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BACKGROUND: In rheumatoid arthritis (RA), the activation of T and B cell clones specific for self-antigens leads to the chronic inflammation of the synovium. Here, we perform an in-depth quantitative analysis of the seven chains that comprise the adaptive immune receptor repertoire (AIRR) in RA. RESULTS: In comparison to controls, we show that RA patients have multiple and strong differences in the B cell receptor repertoire including reduced diversity as well as altered isotype, chain, and segment frequencies. We demonstrate that therapeutic tumor necrosis factor inhibition partially restores this alteration but find a profound difference in the underlying biochemical reactivities between responders and non-responders. Combining the AIRR with HLA typing, we identify the specific T cell receptor repertoire associated with disease risk variants. Integrating these features, we further develop a molecular classifier that shows the utility of the AIRR as a diagnostic tool. CONCLUSIONS: Simultaneous sequencing of the seven chains of the human AIRR reveals novel features associated with the disease and clinically relevant phenotypes, including response to therapy. These findings show the unique potential of AIRR to address precision medicine in immune-related diseases.
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Artritis Reumatoide , Humanos , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Membrana Sinovial , Linfocitos B , Factor de Necrosis Tumoral alfa , FenotipoRESUMEN
Despite significant advances in cancer diagnosis and treatment, osteosarcoma (OSA), an aggressive primary bone tumor, has eluded attempts at improving patient survival for many decades. The difficulty in managing OSA lies in its extreme genetic complexity, drug resistance, and heterogeneity, making it improbable that a single-target treatment would be beneficial for the majority of affected individuals. Precision medicine seeks to fill this gap by addressing the intra- and inter-tumoral heterogeneity to improve patient outcome and survival. The characterization of differentially expressed genes (DEGs) unique to the tumor provides insight into the phenotype and can be useful for informing appropriate therapies as well as the development of novel treatments. Traditional DEG analysis combines patient data to derive statistically inferred genes that are dysregulated in the group; however, the results from this approach are not necessarily consistent across individual patients, thus contradicting the basis of precision medicine. Spontaneously occurring OSA in the dog shares remarkably similar clinical, histological, and molecular characteristics to the human disease and therefore serves as an excellent model. In this study, we use transcriptomic sequencing of RNA isolated from primary OSA tumor and patient-matched normal bone from seven dogs prior to chemotherapy to identify DEGs in the group. We then evaluate the universality of these changes in transcript levels across patients to identify DEGs at the individual level. These results can be useful for reframing our perspective of transcriptomic analysis from a precision medicine perspective by identifying variations in DEGs among individuals.
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Neoplasias Óseas , Enfermedades de los Perros , Osteosarcoma , Animales , Perros , Humanos , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/genética , Neoplasias Óseas/veterinaria , Enfermedades de los Perros/genética , Osteosarcoma/genética , Osteosarcoma/veterinaria , Medicina de Precisión , Transcriptoma/genéticaRESUMEN
Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties.
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Neoplasias , Epiplón , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Ratones , Neoplasias/patología , Epiplón/patología , Bazo/patología , Linfocitos T ReguladoresRESUMEN
Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC.
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Carcinoma Neuroendocrino , Neoplasias de la Tiroides , Animales , Carcinoma Neuroendocrino/patología , Línea Celular Tumoral , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Ratones , Piridinas , Tiazoles/uso terapéutico , Neoplasias de la Tiroides/metabolismoRESUMEN
Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This study established a method to effectively isolate RNA from normal canine bone cells using the phalanges, without contamination from other tissue types, for downstream transcriptomic analysis.
