ABSTRACT
BACKGROUND: DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer. RESULTS: We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib. CONCLUSIONS: Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Methylation , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cluster Analysis , CpG Islands , Down-Regulation , Drug Resistance, Neoplasm/genetics , Epigenesis, Genetic , Erlotinib Hydrochloride , Gene Expression Profiling , Gene Regulatory Networks , Humans , Lung/metabolism , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Phenotype , Prognosis , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Reproducibility of ResultsABSTRACT
Single-cell technologies enable high-resolution studies of phenotype-defining molecular mechanisms. However, data sparsity and cellular heterogeneity make modeling biological variability across single-cell samples difficult. Here we present SCORPION, a tool that uses a message-passing algorithm to reconstruct comparable gene regulatory networks from single-cell/nuclei RNA-sequencing data that are suitable for population-level comparisons by leveraging the same baseline priors. Using synthetic data, we found that SCORPION outperformed 12 existing gene regulatory network reconstruction techniques. Using supervised experiments, we show that SCORPION can accurately identify differences in regulatory networks between wild-type and transcription factor-perturbed cells. We demonstrate SCORPION's scalability to population-level analyses using a single-cell RNA-sequencing atlas containing 200,436 cells from colorectal cancer and adjacent healthy tissues. The differences between tumor regions detected by SCORPION are consistent across multiple cohorts as well as with our understanding of disease progression, and elucidate phenotypic regulators that may impact patient survival.
Subject(s)
Gene Expression Regulation , Gene Regulatory Networks , Humans , Gene Expression Profiling , Algorithms , RNAABSTRACT
Histone deacetylase inhibitors (HDACi) are currently being explored for the treatment of both solid and hematological malignancies. Although originally thought to exert cytotoxic responses through tumor-intrinsic mechanisms by increasing expression of tumor suppressor genes, several studies have demonstrated that therapeutic responses depend on an intact adaptive immune system: particularly CD8 T cells. It is therefore critical to understand how HDACi directly affects T cells in order to rationally design regimens for combining with immunotherapy. In this study, we evaluated T cell responses to a novel class-selective HDACi (OKI-179, bocodepsin) by assessing histone acetylation levels, which revealed rapid responsiveness accompanied by an increase in CD4 and CD8 T cell frequencies in the blood. However, these rapid responses were transient, as histone acetylation and frequencies waned within 24 hours. This contrasts with in vitro models where high acetylation was sustained and continuous exposure to HDACi suppressed cytokine production. In vivo comparisons demonstrated that stopping OKI-179 treatment during PD-1 blockade was superior to continuous treatment. These findings provide novel insight into the direct effects of HDAC inhibitors on T cells and that treatment schedules that take into account acute T cell effects should be considered when combined with immunotherapies in order to fully harness the tumor-specific T cell responses in patients.
Subject(s)
Histone Deacetylase Inhibitors , Histones , Humans , Histone Deacetylase Inhibitors/pharmacology , Immunotherapy , Clinical Protocols , CD8-Positive T-LymphocytesABSTRACT
Dermatomyositis is an idiopathic inflammatory disease that affects the skin and proximal skeletal muscles. To our best knowledge, there are very few cases of Dermatomyositis reported in Nepal and almost none with respiratory manifestation. We present to you the case of dermatomyositis complicated with interstitial lung disease of a 74 years old male patient with a three-week history of generalized weakness, weight loss, Gottron's papule, and heliotrope rash, with features of interstitial lung disease such as shortness of breath, fever, and productive cough, later confirmed by high-resolution computed tomography. We monitored his serum creatine kinase/creatine phosphokinase level for two consecutive days which showed a progressive increase (281 U/L to 290 U/L). His antinuclear antibody test was positive. He was managed with standard treatment of dermatomyositis. This report might help understand the presentation of disease in our setting which can help in early diagnosis and early initiation of treatment. Keywords: case reports; dermatomyositis; interstitial; lung diseases; Nepal.
Subject(s)
Dermatomyositis , Lung Diseases, Interstitial , Aged , Cough , Dermatomyositis/complications , Dermatomyositis/diagnosis , Dyspnea , Humans , Lung Diseases, Interstitial/complications , Lung Diseases, Interstitial/diagnosis , Male , SkinABSTRACT
The heterogeneity and aggressiveness of triple-negative breast cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective therapeutic targets, TNBC remains elusive in terms of improving patient outcomes. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth both in vitro and in vivo. JNK-IN-8 reduced colony formation, cell viability, and organoid growth in vitro and slowed patient-derived xenograft and syngeneic tumor growth in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy transcription factor EB (TFEB) and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of unphosphorylated TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8-driven upregulation of lysosome biogenesis and/or autophagy markers. TFEB and TFE3 are phosphorylated by a number of kinases, including mTOR. JNK-IN-8 reduced phosphorylation of mTOR targets in a concentration-dependent manner. Knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8 but inhibited colony formation. Similarly, reexpression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8-induced TFEB dephosphorylation. In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB/TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.
