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CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.
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Linfocitos T CD8-positivos/metabolismo , ARN Helicasas/genética , Animales , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Memoria Inmunológica , Inmunoterapia , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , ARN Helicasas/deficiencia , ARN Guía de Kinetoplastida/metabolismo , TranscriptomaRESUMEN
Pyroptosis, an inflammatory form of cell death, promotes the release of immunogenic substances and stimulates immune cell recruitment, a process, which could turn cold tumors into hot ones. Thus, instigating pyroptosis in triple-negative breast cancer (TNBC) serves as a viable method for restoring antitumor immunity. We analyzed the effects of Histone Deacetylase Inhibitors (HDACi) on TNBC cells using the Cell Counting Kit-8 and colony formation assay. Apoptosis and lactate dehydrogenase (LDH) release assays were utilized to determine the form of cell death. The pyroptotic executor was validated by quantitative real-time polymerase chain reaction and western blot. Transcriptome was analyzed to investigate pyroptosis-inducing mechanisms. A subcutaneously transplanted tumor model was generated in BALB/c mice to evaluate infiltration of immune cells. HDACi significantly diminished cell proliferation, and pyroptotic "balloon"-like cells became apparent. HDACi led to an intra and extracellular material exchange, signified by the release of LDH and the uptake of propidium iodide. Among the gasdermin family, TNBC cells expressed maximum quantities of GSDME, and expression of GSDMA, GSDMB, and GSDME were augmented post HDACi treatment. Pyroptosis was instigated via the activation of the caspase 3-GSDME pathway with the potential mechanisms being cell cycle arrest and altered intracellular REDOX balance due to aberrant glutathione metabolism. In vivo experiments demonstrated that HDACi can activate pyroptosis, limit tumor growth, and escalate CD8+ lymphocyte and CD11b+ cell infiltration along with an increased presence of granzyme B in tumors. HDACi can instigate pyroptosis in TNBC, promoting infiltration of immune cells and consequently intensifying the efficacy of anticancer immunity.
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Inhibidores de Histona Desacetilasas , Ratones Endogámicos BALB C , Piroptosis , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Piroptosis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Femenino , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
BACKGROUND: Recurrent and metastatic thyroid cancer is more invasive and can transform to dedifferentiated thyroid cancer, thus leading to a severe decline in the 10-year survival. The thyroid-stimulating hormone receptor (TSHR) plays an important role in differentiation process. We aim to find a therapeutic target in redifferentiation strategies for thyroid cancer. METHODS: Our study integrated the differentially expressed genes acquired from the Gene Expression Omnibus database by comparing TSHR expression levels in the Cancer Genome Atlas database. We conducted functional enrichment analysis and verified the expression of these genes by RT-PCR in 68 pairs of thyroid tumor and paratumor tissues. Artificial intelligence-enabled virtual screening was combined with the VirtualFlow platform for deep docking. RESULTS: We identified five genes (KCNJ16, SLC26A4, TG, TPO, and SYT1) as potential cancer treatment targets. TSHR and KCNJ16 were downregulated in the thyroid tumor tissues, compared with paired normal tissues. In addition, KCNJ16 was lower in the vascular/capsular invasion group. Enrichment analyses revealed that KCNJ16 may play a significant role in cell growth and differentiation. The inward rectifier potassium channel 5.1 (Kir5.1, encoded by KCNJ16) emerged as an interesting target in thyroid cancer. Artificial intelligence-facilitated molecular docking identified Z2087256678_2, Z2211139111_1, Z2211139111_2, and PV-000592319198_1 (-7.3 kcal/mol) as the most potent commercially available molecular targeting Kir5.1. CONCLUSION: This study may provide greater insights into the differentiation features associated with TSHR expression in thyroid cancer, and Kir5.1 may be a potential therapeutic target in the redifferentiation strategies for recurrent and metastatic thyroid cancer.
