activity was studied in enzalutamide-resistant models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Changes in prostate-specific antigen (PSA) levels and progression-free survival were used to determine the activity of cabazitaxel treatment. Cell proliferation, immunofluorescence, and AR transactivation assay were used in enzalutamide-resistant models. RESULTS AND LIMITATIONS: A total of 79 patients who had progressive mCRPC after docetaxel (median: 8 cycles; range: 4-12 mo), and AA (median: 4.8 mo; range:1-55 mo) received cabazitaxel 25mg/m(2) every 3 weeks (median: 6 cycles; range:1-15 cycles). A PSA decline ≥30% was achieved in 48 patients (62%; 95% confidence interval [CI], 51-73), and a decline ≥50% was achieved in 28 patients (35%; 95% CI, 25-47). The median progression-free survival and overall survival were 4.4 and 10.9 mo, respectively. In vitro, cabazitaxel decreased cell viability in both enzalutamide-sensitive and enzalutamide-resistant prostate cancer cells within the same range of concentrations. PC3, an AR-negative cell line, exhibited similar sensitivity to cabazitaxel. CONCLUSIONS: Cabazitaxel and AR-pathway inhibitors are not cross-resistant. Preclinical data suggest that cabazitaxel activity does not act mainly through AR axis inhibition. PATIENT SUMMARY: The antitumour activity of cabazitaxel, a chemotherapy agent, was studied in prostate cancer resistant to conventional hormonal therapy and to more recent endocrine therapies (abiraterone or enzalutamide). Cabazitaxel retained anticancer activity in more than half of the cases.
Asunto(s)
Antagonistas de Andrógenos/uso terapéutico , Androstenos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/efectos de los fármacos , Taxoides/uso terapéutico , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/efectos adversos , Androstenos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Benzamidas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia sin Enfermedad , Docetaxel , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Francia , Humanos , Calicreínas/sangre , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Nitrilos , Feniltiohidantoína/efectos adversos , Feniltiohidantoína/uso terapéutico , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/mortalidad , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/metabolismo , Estudios Retrospectivos , Transducción de Señal/efectos de los fármacos , Taxoides/efectos adversos , Factores de Tiempo , Resultado del TratamientoRESUMEN
PURPOSE: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression. EXPERIMENTAL DESIGN: To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. RESULTS: ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. CONCLUSIONS: These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Oligonucleótidos Antisentido/farmacología , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética , Animales , Benzamidas , Western Blotting , Humanos , Inmunohistoquímica , Masculino , Ratones , Nitrilos , Feniltiohidantoína/farmacología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Isoformas de Proteínas , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
Asunto(s)
Antígenos de Protozoos/genética , Sulfatos de Condroitina/metabolismo , Melanoma Experimental/terapia , Placenta/metabolismo , Proteínas Recombinantes/administración & dosificación , Neoplasias Cutáneas/terapia , Animales , Antígenos de Protozoos/metabolismo , Línea Celular Tumoral , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Receptores de Hialuranos/metabolismo , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Terapia Molecular Dirigida , Oligopéptidos/genética , Oligopéptidos/metabolismo , Especificidad de Órganos , Embarazo , Proteínas Recombinantes/farmacología , Neoplasias Cutáneas/metabolismoRESUMEN
Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.