RESUMEN
Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most patients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1+ T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8+ T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogramming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1+ stem-like T cells.
Asunto(s)
Linfocitos T CD8-positivos , Inmunoterapia , Macrófagos , Neoplasias , Animales , Ratones , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Inmunoterapia/métodos , Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias/terapia , Microambiente Tumoral , Anticuerpos Biespecíficos/inmunología , Interleucina-2 , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP's immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators-interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP's cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Evasión Inmune , Tolerancia Inmunológica , Neoplasias , Animales , Humanos , Ratones , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neoplasias/inmunología , Quimiocina CCL7/metabolismo , Interleucina-33 , Proteína S/metabolismoRESUMEN
Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
Asunto(s)
Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Receptores de Interleucina-2 , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Infecciones/tratamiento farmacológico , Infecciones/inmunología , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/agonistas , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores de Interleucina-2/agonistasRESUMEN
Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via inhibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mechanism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.
Asunto(s)
Glioblastoma , Animales , Antidepresivos Tricíclicos/metabolismo , Antidepresivos Tricíclicos/uso terapéutico , Autofagia , Antígeno B7-H1/metabolismo , Glioblastoma/patología , Imipramina/metabolismo , Imipramina/uso terapéutico , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Macrófagos/metabolismo , Ratones , Recurrencia Local de Neoplasia/tratamiento farmacológico , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cancers induced by human papillomaviruses (HPV) should be responsive to immunotherapy by virtue of expressing the immunogenic oncoproteins E6/E7. However, advanced forms of cervical cancer, driven by HPV, are poorly responsive to immune response-enhancing treatments involving therapeutic vaccination against these viral neoantigens. Leveraging a transgenic mouse model of HPV-derived cancers, K14HPV16/H2b, we demonstrated that a potent nanoparticle-based E7 vaccine, but not a conventional "liquid" vaccine, induced E7 tumor antigen-specific CD8+ T cells in cervical tumor-bearing mice. Vaccination alone or in combination with anti-PD-1/anti-CTLA4 did not elicit tumor regression nor increase CD8+ T cells in the tumor microenvironment (TME), suggesting the presence of immune-suppressive barriers. Patients with cervical cancer have poor dendritic cell functions, have weak cytotoxic lymphocyte responses, and demonstrate an accumulation of myeloid cells in the periphery. Here, we illustrated that myeloid cells in K14HPV16/H2b mice possess potent immunosuppressive activity toward antigen-presenting cells and CD8+ T cells, dampening antitumor immunity. These immune-inhibitory effects inhibited synergistic effects of combining our oncoprotein vaccine with immune checkpoint-blocking antibodies. Our data highlighted a link between HPV-induced cancers, systemic amplification of myeloid cells, and the detrimental effects of myeloid cells on CD8+ T-cell activation and recruitment into the TME. These results established immunosuppressive myeloid cells in lymphoid organs as an HPV+ cancer-induced means of circumventing tumor immunity that will require targeted abrogation to enable the induction of efficacious antitumor immune responses.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD8-positivos/inmunología , Células Mieloides/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/inmunología , Animales , Antígeno CTLA-4/antagonistas & inhibidores , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Terapia de Inmunosupresión , Inmunoterapia/métodos , Ratones , Células Mieloides/efectos de los fármacos , Nanopartículas/administración & dosificación , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/administración & dosificación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microambiente Tumoral , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Activinas/metabolismo , Neoplasias/metabolismo , Animales , Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Femenino , Xenoinjertos , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones SCID , Metástasis de la Neoplasia , Neoplasias/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Microambiente TumoralRESUMEN
Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Nanopartículas/química , Proteínas E7 de Papillomavirus/química , Animales , Anticuerpos/farmacología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/química , Femenino , Neoplasias Pulmonares/secundario , Ratones Endogámicos C57BL , Ratones Transgénicos , Recurrencia Local de Neoplasia , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/mortalidad , Neoplasias Experimentales/terapia , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/farmacología , Linfocitos T Reguladores/inmunología , Resultado del Tratamiento , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Neoplasias Vaginales/inmunología , Neoplasias Vaginales/patología , Neoplasias Vaginales/prevención & controlRESUMEN
Advanced cutaneous melanoma is one of the most challenging cancers to treat because of its high plasticity, metastatic potential, and resistance to treatment. New targeted therapies and immunotherapies have shown remarkable clinical efficacy. However, such treatments are limited to a subset of patients and relapses often occur, warranting validation of novel targeted therapies. Posttranslational modification of proteins by ubiquitin coordinates essential cellular functions, including ubiquitin-proteasome system (UPS) function and protein homeostasis. Deubiquitinating enzymes (DUB) have been associated to multiple diseases, including cancer. However, their exact involvement in melanoma development and therapeutic resistance remains poorly understood. Using a DUB trap assay to label cellular active DUBs, we have observed an increased activity of the proteasome-associated DUB, USP14 (Ubiquitin-specific peptidase 14) in melanoma cells compared with melanocytes. Our survey of public gene expression databases indicates that high expression of USP14 correlates with melanoma progression and with a poorer survival rate in metastatic melanoma patients. Knockdown or pharmacologic inhibition of USP14 dramatically impairs viability of melanoma cells irrespective of the mutational status of BRAF, NRAS, or TP53 and their transcriptional cell state, and overcomes resistance to MAPK-targeting therapies both in vitro and in human melanoma xenografted mice. At the molecular level, we find that inhibition of USP14 rapidly triggers accumulation of poly-ubiquitinated proteins and chaperones, mitochondrial dysfunction, ER stress, and a ROS production leading to a caspase-independent cell death. Our results provide a rationale for targeting the proteasome-associated DUB USP14 to treat and combat melanomas. Mol Cancer Ther; 17(7); 1416-29. ©2018 AACR.
Asunto(s)
Enzimas Desubicuitinizantes/genética , Melanoma/tratamiento farmacológico , Terapia Molecular Dirigida , Ubiquitina Tiolesterasa/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/genética , Melanocitos/efectos de los fármacos , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Ratones , Inhibidores de Proteasoma/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/ß1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/patología , Receptores sigma/biosíntesis , Animales , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiología , Movimiento Celular/fisiología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Células HCT116 , Células HEK293 , Humanos , Células K562 , Ratones , Células 3T3 NIH , Invasividad Neoplásica , Neoplasias/genética , Receptores sigma/genética , Transducción de Señal , Receptor Sigma-1RESUMEN
Disruption of the endothelial barrier by tumour-derived secreted factors is a critical step in cancer cell extravasation and metastasis. Here, by comparative proteomic analysis of melanoma secretomes, we identify the matricellular protein SPARC as a novel tumour-derived vascular permeability factor. SPARC deficiency abrogates tumour-initiated permeability of lung capillaries and prevents extravasation, whereas SPARC overexpression enhances vascular leakiness, extravasation and lung metastasis. SPARC-induced paracellular permeability is dependent on the endothelial VCAM1 receptor and p38 MAPK signalling. Blocking VCAM1 impedes melanoma-induced endothelial permeability and extravasation. The clinical relevance of our findings is highlighted by high levels of SPARC detected in tumour from human pulmonary melanoma lesions. Our study establishes tumour-produced SPARC and VCAM1 as regulators of cancer extravasation, revealing a novel targetable interaction for prevention of metastasis.
