RESUMEN
Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype in vitro and in vivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.
Asunto(s)
Carcinoma de Células Escamosas , Receptores ErbB , Vesículas Extracelulares , Isoformas de Proteínas , Inhibidores de Proteínas Quinasas , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Vesículas Extracelulares/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Animales , Línea Celular Tumoral , Ratones , Transducción de Señal/efectos de los fármacos , Fosforilación/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
Profiling tumors at single-cell resolution provides an opportunity to understand complexities underpinning lymph-node metastases in head and neck squamous-cell carcinoma. Single-cell RNAseq (scRNAseq) analysis of cancer-cell trajectories identifies a subpopulation of pre-metastatic cells, driven by actionable pathways including AXL and AURK. Blocking these two proteins blunts tumor invasion in patient-derived cultures. Furthermore, scRNAseq analyses of tumor-infiltrating CD8 + T-lymphocytes show two distinct trajectories to T-cell dysfunction, corroborated by their clonal architecture based on single-cell T-cell receptor sequencing. By determining key modulators of these trajectories, followed by validation using external datasets and functional experiments, we uncover a role for SOX4 in mediating T-cell exhaustion. Finally, interactome analyses between pre-metastatic tumor cells and CD8 + T-lymphocytes uncover a putative role for the Midkine pathway in immune-modulation and this is confirmed by scRNAseq of tumors from humanized mice. Aside from specific findings, this study demonstrates the importance of tumor heterogeneity analyses in identifying key vulnerabilities during early metastasis.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Ratones , Animales , Carcinoma de Células Escamosas/patología , Evasión Inmune , Neoplasias de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Linfocitos T CD8-positivos , Linfocitos Infiltrantes de TumorRESUMEN
Wild-type KRAS (KRASWT) amplification has been shown to be a secondary means of KRAS activation in cancer and associated with poor survival. Nevertheless, the precise role of KRASWT overexpression in lung cancer progression is largely unexplored. Here, we identify and characterize a KRAS-responsive lncRNA, KIMAT1 (ENSG00000228709) and show that it correlates with KRAS levels both in cell lines and in lung cancer specimens. Mechanistically, KIMAT1 is a MYC target and drives lung tumorigenesis by promoting the processing of oncogenic microRNAs (miRNAs) through DHX9 and NPM1 stabilization while halting the biogenesis of miRNAs with tumor suppressor function via MYC-dependent silencing of p21, a component of the Microprocessor Complex. KIMAT1 knockdown suppresses not only KRAS expression but also KRAS downstream signaling, thereby arresting lung cancer growth in vitro and in vivo. Taken together, this study uncovers a role for KIMAT1 in maintaining a positive feedback loop that sustains KRAS signaling during lung cancer progression and provides a proof of principle that interfering with KIMAT1 could be a strategy to hamper KRAS-induced tumorigenesis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Largo no Codificante/genética , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Nucleofosmina , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities. METHODS: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies. FINDINGS: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival. INTERPRETATION: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors. FUNDING: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Aurora Quinasas/metabolismo , Biomarcadores de Tumor , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Gefitinib/farmacología , Mutación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/genética , Técnica del Anticuerpo Fluorescente , Humanos , Modelos Biológicos , Bibliotecas de Moléculas Pequeñas , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Chromosomal instability (CIN) is a driver of clonal diversification and intratumor heterogeneity, providing genetic diversity that contributes to tumor progression. It is estimated that approximately 80% of solid cancers, including non-small cell lung cancer (NSCLC), exhibit features of CIN, which affects tumor growth and response to therapy. However, the molecular mechanisms connecting CIN to tumor progression are still poorly understood. Through an RNAi screen performed on genes involved in CIN and overexpressed in human lung adenocarcinoma samples, we identified the cytoskeleton-associated protein 2-like (CKAP2L) as a potential oncogene that promotes lung cancer proliferation and growth in vitro and in vivo. Mechanistically, CKAP2L directly interacted with RNA Pol II and regulated transcription elongation of key genes involved in spindle assembly checkpoint, chromosome segregation, cell cycle, and E2F signaling. Furthermore, depletion of CKAP2L increased the sensitivity of NSCLC cells to alvocidib, a pan-CDK inhibitor, leading to a significant reduction of cell proliferation and an increase in cell death. Altogether, these findings shed light on the molecular mechanisms through which CKAP2L, a protein involved in CIN, promotes cancer progression and suggest that its inhibition represents a novel therapeutic strategy in NSCLC. SIGNIFICANCE: These findings demonstrate the oncogenic function of