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Canales de Potasio de Rectificación Interna , Neoplasias de la Tiroides , Humanos , Canales de Potasio de Rectificación Interna/genética , Simulación del Acoplamiento Molecular , Inteligencia Artificial , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Receptores de Tirotropina/metabolismo , Descubrimiento de DrogasRESUMEN
Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.
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Dependovirus/genética , Receptores de Antígenos/genética , Linfocitos T/metabolismo , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Assembling evidences suggested that aberrant expression of tissue differentiation-inducing non-protein coding RNA (TINCR) intimately associated with variety of human cancer. However, the expression pattern and involvement of TINCR in breast cancer has not been fully investigated. Here we set out to analyze expression of TINCR in breast cancer and elucidate its mechanistic involvement in tumor incidence and progression. METHODS: The expression of TINCR was determined by q-PCR. SP1 binding sites were analyzed by ChIP-qPCR. The relative transcription activity was measured with luciferase reporter assay. Cell viability was measured with CCK-8 method. Clonogenic capacity was evaluated by soft agar assay. Cell apoptosis was analyzed by Annexin V/7-AAD staining. The migration and invasion were determined by trans-well assay and wound healing. The tumor growth in vivo was evaluated in xenograft mice model. Protein expression was quantified by immunoblotting. RESULTS: TINCR was aberrantly up-regulated by SP1, which in turn stimulated cell proliferation, anchorage-independent growth and suppressed cell apoptosis in breast cancer. TINCR silencing significantly suppressed migration and invasion in vitro and xenograft tumor growth in vivo. Mechanistically, TINCR modulated KLF4 expression via competing with miR-7, which consequently contributed to its oncogenic potential. MiR-7 inhibition severely compromised TINCR silencing-elicited tumor repressive effects. CONCLUSION: Our data uncovered a crucial role of TINCR-miR-7-KLF4 axis in human breast cancer.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Factor de Transcripción Sp1/metabolismo , Adulto , Animales , Apoptosis/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Xenoinjertos , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Persona de Mediana Edad , Carga Tumoral , Regulación hacia ArribaRESUMEN
Globally, breast cancer (BC) is the predominant malignancy with a significant death rate due to metastasis. The epithelial-mesenchymal transition (EMT) is a fundamental initiator for metastatic progression. Through advanced computational strategies, TCF19 was identified as a critical EMT-associated gene with diagnostic and prognostic significance in BC, based on a novel EMT score. Molecular details and the pro-EMT impact of the TCF19/miR-199a-5p/SP1/LOXL2 axis were explored in BC cell lines through in vitro validations, and the oncogenic and metastatic potential of TCF19 and LOXL2 were investigated using subcutaneous and tail-vein models. Additionally, BC-specific enrichment of TCF19 and LOXL2 was measured using a distribution landscape driven by diverse genomic analysis techniques. Molecular pathways revealed that TCF19-induced LOXL2 amplification facilitated migratory, invasive, and EMT activities of BC cells in vitro, and promoted the growth and metastatic establishment of xenografts in vivo. TCF19 decreases the expression of miR-199a-5p and alters the nuclear dynamics of SP1, modulating SP1's affinity for the LOXL2 promoter, leading to increased LOXL2 expression and more malignant characteristics in BC cells. These findings unveil a novel EMT-inducing pathway, the TCF19/miR-199a-5P/SP1/LOXL2 axis, highlighting the pivotal role of TCF19 and suggesting potential for novel therapeutic approaches for more focused BC interventions.