Asunto(s)
Endotelio Vascular/metabolismo , Melanoma/metabolismo , Metástasis de la Neoplasia , Osteonectina/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Permeabilidad Capilar , Estudios de Casos y Controles , Línea Celular Tumoral , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Pulmonares/secundario , Sistema de Señalización de MAP Quinasas , Melanoma/patología , Ratones Desnudos , Comunicación ParacrinaAsunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Factores Inmunológicos/farmacología , Melanoma/patología , Nevo/patología , Neoplasias Cutáneas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica , Dermoscopía , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Integrina beta3/análisis , Melanoma/química , Esclerosis Múltiple/tratamiento farmacológico , Natalizumab , Nevo/química , Osteonectina/análisis , Neoplasias Cutáneas/químicaRESUMEN
Metformin was reported to inhibit the proliferation of many cancer cells, including melanoma cells. In this report, we investigated the effect of metformin on melanoma invasion and metastasis development. Using different in vitro approaches, we found that metformin inhibits cell invasion without affecting cell migration and independently of antiproliferation action. This inhibition is correlated with modulation of expression of proteins involved in epithelial-mesenchymal transition such as Slug, Snail, SPARC, fibronectin, and N-cadherin and with inhibition of MMP-2 and MMP-9 activation. Furthermore, our data indicate that this process is dependent on activation of AMPK and tumor suppressor protein p53. Finally, we showed that metformin inhibits melanoma metastasis development in mice using extravasation and metastasis models. The presented data reinforce the fact that metformin might be a good candidate for clinical trial in melanoma treatment.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Melanoma/metabolismo , Melanoma/patología , Metformina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Melanoma/genética , Metaloendopeptidasas/metabolismo , Ratones , Invasividad Neoplásica , Metástasis de la NeoplasiaRESUMEN
During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAF(V600E) using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAF(V600E). Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Melanocitos/metabolismo , Melanoma/patología , Invasividad Neoplásica , Osteonectina , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Transducción de Señal , Factores de Transcripción de la Familia Snail , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Chronic myelogenous leukemia (CML) is a cytogenetic disorder resulting from the expression of p210BCR-ABL. Imatinib, an inhibitor of BCR-ABL, has emerged as the leading compound to treat CML patients. Despite encouraging clinical results, resistance to imatinib represents a major drawback for therapy, as a substantial proportion of patients are refractory to this treatment. Recent publications have described the existence of a small cancer cell population with the potential to exhibit the phenotypic switch responsible for chemoresistance. To investigate the existence of such a chemoresistant cellular subpopulation in CML, we used a two-step approach of pulse and continuous selection by imatinib in different CML cell lines that allowed the emergence of a subpopulation of adherent cells (IM-R Adh) displaying an epithelial-mesenchymal transition (EMT)-like phenotype. Overexpression of several EMT markers was observed in this CML subpopulation, as well as in CD34(+) CML primary cells from patients who responded poorly to imatinib treatment. In response to imatinib, this CD44(high)/CD24(low) IM-R Adh subpopulation exhibited increased adhesion, transmigration and invasion in vitro and in vivo through specific overexpression of the αVß3 receptor. FAK/Akt pathway activation following integrin ß3 (ITGß3) engagement mediated the migration and invasion of IM-R Adh cells, whereas persistent activation of ERK counteracted BCR-ABL inhibition by imatinib, promoting cell adhesion-mediated resistance.
Asunto(s)
Benzamidas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mesodermo/efectos de los fármacos , Mesodermo/patología , Piperazinas/farmacología , Pirimidinas/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Mesilato de Imatinib , Integrina alfaVbeta3/metabolismo , Ratones , FenotipoRESUMEN
Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell-matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial/mesenchymal-like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor-derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2/M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21(Cip1/Waf1) acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC-silenced cells are independent of p21(Cip1/Waf1) induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well-known function as a mediator of melanoma cell migration and tumor-host interactions, SPARC regulates, in a cell-autonomous manner, cell cycle progression and proliferation through the p53/p21(Cip1/Waf1) pathway.
Asunto(s)
Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Melanoma/patología , Osteonectina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Fase G2 , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Ratones , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mitosis , Modelos Biológicos , Invasividad Neoplásica , Osteonectina/genética , Interferencia de ARN , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8(+) T cell-dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-gamma requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.