CKAP2L through regulation of transcription elongation and suggest that targeting CKAP2L could enhance therapeutic response in patients with NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas del Citoesqueleto/fisiología , Neoplasias Pulmonares/patología , Elongación de la Transcripción Genética , Células A549 , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Elongación de la Transcripción Genética/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: We have previously identified and validated a panel of molecular prognostic markers (ATP13A3, SSR3, and ANO1) for Head and Neck Squamous Cell Carcinoma (HNSCC). The aim of this study was to investigate the consequence of ATP13A3 dysregulation on signaling pathways, to aid in formulating a therapeutic strategy targeting ATP13A3-overexpressing HNSCC. MATERIALS AND METHODS: Gene Set Enrichment Analysis (GSEA) was performed on HNSCC microarray expression data (Internal local dataset [n = 92], TCGA [n = 232], EMBL [n = 81]) to identify pathways associated with high expression of ATP13A3. Validation was performed using immunohistochemistry (IHC) on tissue microarrays (TMAs) of head and neck cancers (n = 333), staining for ATP13A3 and phosphorylated Aurora kinase A (phospho-T288). Short interfering RNA was used to knockdown ATP13A3 expression in patient derived HNSCC cell lines. Protein expression of ATP13A3 and Aurora kinase A was then assessed by immunoblotting. RESULTS: GSEA identified Aurora kinase pathway to be associated with high expression of ATP13A3 (p = 0.026). The Aurora kinase pathway was also associated with a trend towards poor prognosis and tumor aggressiveness (p = 0.086, 0.094, respectively). Furthermore, the immunohistochemical staining results revealed a significant association between Aurora kinase activity and high ATP13A3 expression (p < 0.001). Knockdown of ATP13A3 in human head and neck cell lines showed decrease in Aurora kinase A levels. CONCLUSION: Tumors with high ATP13A3 are associated with high Aurora kinase activity. This suggests a potential therapeutic role of Aurora kinase inhibitors in a subset of poor prognosis HNSCC patients with overexpression of ATP13A3.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aurora Quinasa A/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Adenosina Trifosfatasas/genética , Aurora Quinasa A/antagonistas & inhibidores , Línea Celular Tumoral , Femenino , Silenciador del Gen , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Masculino , Proteínas de Transporte de Membrana/genética , Terapia Molecular Dirigida/métodos , Pronóstico , ARN Interferente Pequeño , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Análisis de Matrices TisularesRESUMEN
A subset of lung adenocarcinomas is driven by the EML4-ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on-target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4-ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti-apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first-, second- and third-generation ALK inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Humanos , Ratones , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.
Asunto(s)
Everolimus/farmacología , Histona Demetilasas/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Translocación Genética , Tranilcipromina/farmacología , Animales , Antidepresivos/farmacología , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Quimioterapia Combinada , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: SCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring. METHODS: We applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran's I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample. RESULTS: Genome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival. CONCLUSIONS: We demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Carcinoma Pulmonar de Células Pequeñas , Biomarcadores de Tumor , Ácidos Nucleicos Libres de Células/genética , ADN , Humanos , Neoplasias Pulmonares/genética , Mutación , Carcinoma Pulmonar de Células Pequeñas/genéticaRESUMEN
Management of locally advanced head and neck squamous cell carcinoma (HNSCC) requires a multi-prong approach comprising surgery, radiation and/or chemotherapy, yet outcomes are limited. This is largely due to a paucity of biomarkers that can predict response to specific treatment modalities. Here, we evaluated TGFß3 protein levels in extracellular vesicles (EVs) released by HNSCC cells as a predictor for response to chemoradiation therapy (CRT). To this end, specific EV-fractions were isolated from cell lines or HNSCC patient plasma, and TGFß3 protein was quantified. In patients treated with CRT, TGFß3 levels were found to be significantly higher in plasma EV-fractions or non-responders compared with responders. High levels of TGFß3 levels in Annexin V-EVs were associated with the worst progression-free survival. In vitro experiments demonstrated that TGFß3 silencing sensitized HNSCC cells to cytotoxic therapies, and this phenotype could be rescued by treatment with exogenous. In addition, specific EV-fractions shed by cisplatin-resistant cells were sufficient to transfer the resistant phenotype to sensitive cells through activation of TGFß-signaling pathway. Therefore, our data show that TGFß3 transmitted through EV plays a significant role in response to cytotoxic therapy, which can be exploited as a potential biomarker for CRT response in HNSCC patients treated with curative intent.