Subject(s)
Triple Negative Breast Neoplasms , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/pharmacology , Benzamides , Humans , Lysosomes , Pyridines , Pyrimidines , TOR Serine-Threonine Kinases , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/geneticsABSTRACT
Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non-small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. IMPLICATIONS: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.
Subject(s)
2-Chloroadenosine/analogs & derivatives , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , 2-Chloroadenosine/pharmacology , 2-Chloroadenosine/therapeutic use , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation , Homeostasis , Humans , Lung Neoplasms/pathology , Mutation , Oxidation-Reduction , Signal Transduction , TransfectionABSTRACT
Check point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.
Subject(s)
Antineoplastic Agents/pharmacology , Mitosis/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Radiation Tolerance/drug effects , Apoptosis/drug effects , Cell Line, Tumor , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Histones/metabolism , Humans , Immunohistochemistry , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/drug effects , Tubulin/metabolismABSTRACT
PURPOSE: Radiotherapy with or without chemotherapy is a mainstay of treatment for locally advanced non-small cell lung cancer (NSCLC), but no predictive markers are currently available to select patients who will benefit from these therapies. In this study, we investigated the association between alterations in STK11/LKB1, the second most common tumor suppressor in NSCLC, and response to radiotherapy as well as potential therapeutic approaches to improve outcomes. EXPERIMENTAL DESIGN: We conducted a retrospective analysis of 194 patients with stage I-III NSCLC, including 164 stage III patients bearing mutant or wild-type STK11/LKB1 treated with radiotherapy, and assessed locoregional recurrence (LRR), distant metastasis rates, disease-free survival (DFS), and overall survival (OS), and we investigated the causal role of LKB1 in mediating radiotherapy resistance using isogenic pairs of NSCLC cell lines with LKB1 loss or gain. RESULTS: In stage III patients, with 4 years median follow-up, STK11/LKB1 mutations were associated with higher LRR (P = 0.0108), and shorter DFS (HR 2.530, P = 0.0029) and OS (HR 2.198, P = 0.0263). LKB1 loss promoted relative resistance to radiotherapy, which was dependent on the KEAP1/NRF2 pathway for redox homeostasis. Suppression of the KEAP1/NRF2 pathway via KEAP1 expression, or pharmacologic blockade of glutaminase (GLS) 1 sensitized LKB1-deficient tumors to radiotherapy. CONCLUSIONS: These data provide evidence that LKB1 loss is associated with LRR and poor clinical outcomes in patients with NSCLC treated with radiotherapy and that targeting the KEAP1/NRF2 pathway or GLS inhibition are potential approaches to radiosensitize LKB1-deficient tumors.
Subject(s)
AMP-Activated Protein Kinase Kinases/genetics , Carcinoma, Non-Small-Cell Lung/radiotherapy , Glutaminase/antagonists & inhibitors , Kelch-Like ECH-Associated Protein 1/metabolism , Lung Neoplasms/radiotherapy , Mutation , NF-E2-Related Factor 2/metabolism , Radiation Tolerance/drug effects , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation , Female , Follow-Up Studies , Gamma Rays/adverse effects , Gene Expression Regulation, Neoplastic , Humans , Kelch-Like ECH-Associated Protein 1/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Mice , Mice, Nude , Middle Aged , NF-E2-Related Factor 2/genetics , Prognosis , Radiotherapy/adverse effects , Retrospective Studies , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
MicroRNAs (miRNAs) are short single-stranded RNAs, measuring 21 to 23 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of miRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We performed microRNA microarray profiling of HNSCC tumor samples obtained from a prospective clinical trial evaluating the role of postoperative radiotherapy in head and neck cancer. We also mined through The Cancer Genome Atlas to evaluate expression and survival data. Biological experiments, including cell proliferation, flow cytometry, cell migration and invasion, clonogenic survival, and fluorescent microscopy, were conducted using HN5 and UM-SCC-22B cell lines. miR-125a-5p downregulation was associated with recurrent disease in a panel of high-risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via the Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G1/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2, EIF4EBP1, and TXNRD1, which support the tumor suppressive mechanism of miR-125a-5p. Functional validation of ERBB2 suggests that miR-125a-5p affects cell proliferation and sensitivity to ionizing radiation, in part, through ERBB2. Our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.
