Downregulation of TRIB3 enhances the sensitivity of lung cancer cells to amino acid deprivation by suppressing AKT activation.

Tribbles pseudokinase 3 (TRIB3), a member of the mammalian Tribbles family, is implicated in multiple biological processes. This study aimed to investigate the biological functions of TRIB3 in lung cancer and its effect on amino acid-deprived lung cancer cells. TRIB3 mRNA expression was elevated in lung cancer tissues and cell lines compared to normal lung tissues and cells. TRIB3 knockdown markedly reduced the viability and proliferation of H1299 lung cancer cells. Deprivation of amino acids, particularly arginine, glutamine, lysine, or methionine, strongly increased TRIB3 expression via ATF4 activation in H1299 lung cancer cells. Knockdown of TRIB3 led to transcriptional downregulation of ATF4 and reduced AKT activation induced by amino acid deprivation, ultimately increasing the sensitivity of H1299 lung cancer cells to amino acid deprivation. Additionally, TRIB3 knockdown enhanced the sensitivity of H1299 cells to V-9302, a competitive antagonist of transmembrane glutamine flux. These results suggest that TRIB3 is a pro-survival regulator of cell viability in amino acid-deficient tumor microenvironments and a promising therapeutic target for lung cancer treatment.

The combination of osimertinib with Raf inhibitor overcomes osimertinib resistance induced by KRAS amplification in EGFR-mutated lung cancer cells.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR)1 tyrosine kinase inhibitor (TKI) approved for the treatment of EGFR-positive patients exhibiting a T790 M resistance mutation after treatment with an earlier generation of EGFR TKIs. However, resistance to osimertinib inevitably develops despite its efficacy, and the resistance mechanisms are complex and not fully understood. We established cell lines with acquired resistance to osimertinib from gefitinib- or erlotinib-resistant NSCLC cells using a dose-escalation method, and found that they had upregulated levels of phosphorylated ERK1/2. Targeted next-generation sequencing of 143 genes was performed, and interestingly, amplification of KRAS was observed in osimertinib-resistant cells. Transfection of siRNA against the KRAS gene notably reduced the activation of ERK1/2 and AKT and significantly enhanced the induction of apoptosis by osimertinib treatment in osimertinib-resistant cells. LY3009120, a RAF inhibitor, showed a significant synergistic effect with osimertinib on apoptotic cell death in osimertinib-resistant cells. Combined treatment with osimertinib and LY3009120 also demonstrated remarkable synergistic anti-tumor activity in mouse xenografts of these cells. This could be a potential new treatment option for KRAS amplification-induced osimertinib failure.

The Combination of Trametinib, a MEK Inhibitor, and Temsirolimus, an mTOR Inhibitor, Radiosensitizes Lung Cancer Cells.

BACKGROUND/AIM: We evaluated the radiosensitizing effect of the combination treatment of trametinib, a MEK inhibitor, and temsirolimus, an mTOR inhibitor, on non-small-cell lung carcinoma (NSCLC) cells. MATERIALS AND METHODS: The effects of combining trametinib and temsirolimus with radiation in NSCLC cell lines were evaluated using clonogenic survival and apoptosis assays. DNA double-strand breaks and cell cycle distribution were analyzed using flow cytometry. Tumor volume was measured to determine the radiosensitivity in lung cancer xenograft models. RESULTS: Exposure of lung cancer cells to a combination of trametinib and temsirolimus reduced clonogenic survival and promoted radiation-induced apoptosis. Combined inhibition of MEK and mTOR induced prolonged expression of γH2AX after irradiation and resulted in prolonged G2/M cell cycle arrest after irradiation in A549 cells. In vivo studies revealed that co-administration of the drugs sensitizes lung cancer xenografts to radiotherapy. CONCLUSION: The combination of trametinib and temsirolimus can enhance lung cancer radiosensitivity in vitro and in vivo.

Radiation Induces Autophagy via Histone H4 Lysine 20 Trimethylation in Non-small Cell Lung Cancer Cells.