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Huesos/química , Biología Molecular/métodos , ARN/aislamiento & purificación , Transcriptoma/genética , Animales , Perros , Regulación de la Expresión Génica/genética , Osteoblastos/química , Osteoclastos/química , Osteocitos/química , ARN/química , ARN/genéticaRESUMEN
BACKGROUND: The Wnt/ß-catenin pathway is linked to tumorigenesis in a variety of tumors and promotes T cell exclusion and resistance to checkpoint inhibitors. We sought to determine whether a small molecule inhibitor of this pathway, WNT974, would impair tumor growth, affect gene expression patterns, and improve the immune response in human and murine ovarian cancer models. METHODS: Human ovarian cancer cells were treated with WNT974 in vitro. RNAseq libraries were constructed and differences in gene expression patterns between responders and nonresponders were compared to The Cancer Genome Atlas (TCGA). Mice with subcutaneous or intraperitoneal ID8 ovarian cancer tumors were treated with WNT974, paclitaxel, combination, or control. Tumor growth and survival were measured. Flow cytometry and ß-TCR repertoire analysis were used to determine the immune response. RESULTS: Gene expression profiling revealed distinct signatures in responders and nonresponders, which strongly correlated with T cell infiltration patterns in the TCGA analysis of ovarian cancer. WNT974 inhibited tumor growth, prevented ascites formation, and prolonged survival in mouse models. WNT974 increased the ratio of CD8+ T cells to T regulatory cells (Tregs) in tumors and enhanced the effector functions of infiltrating CD4+ and CD8+ T cells. Treatment also decreased the expression of inhibitory receptors on CD8+ T cells. Combining WNT974 with paclitaxel further reduced tumor growth, prolonged survival, and expanded the T cell repertoire. CONCLUSIONS: These findings suggest that inhibiting the Wnt/ß-catenin pathway may have a potent immunomodulatory effect in the treatment of ovarian cancer, particularly when combined with paclitaxel.
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The mitochondrial HSP70 chaperone mortalin (HSPA9/GRP75) is often upregulated and mislocalized in MEK/ERK-deregulated tumors. Here, we show that mortalin depletion can selectively induce death of immortalized normal fibroblasts IMR90E1A when combined with K-RasG12V expression, but not with wild-type K-Ras expression, and that K-RasG12V-driven MEK/ERK activity is necessary for this lethality. This cell death was attenuated by knockdown or inhibition of adenine nucleotide translocase (ANT), cyclophilin D (CypD), or mitochondrial Ca2+ uniporter (MCU), which implicates a mitochondria-originated death mechanism. Indeed, mortalin depletion increased mitochondrial membrane permeability and induced cell death in KRAS-mutated human pancreatic ductal adenocarcinoma (PDAC) and colon cancer lines, which were attenuated by knockdown or inhibition of ANT, CypD, or MCU, and occurred independently of TP53 and p21CIP1. Intriguingly, JG-98, an advanced MKT-077 derivative, phenocopied the lethal effects of mortalin depletion in K-RasG12V-expressing IMR90E1A and KRAS-mutated tumor cell lines in vitro. Moreover, JG-231, a JG-98 analog with improved microsomal stability effectively suppressed the xenograft of MIA PaCa-2, a K-RasG12C-expressing human PDAC line, in athymic nude mice. These data demonstrate that oncogenic KRAS activity sensitizes cells to the effects of mortalin depletion, suggesting that mortalin has potential as a selective therapeutic target for KRAS-mutated tumors.