Subject(s)
Biomarkers, Tumor , Genes, Tumor Suppressor , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , MicroRNAs/genetics , 3' Untranslated Regions , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cell Survival/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Head and Neck Neoplasms/pathology , Humans , Neoplasm Recurrence, Local , Prognosis , RNA Interference , RNA, Messenger/genetics , Radiation, IonizingABSTRACT
Purpose: Although postoperative radiotherapy is often used to maintain local control after surgical resection and chemotherapy for locally advanced non-small cell lung cancer (NSCLC), both locoregional failure and distant metastasis remain problematic. The mechanisms of therapeutic resistance remain poorly understood.Experimental Design: We used reverse-phase protein arrays (RPPA) to profile the baseline expression of 170 total and phosphorylated proteins in 70 NSCLC cell lines to categorize pathways that may contribute to radiation resistance. Significant markers identified by RPPA were further analyzed in tissue microarrays (TMA) of specimens from 127 patients with NSCLC who had received surgery before receiving postoperative radiotherapy. Cox regression analysis and log-rank tests were used to identify potential predictive factors. We then validated the biological function of the markers in NSCLC cell lines in vitroResults: Of the 170 proteins or phospho-proteins profiled, a subset of 12 proteins was found to correlate with radiation response parameters. TMA analysis of the 12 proteins showing the greatest differences in expression in the RPPA analysis demonstrated that RAD50 had the strongest correlation with distant relapse-free survival, locoregional relapse-free survival, and disease-free survival in patients with NSCLC. We confirmed that knockdown of RAD50 sensitized NSCLC cells to radiation and that upregulation of RAD50 increased radioresistance in in vitro experiments.Conclusions: Upregulated RAD50 may be a predictor of radioresistance in patients with lung cancer who received radiotherapy. Clin Cancer Res; 24(2); 341-50. ©2017 AACR.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Gene Expression , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Acid Anhydride Hydrolases , Biomarkers, Tumor , Carcinoma, Non-Small-Cell Lung/therapy , Combined Modality Therapy , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Lung Neoplasms/therapy , Male , Neoplasm Staging , Prognosis , Proportional Hazards Models , Radiation Tolerance/genetics , Treatment OutcomeABSTRACT
Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1-low expression group (P = 0.01, 1.9 × 10-3, and 9 × 10-4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10-4 and P = 4 × 10-4, respectively).Conclusions: Taken together, our data point to a targetable Axl-PI3 kinase-PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713-22. ©2017 AACR.
Subject(s)
B7-H1 Antigen/genetics , Carcinoma, Squamous Cell/radiotherapy , Head and Neck Neoplasms/radiotherapy , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Aged , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/radiation effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Lymphocytes, Tumor-Infiltrating , Male , Middle Aged , Papillomaviridae/pathogenicity , Proteomics , RNA, Messenger/radiation effects , Radiation Tolerance/genetics , Signal Transduction/radiation effects , Axl Receptor Tyrosine KinaseABSTRACT
Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Drug Resistance, Neoplasm , Hepatocyte Growth Factor/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Neovascularization, Pathologic/metabolism , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line, Tumor , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Gene Expression Profiling , Humans , Hypoxia/genetics , Hypoxia/metabolism , Kaplan-Meier Estimate , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Male , Mice , Molecular Targeted Therapy , Multicenter Studies as Topic , Neovascularization, Pathologic/genetics , Prognosis , Protein Kinase Inhibitors/pharmacology , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: The local-regional control rate for advanced head-and-neck squamous cell carcinoma (HNSCC) remains poor and is unpredictable for a given individual. This study examined whether gene expression patterns developed from tumors from surgicopathologic, criteria-defined, high-risk HNSCC patients could be correlated with clinical outcomes, namely, metastasis or nonrecurrent disease. METHODS AND MATERIALS: Fifteen primary tumors from patients treated with a consistent protocol of surgery followed by radiotherapy were examined. Seven of these tumors were from high-risk patients who developed distant metastasis (DM), and eight tumors were from patients with no recurrence (NR) (median follow-up, 59 months). RESULTS: Unsupervised clustering of gene expression did not separate the two groups from one another, but when supervised methodologies were applied, 205 genes discriminated the two groups. Within the DM group, genes associated with cell growth and proliferation; DNA replication, recombination, and repair; antiapoptotic pathways; cell adhesion; and angiogenesis were identified. For NR samples, discriminatory genes were associated with the onset of apoptosis. CONCLUSIONS: Our data suggest that gene expression analysis of surgically excised HNSCC tumors from patients considered at high risk for recurrence has the potential to identify individuals susceptible to metastasis on the basis of distinct gene-expression patterns. These patients would be ideal candidates for testing systemic therapy.