RESUMO
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.
RESUMO
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.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Camundongos , Animais , Carcinoma de Células Escamosas/patologia , Evasão da Resposta Imune , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Linfócitos T CD8-Positivos , Linfócitos do Interstício TumoralRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nucleofosmina , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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).
Assuntos
Aurora Quinases/antagonistas & inibidores , Aurora Quinases/metabolismo , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Gefitinibe/farmacologia , Mutação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/genética , Imunofluorescência , Humanos , Modelos Biológicos , Bibliotecas de Moléculas Pequenas , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas do Citoesqueleto/fisiologia , Neoplasias Pulmonares/patologia , Elongação da Transcrição Genética , Células A549 , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Elongação da Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Aurora Quinase A/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Adenosina Trifosfatases/genética , Aurora Quinase A/antagonistas & inibidores , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana Transportadoras/genética , Terapia de Alvo Molecular/métodos , Prognóstico , RNA Interferente Pequeno , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise Serial de TecidosRESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Feminino , Humanos , Camundongos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
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.
Assuntos
Everolimo/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/genética , Translocação Genética , Tranilcipromina/farmacologia , Animais , Antidepressivos/farmacologia , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Quimioterapia Combinada , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Carcinoma de Pequenas Células do Pulmão , Biomarcadores Tumorais , Ácidos Nucleicos Livres/genética , DNA , Humanos , Neoplasias Pulmonares/genética , Mutação , Carcinoma de Pequenas Células do Pulmão/genéticaRESUMO
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.
Assuntos
Biomarcadores Tumorais/sangue , Vesículas Extracelulares/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Fator de Crescimento Transformador beta3/sangue , Adulto , Idoso , Quimiorradioterapia/métodos , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Neoplasias de Cabeça e Pescoço/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Tolerância a Radiação/fisiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangueRESUMO
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.
Assuntos
Genômica/métodos , Imageamento por Ressonância Magnética Multiparamétrica/métodos , Neoplasias da Próstata/diagnóstico por imagem , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/genéticaRESUMO
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.
Assuntos
Carcinoma de Células Escamosas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
RESUMO
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.
Assuntos
Carcinogênese/genética , Neoplasias Pulmonares/genética , MicroRNAs/efeitos adversos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Humanos , Neoplasias Pulmonares/fisiopatologia , CamundongosRESUMO
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.
Assuntos
Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Animais , Antineoplásicos/farmacologia , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/genética , Pessoa de Meia-Idade , Família Multigênica/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Medicina de Precisão/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos Endogâmicos NOD , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fenótipo , Fosfoproteínas/genética , Quinazolinas/farmacologia , Fatores de Transcrição , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Receptores ErbB/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , RNA Longo não Codificante/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Feminino , Gefitinibe , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/genética , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Bucais/genética , Isoformas de RNA , Splicing de RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.