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Proteínas HSP70 de Choque Térmico/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Antineoplásicos/farmacología , Muerte Celular , Femenino , Células HCT116 , Proteínas HSP70 de Choque Térmico/genética , Humanos , Ratones , Ratones Desnudos , Membranas Mitocondriales/patología , Proteínas Mitocondriales/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Permeabilidad , Proteínas Proto-Oncogénicas p21(ras)/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Obesity is a major risk factor for renal cancer, yet our understanding of its effects on antitumor immunity and immunotherapy outcomes remains incomplete. Deciphering these associations is critical, given the growing clinical use of immune checkpoint inhibitors for metastatic disease and mounting evidence for an obesity paradox in the context of cancer immunotherapies, wherein obese patients with cancer have improved outcomes. METHODS: We investigated associations between host obesity and anti-programmed cell death (PD-1)-based outcomes in both renal cell carcinoma (RCC) subjects and orthotopic murine renal tumors. Overall survival (OS) and progression-free survival (PFS) were determined for advanced RCC subjects receiving standard of care anti-PD-1 who had ≥6 months of follow-up from treatment initiation (n=73). Renal tumor tissues were collected from treatment-naive subjects categorized as obese (body mass index, 'BMI' ≥30 kg/m2) or non-obese (BMI <30 kg/m2) undergoing partial or full nephrectomy (n=19) then used to evaluate the frequency and phenotype of intratumoral CD8+ T cells, including PD-1 status, by flow cytometry. In mice, antitumor immunity and excised renal tumor weights were evaluated ±administration of a combinatorial anti-PD-1 therapy. For a subset of murine renal tumors, immunophenotyping was performed by flow cytometry and immunogenetic profiles were evaluated via nanoString. RESULTS: With obesity, RCC patients receiving anti-PD-1 administration exhibited shorter PFS (p=0.0448) and OS (p=0.0288). Treatment-naive renal cancer subjects had decreased frequencies of tumor-infiltrating PD-1highCD8+ T cells, a finding recapitulated in our murine model. Following anti-PD-1-based immunotherapy, both lean and obese mice possessed distinct populations of treatment responders versus non-responders; however, obesity reduced the frequency of treatment responders (73% lean vs 44% obese). Tumors from lean and obese treatment responders displayed similar immunogenetic profiles, robust infiltration by PD-1int interferon (IFN)γ+CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC), yielding favorable CD44+CD8+ T cell to MDSC ratios. Neutralizing interleukin (IL)-1ß in obese mice improved treatment response rates to 58% and reduced MDSC accumulation in tumors. CONCLUSIONS: We find that obesity is associated with diminished efficacy of anti-PD-1-based therapies in renal cancer, due in part to increased inflammatory IL-1ß levels, highlighting the need for continued study of this critical issue.
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Inmunoterapia/métodos , Neoplasias Renales/tratamiento farmacológico , Obesidad/complicaciones , Animales , Femenino , Humanos , Neoplasias Renales/inmunología , Masculino , Ratones , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.
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Anticuerpos Monoclonales/uso terapéutico , Benzamidas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Linfocitos T CD8-positivos/inmunología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Piridinas/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.
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Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Neoplasias de la Tiroides/metabolismo , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Lentivirus/genética , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genéticaRESUMEN
The expression of MHC class II molecules (MHCII) on tumor cells correlates with survival and responsiveness to immunotherapy. However, the mechanisms underlying these observations are poorly defined. Using a murine breast tumor line, we showed that MHCII-expressing tumors grew more slowly than controls and recruited more functional CD4+ and CD8+ T cells. In addition, MHCII-expressing tumors contained more TCR clonotypes expanded to a larger degree than control tumors. Functional CD8+ T cells in tumors depended on CD4+ T cells. However, both CD4+ and CD8+ T cells eventually became exhausted, even in MHCII-expressing tumors. Treatment with anti-CTLA4, but not anti-PD-1 or anti-TIM-3, promoted complete eradication of MHCII-expressing tumors. These results suggest tumor cell expression of MHCII facilitates the local activation of CD4+ T cells, indirectly helps the activation and expansion of CD8+ T cells, and, in combination with the appropriate checkpoint inhibitor, promotes tumor regression.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Neoplasias Mamarias Experimentales/inmunología , Carga Tumoral/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transactivadores/genética , Transactivadores/inmunología , Transactivadores/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