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/secondary , Gene Expression , Head and Neck Neoplasms/genetics , Carcinoma, Squamous Cell/radiotherapy , Carcinoma, Squamous Cell/surgery , Cell Cycle/genetics , Cell Proliferation , Cell Shape/genetics , DNA Repair/genetics , DNA Replication , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/radiotherapy , Head and Neck Neoplasms/surgery , Humans , Lymphatic Metastasis , Neoplasm Recurrence, Local/genetics , Oligonucleotide Array Sequence Analysis/methods , Signal Transduction/genetics , Up-RegulationABSTRACT
Improved therapies are greatly needed for non-small cell lung cancer (NSCLC) that does not harbor targetable kinase mutations or translocations. We previously demonstrated that NSCLC cells that harbor kinase-inactivating BRAF mutations (KIBRAF) undergo senescence when treated with the multitargeted kinase inhibitor dasatinib. Similarly, treatment with dasatinib resulted in a profound and durable response in a patient with KIBRAF NSCLC. However, no canonical pathways explain dasatinib-induced senescence in KIBRAF NSCLC. To investigate the underlying mechanism, we used 2 approaches: gene expression and reverse phase protein arrays. Both approaches showed that DNA repair pathways were differentially modulated between KIBRAF NSCLC cells and those with wild-type (WT) BRAF. Consistent with these findings, dasatinib induced DNA damage and activated DNA repair pathways leading to senescence only in the KIBRAF cells. Moreover, dasatinib-induced senescence was dependent on Chk1 and p21, proteins known to mediate DNA damage-induced senescence. Dasatinib also led to a marked decrease in TAZ but not YAP protein levels. Overexpression of TAZ inhibited dasatinib-induced senescence. To investigate other vulnerabilities in KIBRAF NSCLC cells, we compared the sensitivity of these cells with that of WTBRAF NSCLC cells to 79 drugs and identified a pattern of sensitivity to EGFR and MEK inhibitors in the KIBRAF cells. Clinically approved EGFR and MEK inhibitors, which are better tolerated than dasatinib, could be used to treat KIBRAF NSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KIBRAF NSCLC cells represents a unique vulnerability with potential clinical applications.
Subject(s)
Cellular Senescence/drug effects , DNA Damage , DNA Repair/drug effects , Dasatinib/pharmacology , Mutation , Proto-Oncogene Proteins B-raf/genetics , Blotting, Western , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cellular Senescence/genetics , Checkpoint Kinase 1 , Comet Assay , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective against non-small cell lung cancer (NSCLC) with activating EGFR mutations, but resistance is inevitable. Mechanisms of acquired resistance include T790M mutations and epithelial-mesenchymal transition (EMT). One potential strategy for overcoming this resistance is the inhibition of polo-like kinase 1 (PLK1) based on our previous studies showing that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibition than epithelial cell lines. To determine the extent to which PLK1 inhibition overcomes EGFR TKI resistance we measured the effects of the PLK1 inhibitor volasertib alone and in combination with the EGFR inhibitor erlotinib in vitro and in vivo in EGFR mutant NSCLC cell lines with acquired resistance to erlotinib. Two erlotinib-resistant cell lines that underwent EMT had higher sensitivity to volasertib, which caused G2/M arrest and apoptosis, than their parental cells. In all NSCLC cell lines with T790M mutations, volasertib markedly reduced erlotinib resistance. All erlotinib-resistant NSCLC cell lines with T790M mutations had higher sensitivity to erlotinib plus volasertib than to erlotinib alone, and the combination treatment caused G2/M arrest and apoptosis. Compared with either agent alone, the combination treatment also caused significantly more DNA damage and greater reductions in tumor size. Our results suggest that PLK1 inhibition is clinically effective against NSCLC that becomes resistant to EGFR inhibition through EMT or the acquisition of a T790M mutation. These results uncover new functions of PLK1 inhibition in the treatment of NSCLC with acquired resistance to EGFR TKIs.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Cycle Proteins/antagonists & inhibitors , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride/pharmacology , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pteridines/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Disease-Free Survival , Drug Resistance, Neoplasm , Drug Synergism , Erlotinib Hydrochloride/administration & dosage , Female , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/pathology , Mice , Mice, Nude , Mutation , Protein Kinase Inhibitors/administration & dosage , Pteridines/administration & dosage , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC. EXPERIMENTAL DESIGN: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC. RESULTS: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2-M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas. CONCLUSIONS: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643-50. ©2016 AACR.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Head and Neck Neoplasms/metabolism , Proteome , Proteomics , Radiation Tolerance , Biomarkers, Tumor , Cell Line, Tumor , DNA Damage , Focal Adhesion Kinase 1/genetics , Gene Dosage , Gene Expression , Head and Neck Neoplasms/etiology , Head and Neck Neoplasms/radiotherapy , Humans , Proteomics/methods , Radiation Tolerance/genetics , Treatment OutcomeABSTRACT