BACKGROUND/AIM: Radiotherapy-induced autophagy affects radiation-sensitivity and radiotherapy efficacy. Histone modifications also occur during radiotherapy. This study assessed radiotherapy effects on histone modification and autophagy in non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: NSCLC cells were subjected to γ-irradiation. Autophagy was detected using western blotting and acridine orange staining. Radiation effect on cell growth was evaluated by clonogenic assay. Histone modifications were assessed by western blotting. Next generation sequencings (NGSs) were conducted to identify histone modification target genes. RESULTS: Radio-protective autophagy and histone H4 lysine 20 trimethylation (H4K20me3) were up-regulated after irradiation. By NGSs, genes that are differentially expressed upon irradiation were identified, including the candidate H4K20me3 target gene GABARAPL1. Furthermore, we showed that GABARAPL1 is essential for the radiation-induced autophagy. CONCLUSION: Our findings revealed the regulatory axis of radiation-induced H4K20me3-GABARAPL1 in radio-protective autophagy. Modulation of this axis may be a new strategy to enhance radiotherapy efficacy in NSCLC.

Anti-tumor effect of CDK inhibitors on CDKN2A-defective squamous cell lung cancer cells.

BACKGROUND: Squamous cell lung cancer (SqCLC) is a distinct histologic subtype of non-small cell lung cancer (NSCLC). Although the discovery of driver mutations and their targeted drugs has remarkably improved the treatment outcomes for lung adenocarcinoma, currently no such molecular target is clinically available for SqCLC. The CDKN2A locus at 9p21 encodes two alternatively spliced proteins, p16INK4a (p16) and p14ARF (p14), which function as cell cycle inhibitors. The Cancer Genome Atlas (TCGA) project revealed that CDKN2A is inactivated in 72% of SqCLC cases. In addition, it was found that CDKN2A mutations are significantly more common in SqCLC than in adenocarcinoma. Down-regulation of p16 and p14 by CDKN2A gene inactivation leads to activation of cyclin-dependent kinases (CDKs), thereby permitting constitutive phosphorylation of Rb and subsequent cell cycle progression. Here, we hypothesized that CDK inhibition may serve as an attractive strategy for the treatment of CDKN2A-defective SqCLC. METHODS: We investigated whether the CDK inhibitors flavopiridol and dinaciclib may exhibit antitumor activity in CDKN2A-defective SqCLC cells compared to control cells. The cytotoxic effect of the CDK inhibitors was evaluated using cell viability assays, and the induction of apoptosis was assessed using TUNEL assays and Western blot analyses. Finally, anti-tumor effects of the CDK inhibitors on xenografted cells were investigated in vivo. RESULTS: We found that flavopiridol and dinaciclib induced cytotoxicity by enhancing apoptosis in CDKN2A-defective SqCLC cells, and that epithelial to mesenchymal transition (EMT) decreased and autophagy increased during this process. In addition, we found that autophagy had a cytoprotective role. CONCLUSION: Our data suggest a potential role of CDK inhibitors in managing CDKN2A-defective SqCLC.

Altered expression of cellular proliferation, apoptosis and the cell cycle-related genes in lung cancer cells with acquired resistance to EGFR tyrosine kinase inhibitors.

Non-small cell lung cancers harboring somatic gain-of-function mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain respond well to treatment with EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib. However, all patients who experience a marked improvement with these drugs eventually develop disease progression due to the acquisition of drug resistance. Approximately half of the cases with acquired resistance to EGFR TKIs can be accounted for by a second-site mutation in exon 20 of the EGFR kinase domain (T790M). However, the changes of gene expression involved in EGFR TKI resistance due to the T790M mutation remain poorly defined. The present study established lung cancer cell lines that were resistant to gefitinib or erlotinib, and these cell lines were verified to contain the EGFR T790M mutation. The differential expression of genes associated with acquired resistance was verified in the present study by mRNA microarray analysis. Among the genes whose expression was significantly altered, genes whose expression was altered in gefitinib- and erlotinib-resistant cells were focused on. Notably, a total of 1,617 genes were identified as being differentially expressed in gefitinib- and erlotinib-resistant cells. Indeed, Gene ontology analysis revealed altered expression of genes involved in the regulation of cellular proliferation, apoptosis, and the cell cycle in EGFR TKI-resistant cells. The present results demonstrate distinctive gene expression patterns of EGFR TKI-resistant lung cancer cells with the EGFR T790M mutation. The present study can provide key insights into gene expression profiles involved in conferring resistance to EGFR TKI therapy in lung cancer cells.

Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells.