Asunto(s)
Biomarcadores de Tumor/sangre , Vesículas Extracelulares/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Factor de Crecimiento Transformador beta3/sangre , Adulto , Anciano , Quimioradioterapia/métodos , Cisplatino/administración & dosificación , Resistencia a Antineoplásicos/fisiología , Femenino , Neoplasias de Cabeza y Cuello/sangre , Humanos , Masculino , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Tolerancia a Radiación/fisiología , Carcinoma de Células Escamosas de Cabeza y Cuello/sangreRESUMEN
BACKGROUND: The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification. OBJECTIVE: To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease. DESIGN, SETTING, AND PARTICIPANTS: Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa. INTERVENTION: Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients. OUTCOME MEASUREMENT AND STATISTICAL ANALYSIS: Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication. RESULTS AND LIMITATIONS: A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up. CONCLUSIONS: Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied. PATIENT SUMMARY: Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed.
Asunto(s)
Genómica/métodos , Imágenes de Resonancia Magnética Multiparamétrica/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Anciano , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/genéticaRESUMEN
Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naïve setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.
Asunto(s)
Carcinoma de Células Escamosas/genética , Resistencia a Antineoplásicos/genética , Neoplasias de la Boca/genética , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Precision medicine aims to tailor cancer therapies to target specific tumor-promoting aberrations. For tumors that lack actionable drivers, which occurs frequently in the clinic, extensive molecular characterization and pre-clinical drug efficacy studies will be required. A cell line maintained at low passage and a patient- derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patient's DNA. However, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the PIK3CA and RICTOR genes, respectively. We then conducted pathway analysis, which revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis will significantly aid in identifying actionable alterations in rare tumors and guide patient stratification into early-phase clinical trials.
RESUMEN
Oncogenic KRAS induces tumor onset and development by modulating gene expression via different molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that have been established as main players in tumorigenesis. By overexpressing wild type or mutant KRAS (KRASG12D) and using inducible human and mouse cell lines, we analyzed KRAS-regulated microRNAs in non-small-cell lung cancer (NSCLC). We show that miR-30c and miR-21 are significantly upregulated by both KRAS isoforms and induce drug resistance and enhance cell migration/invasion via inhibiting crucial tumor suppressor genes, such as NF1, RASA1, BID, and RASSF8. MiR-30c and miR-21 levels were significantly elevated in tumors from patients that underwent surgical resection of early stages NSCLC compared to normal lung and in plasma from the same patients. Systemic delivery of LNA-anti-miR-21 in combination with cisplatin in vivo completely suppressed the development of lung tumors in a mouse model of lung cancer. Mechanistically, we demonstrated that ELK1 is responsible for miR-30c and miR-21 transcriptional activation by direct binding to the miRNA proximal promoter regions. In summary, our study defines that miR-30c and miR-21 may be valid biomarkers for early NSCLC detection and their silencing could be beneficial for therapeutic applications.
Asunto(s)
Carcinogénesis/genética , Neoplasias Pulmonares/genética , MicroARNs/efectos adversos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Humanos , Neoplasias Pulmonares/fisiopatología , RatonesRESUMEN
Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess tumor-initiating (TIC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of TIC phenotypes. Many of the implicated GPCRs signal through the G protein GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in patients. GNA13 levels modulate drug resistance and TIC-like phenotypes in patient-derived head and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. Blockade of GNA13 expression, or of select downstream pathways, using small-molecule inhibitors abrogates GNA13-induced TIC phenotypes, rendering cells vulnerable to standard-of-care cytotoxic therapies. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker for tumor progression, and that interfering with GNA13-induced signaling provides a novel strategy to block TICs and drug resistance in HNSCCs.