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Aminoácido Oxidorreductasas , Neoplasias de la Mama , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , MicroARNs , Factor de Transcripción Sp1 , Humanos , Transición Epitelial-Mesenquimal/genética , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Animales , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Ratones , Línea Celular Tumoral , Movimiento Celular , Metástasis de la Neoplasia , Transducción de Señal , Ratones DesnudosRESUMEN
Parathyroid carcinoma (PC) is a rare and aggressive endocrine malignancy with limited treatment options. Current treatments such as chemotherapy and radiotherapy have demonstrated limited efficacy. Here, we report the case of a male patient who presented with symptoms including polydipsia, polyuria, and joint pain. Further examination revealed a neck lump, hypercalcemia, and hyperparathyroidism, leading to a diagnosis of PC after en bloc surgery. Seven months later, the patient developed local recurrence and lung metastases, which were resected via left lateral neck dissection and thoracoscopic wedge resection. A 422-gene panel test revealed the presence of epidermal growth factor receptor (EGFR) p.L858R (c. T2573G) mutation, which may sensitize the EGFR-tyrosine kinase inhibitor response, and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) p.E545KV (c. G1633A) mutation. After multidisciplinary treatment discussions, the patient was treated with the multi-target tyrosine kinase inhibitor, anlotinib, resulting in survival benefits for 19 months. This case highlights the potential of targeted therapy in terms of long-term survival in patients with distant metastatic PC, as well as the importance of precision therapy guided by genome sequencing to identify potential therapeutic targets.
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Receptores ErbB , Mutación , Neoplasias de las Paratiroides , Humanos , Masculino , Receptores ErbB/genética , Receptores ErbB/antagonistas & inhibidores , Neoplasias de las Paratiroides/genética , Neoplasias de las Paratiroides/patología , Persona de Mediana Edad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patologíaRESUMEN
Differentiation of human embryonic stem cells (hESCs) into human embryonic stem cells-derived parathyroid-like cells (hESC-PT) has clinical significance in providing new therapies for congenital and acquired parathyroid insufficiency conditions. However, a highly reproducible, well-documented method for parathyroid differentiation remains unavailable. By imitating the natural process of parathyroid embryonic development, we proposed a new hypothesis about the in vitro differentiation of parathyroid-like cells. Transcriptome, differentiation marker protein detection and parathyroid hormone (PTH) secretion assays were performed after the completion of differentiation. To optimize the differentiation protocol and further improve the differentiation rate, we designed glial cells missing transcription factor 2 (GCM2) overexpression lentivirus transfection assays and constructed hESCs-derived parathyroid organoids. The new protocol enabled hESCs to differentiate into hESC-PT. HESC-PT cells expressed PTH, GCM2 and CaSR proteins, low extracellular calcium culture could stimulate hESC-PT cells to secrete PTH. hESC-PT cells overexpressing GCM2 protein secreted PTH earlier than their counterpart hESC-PT cells. Compared with the two-dimensional cell culture environment, hESCs-derived parathyroid organoids secreted more PTH. Both GCM2 lentiviral transfection and three-dimensional cultures could make hESC-PT cells functionally close to human parathyroid cells. Our study demonstrated that hESCs could differentiate into hESC-PT in vitro, which paves the road for applying the technology to treat hypoparathyroidism and introduces new approaches in the field of regenerative medicine.
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Diferenciación Celular , Células Madre Embrionarias Humanas , Organoides , Glándulas Paratiroides , Hormona Paratiroidea , Factores de Transcripción , Humanos , Organoides/citología , Organoides/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Glándulas Paratiroides/citología , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células Cultivadas , Técnicas de Cultivo de Célula/métodos , Proteínas NuclearesRESUMEN
BACKGROUND: BRAFV600E is the most common genetic mutation in differentiated thyroid cancer (DTC) occurring in 60% of patients and drives malignant tumour cell phenotypes including proliferation, metastasis and immune-escape. BRAFV600E-mutated papillary thyroid cancer (PTC) also displays greatly reduced expression of thyroid differentiation markers, thus tendency to radioactive iodine (RAI) refractory and poor prognosis. Therefore, understanding the molecular mechanisms and main oncogenic events underlying BRAFV600E will guide future therapy development. METHODS: Bioinformatics and clinical specimen analyses, genetic manipulation of BRAFV600E-induced PTC model, functional and mechanism exploration guided with transcriptomic screening, as well as systematic rescue experiments were applied to investigate miR-31 function within BRAFV600E-induced thyroid cancer development. Besides, nanoparticles carrying miR-31 antagomirs were testified to alleviate 131I iodide therapy on PTC models. RESULTS: We identify miR-31 as a significantly increased onco-miR in BRAFV600E-associated PTC that promotes tumour progression, metastasis and RAI refractoriness via sustained Wnt/ß-catenin signalling. Mechanistically, highly activated BRAF/MAPK pathway induces miR-31 expression via c-Jun-mediated transcriptional regulation across in vitro and transgenic mouse models. MiR-31 in turn facilitates ß-catenin stabilisation via directly repressing tumour suppressors CEBPA and DACH1, which direct the expression of multiple essential Wnt/ß-catenin pathway inhibitors. Genetic functional assays showed that thyroid-specific knockout of miR-31 inhibited BRAFV600E-induced PTC progression, and strikingly, enhanced expression of sodium-iodide symporter and other thyroid differentiation markers, thus promoted 131I uptake. Nanoparticle-mediated application of anti-miR-31 antagomirs markedly elevated radio-sensitivity of BRAFV600E-induced PTC tumours to 131I therapy, and efficiently suppressed tumour progression in the pre-clinical mouse model. CONCLUSIONS: Our findings elucidate a novel BRAF/MAPK-miR-31-Wnt/ß-catenin regulatory mechanism underlying clinically BRAFV600E-associated DTC tumourigenesis and dedifferentiation, also highlight a potential adjuvant therapeutic strategy for advanced DTC.
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MicroARNs , Proteínas Proto-Oncogénicas B-raf , Neoplasias de la Tiroides , Animales , Humanos , Ratones , Carcinogénesis/genética , Desdiferenciación Celular/genética , Desdiferenciación Celular/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/metabolismo , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismoRESUMEN
[This corrects the article DOI: 10.7150/jca.20150.].
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Background: The incidence of thyroid cancer in China has rapidly increased in recent decades. As the genetic profiles of thyroid cancer vary dramatically between different geographical regions, a comprehensive genetic landscape of thyroid cancer in the Chinese population is urgently needed. Methods: We retrospectively included thyroid cancer patients from three Chinese medical centers between February 2015 and August 2020. To dissect the genomic profiling of these patients, we performed targeted next-generation sequencing on their tumor tissues using a 1,021-gene panel. Results: A total of 458 Chinese patients with thyroid cancer were enrolled, including four malignant histological subtypes arising from follicular epithelial thyroid cells. BRAF driver mutations were identified in 76.0% of patients, followed by RET rearrangements (7.6%) and RAS driver mutations (4.1%). Tumors with more somatic mutations correlated with worse clinical characteristics, including older age at diagnosis, less differentiation of tumor, larger tumor size, lymph node metastasis and distal metastasis. Subclonal BRAF mutations occurred in 20% (6/30) of patients and were frequent in poorly differentiated or anaplastic tumors (33.3% [2/6] vs. 4.2% [1/24], P = 0.09) and those with distal metastasis (50.0% [2/4] vs. 8.7% [2/23], P = 0.09). Tumors with TERT promoter mutations had significantly more somatic mutations (average: 6.5 vs. 1.8, P < 0.001). Moreover, TERT promoter mutations were not associated with lymph node metastasis but significantly associated with older age at diagnosis and poorly differentiated or anaplastic tumors, regardless of their clonal architecture. Conclusion: Our results shed light on the molecular pathogenesis and clinical characteristics of thyroid cancer in the Chinese population. The number of somatic mutations, TERT promoter mutations, and the clonal architecture of BRAF mutations should be considered in the risk stratification of thyroid cancer.
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Proteínas Proto-Oncogénicas B-raf , Neoplasias de la Tiroides , Humanos , Estudios Retrospectivos , Metástasis Linfática , Proteínas Proto-Oncogénicas B-raf/genética , Pueblos del Este de Asia , Neoplasias de la Tiroides/epidemiología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/diagnóstico , MutaciónRESUMEN
The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.