Inactivation of the fragile histidine triad (Fhit) gene has been reported in the majority of human cancers, particularly in lung cancer. The role of Fhit as a tumor suppressor gene has been well documented, and restoration of Fhit expression suppresses tumorigenicity in tumor cell lines and in mouse models by inducing apoptosis and inhibiting proliferation of tumor cells. Autophagy is a catabolic pathway, whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Although autophagy is necessary for cell survival under stress conditions, recent studies have shown that autophagy can also promote cell death. Due to the fact that both autophagy induction and Fhit expression are commonly associated with nutrient starvation, we hypothesized that Fhit expression may be related to autophagy induction. In the present study, we assessed whether Fhit overexpression by gene transfer induces autophagy in Fhit-deficient non-small cell lung cancer (NSCLC) cells. The results of our study indicate that Fhit protein induces autophagy in NSCLC cells, and that this autophagy prevents apoptotic cell death in vivo and in vitro in a 14-3-3τ protein-dependent manner. To the best of our knowledge, this is the first report to describe Fhit-induced autophagy. Suppressing autophagy might be a promising therapeutic option to enhance the efficacy of Fhit gene therapy in NSCLC.

The combination of irreversible EGFR TKIs and SAHA induces apoptosis and autophagy-mediated cell death to overcome acquired resistance in EGFR T790M-mutated lung cancer.

To overcome T790M-mediated acquired resistance of lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), second generation TKIs such as BIBW2992 (afatinib) and third generation TKIs including WZ4002 have been developed. However, clinical data on their efficacy in treating T790M mutant tumors are lacking. Histone deacetylase (HDAC) inhibitors have been reported to arrest cell growth and to lead to differentiation and apoptosis of various cancer cells, both in vitro and in vivo. In the present study, we assessed whether the combination of suberoylanilide hydroxamic acid (SAHA, vorinostat), a potent HDAC inhibitor, and BIBW2992 or WZ4002 could overcome EGFR TKI resistance associated with T790M mutation in lung cancer cells. While treatment with BIBW2992 or WZ4002 alone slightly reduced the viability of PC-9G and H1975 cells, which possess T790M mutation, combining them with SAHA resulted in significantly decreased cell viability through the activation of the apoptotic pathway. This combination also enhanced autophagy occurrence and inhibition of autophagy significantly reduced the apoptosis induced by the combination treatment, showing that autophagy is required for the enhanced apoptosis. Caspase-independent autophagic cell death was also induced by the combination treatment with SAHA and either BIBW2992 or WZ4002. Finally, the combined treatment with SAHA and either BIBW2992 or WZ4002 showed an enhanced anti-tumor effect on xenografts of H1975 cells in vivo. In conclusion, the combination of new generation EGFR TKIs and SAHA may be a new strategy to overcome the acquired resistance to EGFR TKIs in T790M mutant lung cancer.

Gefitinib induces cytoplasmic translocation of the CDK inhibitor p27 and its binding to a cleaved intermediate of caspase 8 in non-small cell lung cancer cells.

BACKGROUND: The epidermal growth factor receptor (EGFR) represents one of the first rationally selected molecules for targeted therapy in non-small cell lung cancer (NSCLC). Gefitinib is a reversible and highly selective tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of the EGFR. It has been found that treatment with gefitinib induces cell cycle arrest and apoptosis in NSCLC cells harboring activating EGFR mutations. Despite its clinical relevance, however, the mechanism underlying gefitinib-induced apoptosis has remained largely unknown. METHODS: We used the gefitinib-sensitive NSCLC cell line HCC827, which harbors a deletion in exon 19 of the EGFR gene, to examine the effect of gefitinib on the apoptotic machinery. RESULTS: We found that gefitinib treatment caused the NSCLC cells to undergo apoptosis following activation of the caspase 8 cascade. Expression of p27, a cyclin-dependent kinase (CDK) inhibitor whose major target is the cyclin E/CDK2 complex, was found to increase during this process, and this increase was accompanied by translocation of p27 from the nucleus to the cytoplasm. Moreover, we found that cytoplasmic p27 bound to a cleaved intermediate (p43/p41) of caspase 8 and that inhibition of cytoplasmic translocation of p27 reduced gefitinib-induced cell death in HCC827 cells. CONCLUSION: Based on our results, we conclude that gefitinib-induced apoptosis is mediated by the interaction of p27 and caspase 8 in NSCLC cells carrying an activating EGFR mutation.

Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells.

BACKGROUND: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. METHODS: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. RESULTS: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-G1 phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. CONCLUSION: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

Dual Inhibition of PI3K/Akt/mTOR Pathway and Role of Autophagy in Non-Small Cell Lung Cancer Cells.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process. METHODS: We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis. RESULTS: Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process. CONCLUSION: Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.