Sequencing of the T cell receptor (TCR) repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sistema Inmunológico , Receptores de Antígenos de Linfocitos T/genética , Empalme Alternativo , Animales , Neoplasias Encefálicas/metabolismo , Linfocitos T CD4-Positivos/citología , Células Clonales , Análisis por Conglomerados , Simulación por Computador , Glioblastoma/metabolismo , Humanos , Funciones de Verosimilitud , Pulmón/metabolismo , Ratones , Lenguajes de Programación , Receptores de Antígenos de Linfocitos T/química , Sarcoidosis/metabolismo , Programas InformáticosRESUMEN
Although the FDA-approved receptor tyrosine kinases inhibitors, vandetanib and cabozantinib, are used to treat surgically inoperable progressive medullary thyroid carcinoma (MTC), not all patients are responsive while the disease sometimes progresses after an initial response. To better understand MTC drug resistance at molecular and biochemical levels, we have generated drug-resistant subpopulations of the human MTC cell lines, TT and MZ-CRC-1, via prolonged exposure to vandetanib and cabozantinib. These drug-resistant progenies exhibited substantial cross-resistance to vandetanib and cabozantinib, suggesting that these inhibitors may invoke an overlapping resistance mechanism(s) in MTC cells. Of note, vandetanib and cabozantinib increased mitochondrial membrane potential (Δψm) in drug-naïve as well as drug-resistant cells but only drug-naïve cells exhibited substantially altered oxygen consumption and extracellular acidification rates. Therefore, these inhibitors appear to cause a bioenergetics stress to which drug-resistant MTC cells are more tolerant. Given the ability of vandetanib and cabozantinib to increase Δψm, we hypothesized that these inhibitors can augment growth inhibitory effects of mitochondria-targeted carboxy-proxyl and ubiquinone by increasing their Δψm-dependent uptake/retention in MTC cells. Indeed, our in vitro and mouse xenograft data strongly support this possibility.
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Anilidas/farmacología , Carcinoma Neuroendocrino/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinazolinas/farmacología , Neoplasias de la Tiroides/metabolismo , Animales , Carcinoma Neuroendocrino/tratamiento farmacológico , Carcinoma Neuroendocrino/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Dinámicas Mitocondriales/efectos de los fármacos , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Most BRAF-mutated melanomas initially responsive to the FDA-approved inhibitors preferentially targeting B-Raf mutated in Val600 residue eventually relapse, requiring additional therapeutic modalities. Recent studies report the significance of metabolic reprograming in mitochondria for maintenance of BRAF-mutated melanomas and for development of their drug resistance to B-Raf inhibitors, providing a rationale for targeting mitochondria as a potential therapeutic strategy for melanoma. We therefore determined whether mitochondria-targeted metabolism-interfering agents can effectively suppress human B-RafV600E melanoma cell lines and their dabrafenib/PLX4032-resistant progenies using mitochondria-targeted carboxy-proxyl (Mito-CP) and ubiquinone (Mito-Q). These agents exhibited comparable efficacy to PLX4032 in suppressing SK-MEL28, A375, and RPMI-7951 cells in vitro. As determined in SK-MEL28 and A375 cells, Mito-CP induced apoptotic cell death mediated by mitochondrial membrane depolarization and subsequent oxidative stress, which PLX4032 could not induce. Of note, Mito-CP also effectively suppressed PLX4032-resistant progenies of SK-MEL28 and A375. Moreover, when orally administered, Mito-CP suppressed SK-MEL28 xenografts in mice as effectively as PLX4032 without serious adverse effects. These data demonstrate that mitochondria-targeted agents have therapeutic potential to effectively suppress BRAF-mutated melanomas via an effect(s) distinct from those of B-Raf inhibitors.