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Proteínas Bacterianas , Edición Génica , Humanos , Proteínas Bacterianas/genética , Edición Génica/métodos , Linfocitos T CD4-Positivos/metabolismo , ARN , Sistemas CRISPR-Cas/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is a highly malignant subtype of breast cancer (BC) with vicious behaviors. TNBC is usually associated with relatively poor clinical outcomes, earlier recurrence, and high propensity for visceral metastases than other BC types. TNBC has been increasingly recognized to constitute a very molecular heterogeneous subtype, which may offer additional therapeutic opportunities due to newly discovered cancer-causing drivers and targets. At present, there are multiple novel targeted therapeutic drugs in preclinical researches, clinical trial designs, and clinical practices, such as platinum drugs, poly ADP-ribose polymerase (PARP) inhibitors, immunocheckpoint inhibitors, androgen receptor inhibitors as well as PI3K/AKT/mTOR targeted inhibitors. These personalized, single, or combinational therapies based on molecular heterogeneity are currently showing positive results. The scope of this review is to highlight the latest knowledge about these potential TNBC therapeutic drugs, which will provide comprehensive insights into the personalized therapeutic strategies and options for combating TNBC.
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HEADING AIMS: Breast cancer is one of the most common malignant tumors with a high incidence and leading cancer-related death in women worldwide. MiR-205 plays a crucial role in breast cancer initiation and progression. Here, we identified the relationship between miR-205 and lymphoid specific helicase and confirmed the significance of the miR-205/lymphoid specific helicase (miR-205/HELLS) axis. MATERIALS AND METHODS: Data from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were analyzed to investigate the expression level of miR-205 and HELLS in breast cancer. The TargetScan, Starbase and miRWalk databases were used to predict the candidate target genes of miR-205. Proliferation and migration abilities were examined using cell counting kit-8 assay, colony formation assays, transwell assay and wound-healing assay. Dual-luciferase reporter assay was utilized to confirm the binding of miR-205 and HELLS. Quantitative RT-PCR, western blot assays or immunohistochemistry were conducted to detect the expression level of genes in breast cancer cells or tissues. Mice xenograft models were constructed to explore the function of miR-205 and HELLS in vivo. KEY FINDINGS: Overexpressed miR-205 alleviated cancer cell proliferation and migration and influenced patients' prognosis by negatively regulating the HELLS gene. Consistently, animal experiments revealed that both overexpressing miR-205 and knocking down HELLS exhibited significant tumor growth inhibition in vivo. SIGNIFICANCE: Our study demonstrated that miR-205 targets HELLS to regulate tumor progression. MiR-205 and HELLS could be considered a novel diagnosis and therapeutic molecular marker of breast cancer.
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Neoplasias de la Mama , MicroARNs , Humanos , Femenino , Ratones , Animales , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Movimiento Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Oncogenes , Neoplasias de la Mama/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background: Immunotherapy is the most promising treatment in triple-negative breast cancer (TNBC), and its efficiency is largely dependent on the intra-tumoral immune cells infiltrations. Thus, novel ways to assist immunotherapy by increasing immune cell infiltrations were highly desirable. Methods: To find key immune-related genes and discover novel immune-evoking molecules, gene expression profiles of TNBC were downloaded from Gene Expression Omnibus (GEO). Single-sample gene set enrichment analysis (ssGSEA) and Weighted Gene Co-expression Network Analysis (WGCNA) were conducted to identified hub genes. The CMap database was used subsequently to predicate potential drugs that can modulate the overall hub gene expression network. In vitro experiments were conducted to assess the anti-tumor activity and the pyroptosis phenotypes induced by GW-8510. Results: Gene expression profiles of 198 TNBC patients were downloaded from GEO dataset GSE76124, and ssGSEA was used to divide them into Immune Cell Proficiency (ICP) group and Immune Cell Deficiency (ICD) group. Hub differential expressed gene modules between two groups were identified by WGCNA and then annotated by Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A cyclin-dependent kinase (CDK) 2 inhibitor, GW-8510 was then identified by the CMap database and further investigated. Treatment with GW-8510 resulted in potent inhibition of TNBC cell lines. More importantly, in vitro and in vivo studies confirmed that GW-8510 and other CDK inhibitors (Dinaciclib, and Palbociclib) can induce pyroptosis by activating caspase-3 and GSDME, which might be the mechanism for their immune regulation potentials. Conclusion: GW-8510, as well as other CDK inhibitors, might serve as potential immune regulators and pyroptosis promotors in TNBC.