Asunto(s)
Transformación Celular Neoplásica/genética , Resistencia a Antineoplásicos/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Células Tumorales CultivadasRESUMEN
In NSCLC alterations in PDGF receptors are markers of worst prognosis and efficient targeting of these receptors is yet to be achieved. In this study, we explored PDGFR-regulated microRNAs demonstrating that miR-23b cluster and miR-125a-5p are downregulated by increased expression of PDGFR-α or PDGFR-ß in NSCLC cells. Mechanistically, the expression of these microRNAs is positively regulated by p53 and negatively modulated by NF-kB p65. Forced expression of miR-23b cluster or miR-125a-5p enhanced drug sensitivity and suppressed invasiveness of NSCLC cells by silencing several genes involved in oncogenic KRAS and NF-kB pathways, including SOS1, GRB2, IQGAP1, RALA, RAF-1, IKKß, AKT2, ERK2 and KRAS itself. Of note, an inverse correlation between miR-23b cluster, miR-125a-5p and respective target genes was also found in vivo in a large dataset of lung adenocarcinoma samples. Furthermore, in vivo delivery of miR-23b cluster or miR-125a-5p significantly repressed tumour growth in a highly aggressive NSCLC circulating tumour cell (CTC) patient derived explant (CDX) mouse model. In conclusion, our finding sheds light on the PDGFR signaling and endorses the possibility to employ miR-23b cluster and miR-125a-5p as therapeutic tools to silence simultaneously a range of redundant pathways and main effectors of tumorigenesis in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , MicroARNs/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/genética , Animales , Antineoplásicos/farmacología , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Masculino , Ratones , MicroARNs/administración & dosificación , MicroARNs/genética , Persona de Mediana Edad , Familia de Multigenes/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a "phenotype-driven precision-oncology" approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of "screenable" patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive "-omics" interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Medicina de Precisión/métodos , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Biomarcadores de Tumor , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Cisplatino/farmacología , Resistencia a Antineoplásicos , Gefitinib , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones Endogámicos NOD , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Fenotipo , Fosfoproteínas/genética , Quinazolinas/farmacología , Factores de Transcripción , Resultado del Tratamiento , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAPRESUMEN
Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de la Boca/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/uso terapéutico , ARN Largo no Codificante/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/genética , Proliferación Celular , Resistencia a Antineoplásicos/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago , Femenino , Gefitinib , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/genética , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Neoplasias de la Boca/genética , Isoformas de ARN , Empalme del ARN , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RUNX1 is crucial for the regulation of megakaryocyte specification, maturation, and thrombopoiesis. Runx1 possesses 2 promoters: the distal P1 and proximal P2 promoters. The major protein isoforms generated by P1 and P2 are RUNX1C and RUNX1B, respectively, which differ solely in their N-terminal amino acid sequences. RUNX1C is the most abundantly expressed isoform in adult hematopoiesis, present in all RUNX1-expressing populations, including the cKit+ hematopoietic stem and progenitor cells. RUNX1B expression is more restricted, being highly expressed in the megakaryocyte lineage but downregulated during erythropoiesis. We generated a Runx1 P1 knock-in of RUNX1B, termed P1-MRIPV This mouse line lacks RUNX1C expression but has normal total RUNX1 levels, solely comprising RUNX1B. Using this mouse line, we establish a specific requirement for the P1-RUNX1C isoform in megakaryopoiesis, which cannot be entirely compensated for by RUNX1B overexpression. P1 knock-in megakaryocyte progenitors have reduced proliferative capacity and undergo increased cell death, resulting in thrombocytopenia. P1 knock-in premegakaryocyte/erythroid progenitors demonstrate an erythroid-specification bias, evident from increased erythroid colony-forming ability and decreased megakaryocyte output. At a transcriptional level, multiple erythroid-specific genes are upregulated and megakaryocyte-specific transcripts are downregulated. In addition, proapoptotic pathways are activated in P1 knock-in premegakaryocyte/erythroid progenitors, presumably accounting for the increased cell death in the megakaryocyte progenitor compartment. Unlike in the conditional adult Runx1 null models, megakaryocytic maturation is not affected in the P1 knock-in mice, suggesting that RUNX1B can regulate endomitosis and thrombopoiesis. Therefore, despite the high degree of structural similarity, RUNX1B and RUNX1C isoforms have distinct and specific roles in adult megakaryopoiesis.