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Melanoma/terapia , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Ubiquinona/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos , Femenino , Humanos , Melanoma/patología , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A single nucleotide polymorphism in the ABCC11 gene, 538G>A (rs17822931), is known to determine human ear wax type. The G/G and G/A genotypes correspond to the wet type, while the A/A genotype corresponds to the dry type. Another earwax determinant, a 27-bp deletion (Δ27) downstream from the rs17822931 site, is a rare variant that leads to the dry phenotype. In a previous report, we found an individual with the G allele who unexpectedly showed the dry type of earwax, leading to the identification of Δ27. We also demonstrated that the Δ27 allele was present in individuals of Japanese, Thai, native North American, Andean, and Bolivian ancestry but absent in those of European and African ancestry. Here, we assessed the Δ27 allele frequency among Japanese and Ukrainian individuals and identified a novel association between the Δ27 and 538G>A mutations. The Δ27 allele frequency was 0.002 (3/1,520; one individual is heterozygous, and another is homozygous) among Japanese individuals and 0 (0/794) among Ukrainians. We also found a previously unreported homozygous genotype for both the Δ27 and A alleles. Our findings suggest that the Δ27 deletion may have occurred in an ABCC11 gene with the 538G>A mutation.
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Transportadoras de Casetes de Unión a ATP/genética , Etnicidad/genética , Eliminación de Secuencia/genética , Alelos , Cerumen/metabolismo , Frecuencia de los Genes , Genotipo , Humanos , Mutación , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the rearranged during transfection (RET) proto-oncogene. Not all patients with progressive MTC respond to current therapy inhibiting RET, demanding additional therapeutic strategies. We recently demonstrated that disrupting mitochondrial metabolism using a mitochondria-targeted agent or by depleting a mitochondrial chaperone effectively suppressed human MTC cells in culture and in mouse xenografts by inducing apoptosis and RET downregulation. These observations led us to hypothesize that mitochondria are potential therapeutic targets for MTC. This study further tests this hypothesis using1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride (MKT-077), a water-soluble rhodocyanine dye analogue, which can selectively accumulate in mitochondria. METHODS: The effects of MKT-077 on cell proliferation, survival, expression of RET and tumor protein 53 (TP53), and mitochondrial activity were determined in the human MTC lines in culture and in mouse xenografts. RESULTS: MKT-077 induced cell cycle arrest in TT and MZ-CRC-1. Intriguingly, MKT-077 also induced RET downregulation and strong cell death responses in TT cells, but not in MZ-CRC-1 cells. This discrepancy was mainly due to the difference between the capacities of these cell lines to retain MKT-077 in mitochondria. The cytotoxicity of MKT-077 in TT cells was mainly attributed to oxidative stress while being independent of TP53. MKT-077 also effectively suppressed tumor growth of TT xenografts. CONCLUSION: MKT-077 can suppress cell survival of certain MTC subtypes by accumulating in mitochondria and interfering with mitochondrial activity although it can also suppress cell proliferation via other mechanisms. These results consistently support the hypothesis that mitochondrial targeting has therapeutic potential for MTC.
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Medullary thyroid carcinoma (MTC), which originates from thyroid parafollicular C cells, accounts for 3 to 5% of thyroid malignancies. MTC occurs either sporadically or in an inherited autosomal dominant manner. Hereditary MTC occurs as a familial MTC or as a part of multiple endocrine neoplasia (MEN) type 2A and B syndromes. A strong genotype-phenotype correlation has been observed between hereditary MTC and germ-line "gain of function" mutations of the RET proto-oncogene. Most cases of pediatric MTC are hereditary whereas sporadic MTC is rare in children and is usually diagnosed in adults. Therefore, MTC in children is most often diagnosed in the course of a familial genetic investigation. The standard treatment of MTC mainly requires surgery involving total thyroidectomy and central neck node dissection before extrathyroidal extension occurs. To prevent MTC development in hereditary syndromes, prophylactic thyroidectomy is performed in presymptomatic patients. An appropriate age at which the surgery should take place is determined based upon the data from genotyping, serum calcitonin measurements, and ultrasonography. For the treatment of advanced MTC cases, the broad spectrum receptor tyrosine kinase inhibitors vandetanib and cabozantinib, which also inhibit RET, are used although they are not always effective.
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Neuroendocrine (NE) lung tumors comprise 20-25% of all invasive lung malignancies. Currently, no effective treatments are available to cure these tumors, and it is necessary to identify a molecular alteration(s) that characterizes NE lung tumor cells. We aimed to identify a kinase mutation(s) associated with NE lung tumor by screening 517 kinase-encoding genes in human lung cancer cell lines. Our next-generation sequencing analysis of six NE lung tumor cell lines (four small cell lung cancer lines and two non-small cell lung cancer lines) and three non-NE lung tumor lines revealed various kinase mutations, including a nonsynonymous mutation in the proto-oncogene RET (c.2071G>A; p.G691S). Further evaluation of the RET polymorphism in total 15 lung cancer cell lines by capillary sequencing suggested that the frequency of the minor allele (A-allele) in NE lung tumor lines was significantly higher than its frequency in a reference population (p = 0.0001). However, no significant difference between non-NE lung tumor lines and a reference group was detected (p = 1.0). Nevertheless, neither RET expression levels were correlated with the levels of neuron-specific enolase (NSE), a key NE marker, nor vandetanib and cabozantinib, small molecule compounds that inhibit RET, affected NSE levels in lung cancer cells. Our data suggest a potential association of G691S RET polymorphism with NE lung tumor, proposing the necessity of more thorough evaluation of this possibility. The dataset of kinase mutation profiles in this report may help choosing cell line models for study of lung cancer.
RESUMEN
Thyroid cancer is the most common endocrine malignancy and its global incidence rates are rapidly increasing. Although the mortality of thyroid cancer is relatively low, its rate of recurrence or persistence is relatively high, contributing to incurability and morbidity of the disease. Thyroid cancer is mainly treated by surgery and radioiodine remnant ablation, which is effective only for non-metastasized primary tumors. Therefore, better understanding of the molecular targets available in this tumor is necessary. Similarly to many other tumor types, oncogenic molecular alterations in thyroid epithelium include aberrant signal transduction of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase/AKT (also known as protein kinase B), NF-кB, and WNT/ß-catenin pathways. However, the role of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT3) pathway, a well-known mediator of tumorigenesis in different tumor types, is relatively less understood in thyroid cancer. Intriguingly, recent studies have demonstrated that, in thyroid cancer, the JAK/STAT3 pathway may function in the context of tumor suppression rather than promoting tumorigenesis. In this review, we provide an update of STAT3 function in thyroid cancer and discuss some of the evidences that support this hypothesis.
RESUMEN
Neuroendocrine (NE) phenotypes characterize a spectrum of lung tumors, including low-grade typical and intermediate-grade atypical carcinoid, high-grade large-cell NE carcinoma and small cell lung carcinoma. Currently, no effective treatments are available to cure NE lung tumors, demanding identification of biological features specific to these tumors. Here, we report that autophagy has an important role for NE lung tumor cell proliferation and survival. We found that the expression levels of the autophagy marker LC3 are relatively high in a panel of lung tumor cell lines expressing high levels of neuron-specific enolase (NSE), a key NE marker in lung tumors. In response to bafilomycin A1 and chloroquine, NE lung tumor cells exhibited cytotoxicity whereas non-NE lung tumor cells exhibited cytostasis, indicating a distinct role of autophagy for NE lung tumor cell survival. Intriguingly, in certain NE lung tumor cell lines, the levels of processed LC3 (LC3-II) were inversely correlated with AKT activity. When AKT activity was inhibited using AKTi or MK2206, the levels of LC3-II and SQSTM1/p62 were increased. In contrast, torin 1, rapamycin or mTOR knockdown increased p62 levels, suggesting that these two pathways have opposing effects on autophagy in certain NE lung tumors. Moreover, inhibition of one pathway resulted in reduced activity of the other, suggesting that these two pathways crosstalk in the tumors. These results suggest that NE lung tumor cells share a common feature of autophagy and are more sensitive to autophagy inhibition than non-NE lung tumor cells.