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BACKGROUND: Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in hematological cancers but have not yet translated in treating solid tumors. The significant hurdles limiting CAR-T therapy were from a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present TSH receptor (TSHR) as a putative target for CAR-T therapy of differentiated thyroid cancer (DTC). METHODS: We undertook a large-scale screen on thyroid cancer tissues and multiple internal organs through bioinformatical analysis and immunohistochemistry to date TSHR expression. Using 3 previously described monoclonal antibodies, we generated 3 third-generation CAR-Ts. We tested anti-TSHR CAR-T in vitro activity by T-cell function and killing assay. Then we tested preclinical therapeutical efficacy in a xenograft mouse model of DTC and analyzed mice's physical conditions and histological abnormalities to evaluate anti-TSHR CAR-T's safety. RESULTS: TSHR is highly and homogeneously expressed on 90.8% (138/152) of papillary thyroid cancer, 89.2% (33/37) of follicular thyroid cancer, 78.2% (18/23) of cervical lymph node metastases, and 86.7% of radioactive iodine resistance diseases. We developed 3 novel anti-TSHR CAR-Ts from monoclonal antibodies M22, K1-18, and K1-70; all 3 CAR-Ts mediate significant antitumor activity in vitro. Among these, we demonstrate that K1-70 CAR-T can have therapeutical efficacy in vivo, and no apparent toxicity has been observed. CONCLUSION: TSHR is a latent target antigen of CAR-T therapy for DTC. Anti-TSHR CAR-T could represent a therapeutic option for patients with locoregional relapsed or distant metastases of thyroid cancer and should be tested in carefully designed clinical trials.
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Receptores Quiméricos de Antígenos , Neoplasias de la Tiroides , Animales , Anticuerpos Monoclonales/uso terapéutico , Humanos , Radioisótopos de Yodo , Ratones , Receptores Quiméricos de Antígenos/uso terapéutico , Receptores de Tirotropina/metabolismo , Linfocitos T , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/terapiaRESUMEN
Papillary thyroid cancer (PTC) remains the most common endocrine malignancy, despite marked achieves in recent decades, and the mechanisms underlying the pathogenesis and progression for PTC are incompletely elucidated. Accumulating evidence show that γ-glutamylcyclotransferase (GGCT), an enzyme participating in glutathione homeostasis and is elevated in multiple types of tumors, represents an attractive therapeutic target. Using bioinformatics, immunohistochemistry, qRT-PCR, and Western blot assays, we found that GGCT expression was upregulated in PTC and correlated with more aggressive clinicopathological characteristics and worse prognosis. GGCT knockdown inhibited the growth and metastasis ability of PTC cells both in vitro and in vivo and reduced the expression of mesenchymal markers (N-cadherin, CD44, MMP2, and MMP9) while increasing epithelial marker (E-cadherin) in PTC cells. We confirmed binding of microRNA-205-5p (miR-205-5p) on the 3'-UTR regions of GGCT by dual-luciferase reporter assay and RNA-RNA pull-down assay. Delivery of miR-205-5p reversed the pro-malignant capacity of GGCT both in vitro and in vivo. Lastly, we found that GGCT interacted with and stabilized CD44 in PTC cells by co-immunoprecipitation and immunohistochemistry assays. Our findings illustrate a novel signaling pathway, miR-205-5p/GGCT/CD44, that involves in the carcinogenesis and progression of PTC. Development of miR-205-mimics or GGCT inhibitors as potential therapeutics for PTC may have remarkable applications.
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MicroARNs , Neoplasias de la Tiroides , Regiones no Traducidas 3' , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/patología , gamma-Glutamilciclotransferasa/genética , gamma-Glutamilciclotransferasa/metabolismoRESUMEN
Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Linfocitos T CD8-positivos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Mutación con Ganancia de Función , Humanos , Prolina , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismoRESUMEN
Nutritional risk index (NRI) is an index based on ideal body weight that aims to present body weight and serum albumin levels. It has been utilized to discriminate patients at risk of postoperative complications and predict the postoperative outcome of major surgeries. However, this index remains limited for breast cancer patients treated with neoadjuvant chemotherapy (NACT). The research explores the clinical and prognostic significance of NRI in breast cancer patients. This study included 785 breast cancer patients (477 cases received NACT and 308 cases did not) were enrolled in this retrospective study. The optimal NRI cutoff value was evaluated by receiver operating characteristic (ROC) curve, then reclassified as low NRI group (<112) and high NRI group (≥112). The results demonstrated that NRI independently predicted survival on disease-free survival (DFS) and overall survival (OS) by univariate and multivariate Cox regression survival analyses [P = 0.019, hazard ratio (HR): 1.521, 95% CI: 1.071-2.161 and P = 0.004, HR: 1.415, 95% CI: 1.119-1.789; and P = 0.026, HR:1.500, 95% CI: 1.051-2.143 and P < 0.001, HR: 1.547, 95% CI: 1.221-1.959]. According to the optimal cutoff value of NRI, the high NRI value patients had longer mean DFS and OS time in contrast to those with low NRI value patients (63.47 vs. 40.50 months; 71.50 vs. 56.39 months). Furthermore, the results demonstrated that the high NRI score patients had significantly longer mean DFS and OS time than those with low NRI score patients in early-stage breast cancer (χ2 = 9.0510, P = 0.0026 and χ2 = 9.2140, P = 0.0024) and advanced breast cancer (χ2 = 6.2500, P = 0.0124 and χ2 = 5.8880, P = 0.0152). The mean DFS and OS values in patients with high NRI scores were significantly longer in contrast to those with low NRI scores in different molecular subtypes. The common toxicities after NACT were hematologic and gastrointestinal reactions, and the NRI had no statistically significant effects on toxicities, except in nausea (χ2 = 9.2413, P = 0.0024), mouth ulcers (χ2 = 4.8133, P = 0.0282), anemia (χ2 = 8.5441, P = 0.0140), and leukopenia (χ2 = 11.0951, P = 0.0039). NRI serves as a minimally invasive, easily accessible and convenient prognostic tool for evaluating breast cancer prognoses and treatment efficacy, and may help doctors in terms of selecting measures of greater efficiency or appropriateness to better treat breast cancer.
RESUMEN
CONTEXT: Although the incidence of papillary thyroid carcinoma (PTC) is significantly higher in females than in males, the prognosis of male PTC is more unfavorable. However, the cause of higher malignancy of PTC in male patients remains unclear. OBJECTIVE: We conducted our analysis on microarrays datasets, tissue samples from PTC patients and the RNAseq datasets from TCGA with survival data. METHODS: We searched all publicly available microarray datasets and performed a genome-wide meta-analysis comparing PTC and normal samples. Gene Ontology analysis was then conducted. The candidate genes were tested by quantitative real-time polymerase chain reaction. The analysis of prognostic value of genes was performed with datasets from The Cancer Genome Atlas. RESULTS: After meta-analyses, 150 significantly differentially expressed genes (DEGs) were specifically found in male subjects. Gene Ontology analysis of these 150 genes revealed that the viral process was activated. Seven genes involved in the viral process in male patients showed a significantly differential expression between PTC and normal tissue. Survival analysis exhibited that the 7 genes, used in combination, were prognostically valuable and, of them, PSMB1 possessed a conspicuous prognostic value, especially in males. CONCLUSION: In this study, we searched all publicly available microarray datasets and conducted a comprehensive analysis to understand the male propensity for higher malignancy. We found that markers of viral infection showed significantly differential expression only in male patients compared with their female counterparts and had a sex-sensitive prognostic value in PTC.