PURPOSE: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non-small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDRis typically localized to the vasculature, amplification ofKDRhas reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream ofKDRand whetherKDRamplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. METHODS: NSCLC cell lines with or withoutKDRamplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N= 294) were screened forKDRamplification by FISH. RESULTS: KDRamplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines withKDRamplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α inKDR-amplified NSCLC cells. In the ZODIAC study,KDRamplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. CONCLUSIONS: Preclinical studies suggestKDRactivates invasion but not survival pathways inKDR-amplified NSCLC models. Patients with NSCLC whose tumor hadKDRamplification were not associated with clinical benefit for vandetanib in combination with docetaxel.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , TOR Serine-Threonine Kinases/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/mortality , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/mortality , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-met/metabolism , Quinazolines/therapeutic use , Signal Transduction/drug effects , Treatment Outcome , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
PURPOSE: To identify new therapeutic targets for non-small cell lung cancer (NSCLC), we systematically searched two cancer cell line databases for sensitivity data on a broad range of drugs. We identified polo-like kinase 1 (PLK1) as the most promising target for further investigation based on a subset of sensitive NSCLC cell lines and inhibitors that were in advanced clinical development. EXPERIMENTAL DESIGN: To identify potential biomarkers of response of NSCLC to PLK1 inhibition and mechanisms of PLK1 inhibitor-induced apoptosis, integrated analysis of gene and protein expression, gene mutations, and drug sensitivity was performed using three PLK1 inhibitors (volasertib, BI2536, and GSK461364) with a large panel of NSCLC cell lines. RESULTS: The NSCLC cell lines had different sensitivities to PLK1 inhibition, with a minority demonstrating sensitivity to all three inhibitors. PLK1 inhibition led to G2-M arrest, but only treatment-sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. NSCLC lines with high epithelial-mesenchymal transition (EMT) gene signature scores (mesenchymal cell lines) were more sensitive to PLK1 inhibition than epithelial lines (P< 0.02). Likewise, proteomic profiling demonstrated that E-cadherin expression was higher in the resistant cell lines than in the sensitive ones (P< 0.01). Induction of an epithelial phenotype by expression of the miRNA miR-200 increased cellular resistance to PLK1 inhibition. Also, KRAS mutation and alterations in the tight-junction, ErbB, and Rho signaling pathways correlated with drug response of NSCLC. CONCLUSIONS: In this first reported large-scale integrated analysis of PLK1 inhibitor sensitivity, we demonstrated that EMT leads to PLK1 inhibition sensitivity of NSCLC cells. Our findings have important clinical implications for mesenchymal NSCLC, a significant subtype of the disease that is associated with resistance to currently approved targeted therapies.
Subject(s)
Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle Proteins/antagonists & inhibitors , Epithelial-Mesenchymal Transition , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Animals , Apoptosis/genetics , Benzimidazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Chromosomal Instability , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition/genetics , Humans , Lung Neoplasms/genetics , Mice , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Pteridines/pharmacology , Thiophenes/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
PURPOSE: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non-small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management. EXPERIMENTAL DESIGN: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). RESULTS: T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate. CONCLUSIONS: Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patient's cancer, which should be validated in predicting response to T790M-targeted inhibitors.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Protein Kinase Inhibitors/administration & dosage , Adult , Afatinib , Aged , Aged, 80 and over , Biopsy , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/blood , Erlotinib Hydrochloride/administration & dosage , Female , Gefitinib , Genotype , Humans , Male , Middle Aged , Mutation , Neoplastic Cells, Circulating/drug effects , Quinazolines/administration & dosageABSTRACT
The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC50 ranging from 2 µM in the most sensitive CAKI-1 to 36 µM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway.