Radioresistance Of Non-small Cell Lung Cancer Cells Research Articles

RSPO3 regulates the radioresistance of Non-Small cell lung cancer cells via NLRP3 Inflammasome-Mediated pyroptosis

PurposeRadioresistance is a significant challenge in the radiotherapy of non-small cell lung cancer (NSCLC). This study aimed to investigate the role of R-spondin 3 (RSPO3) in regulating NSCLC radioresistance. Methods and MaterialsRNA sequencing was performed to analyze genes that are differentially expressed in radioresistant NSCLC cell lines. RSPO3 overexpression and knockdown experiments were conducted to assess its impact on radiosensitivity. The involvement of the β-catenin-NF-κB signaling pathway and the NLRP3 inflammasome in RSPO3-mediated radiosensitivity was also evaluated. In vivo experiments were conducted using a clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) to assess its impact on radiation-induced pyroptosis and subsequent anti-tumor immunity. ResultsRSPO3 expression was downregulated in radioresistant NSCLC cells. Overexpression of RSPO3 increased NSCLC radiosensitivity through the induction of pyroptosis, which was mediated by the β-catenin-NF-κB signaling pathway and the NLRP3 inflammasome. The anti-RSPO3 antibody effectively blocked radiation-induced pyroptosis and anti-tumor immunity in vivo. Conversely, upregulation of RSPO3 enhanced NSCLC tumor radiosensitivity. ConclusionsThe findings demonstrated that RSPO3 plays a crucial role in regulating NSCLC radioresistance via NLRP3 mediated pyroptosis. Targeting the RSPO3-NLRP3 inflammasome axis may offer a potential therapeutic strategy to enhance the efficacy of radiotherapy for NSCLC patients.

Suppression of cyclin-dependent kinase 1 synergizes with checkpoint kinase 2 inhibitor to promote death of non-small cell lung cancer cells with radiotherapeutic resistance

ObjectiveRadiotherapy resistance poses a prevalent clinical challenge in non-small cell lung cancer (NSCLC), necessitating the development of ongoing treatment protocols following the emergence of radiotherapy resistance, as well as the identification of radiotherapy sensitization drugs. The objective of this study was to exclusively investigate therapeutic targets associated with radiotherapy resistance in NSCLC, thereby expanding the range of treatment alternatives available to patients grappling with radiotherapy resistance. MethodsVarious databases were employed to screen for therapeutic targets associated with radiotherapeutic resistance. The impact of administering inhibitors for cyclin-dependent kinase 1 (CDK1) and checkpoint kinase 2 (CHEK2) on cell proliferation and apoptosis in radioresistant NSCLC cell lines was assessed. ResultsThe potential of CDK1 and CHEK2 kinase as a novel biomarker and therapeutic target in radioresistant NSCLC cells is evident. The up-regulation of CDK1 and CHEK2 expression in NSCLC tissues, as well as their activation in NSCLC cells exposed to X-irradiation, leads to radiotherapeutic resistance. Additionally, the heightened expression of CDK1 and CHEK2 may contribute to an increased score of cancer stem cells (CSCs) in NSCLC patients. The suppression of CDK1 in combination with a CHEK2 inhibitor synergistically promotes the death of NSCLC cells with radiotherapeutic resistance. Notably, the sensitivity of radioresistant NSCLC cell lines to the CHEK2 inhibitor (PV-1019) is enhanced through the pharmacological or genetic inhibition of CDK1. ConclusionsThe current study provides new perspectives on the synergistic effects of CDK1 and CHEK2 inhibitors in inducing apoptosis in NSCLC cells that have acquired resistance to radiotherapy.

Hsa-miR-1301-3p Promotes the Proliferation and Migration of Nonsmall Cell Lung Cancer Cells and Reduces Radiosensitivity via Targeting Homeodomain-Only Protein Homeobox.

Background: There is increasing evidence that abnormal expression of microRNAs is involved in the occurrence and progression of tumors. In previous experiments, we found that the content of hsa-miR-1301-3p in tumor tissues of patients with nonsmall cell lung cancer (NSCLC) showed an obvious upward trend compared with that in normal tissues. We performed a detailed study on the impact and underlying mechanism of hsa-miR-1301-3p in NSCLC cells. Methods: The impact of hsa-miR-1301-3p on NSCLC cell proliferation, apoptosis, migration, and invasion was examined using colony formation, flow cytometry, modified Boyden chamber, and wound healing assays. Different doses of radiation were applied to NSCLC cells to investigate their sensitivity to radiotherapy. The potential target gene of hsa-miR-1301-3p was determined by dual-luciferase reporter assay and immunoblotting. Result: hsa-miR-1301-3p was upregulated in NSCLC tissues and cells. hsa-miR-1301-3p effectively promoted the rapid proliferation, migration, and invasion of NSCLC cells, while inhibiting apoptosis. It also induced radioresistance in NSCLC cells. hsa-miR-1301-3p targeted the homeodomain-only protein homeobox (HOPX) mRNA 3' untranslated region and inhibited its transcription in NSCLC cells. Exogenous HOPX overexpression antagonized the mechanism by which hsa-miR-1301-3p regulates NSCLC cell proliferation, metastasis, and apoptosis. Conclusions: hsa-miR-1301-3p plays an oncogenic role in the occurrence and development of NSCLC. By targeting HOPX, hsa-miR-1301-3p can not only promote the proliferation and metastasis of NSCLC cells, but also alleviate apoptosis and reduce radiosensitivity.

GALNT2, an O-glycosylating enzyme, is a critical regulator of radioresistance of non-small cell lung cancer: evidence from an integrated multi-omics analysis.

Radioresistance is the primary reason for radiotherapy failure in non-small cell lung cancer (NSCLC) patients. Glycosylation-related alterations are critically involved in tumor radioresistance. However, the relationship between glycosylation and NSCLC radioresistance is unclear. Here, we generated radioresistant NSCLC cell models by using fractionated irradiation. The aberrant glycosylation involved in NSCLC-related radioresistance was elucidated by transcriptomic, proteomic, and glycomic analyses. We conducted in vitro and in vivo investigations for determining the biological functions of glycosylation. Additionally, its downstream pathways and upstream regulators were inferred and verified. We demonstrated that mucin-type O-glycosylation and the O-glycosylating enzyme GALNT2 were highly expressed in radioresistant NSCLC cells. GALNT2 was found to be elevated in NSCLC tissues; this elevated level showed a remarkable association with response to radiotherapy treatment as well as overall survival. Functional experiments showed that GALNT2 knockdown improved NSCLC radiosensitivity via inducing apoptosis. By using a lectin pull-down system, we revealed that mucin-type O-glycans on IGF1R were modified by GALNT2 and that IGF1R could affect the expression of apoptosis-related genes. Moreover, GALNT2 knockdown-mediated in vitro radiosensitization was enhanced by IGF1R inhibition. According to a miRNA array analysis and a luciferase reporter assay, miR-30a-5p negatively modulated GALNT2. In summary, our findings established GALNT2 as a key contributor to the radioresistance of NSCLC. Therefore, targeting GALNT2 may be a promising therapeutic strategy for NSCLC.

Transcriptome-Based Traits of Radioresistant Sublines of Non-Small Cell Lung Cancer Cells.

Radioresistance is a major obstacle for the successful therapy of many cancers, including non-small cell lung cancer (NSCLC). To elucidate the mechanism of radioresistance of NSCLC cells and to identify key molecules conferring radioresistance, the radioresistant subclones of p53 wild-type A549 and p53-deficient H1299 cell cultures were established. The transcriptional changes between parental and radioresistant NSCLC cells were investigated by RNA-seq. In total, expression levels of 36,596 genes were measured. Changes in the activation of intracellular molecular pathways of cells surviving irradiation relative to parental cells were quantified using the Oncobox bioinformatics platform. Following 30 rounds of 2 Gy irradiation, a total of 322 genes were differentially expressed between p53 wild-type radioresistant A549IR and parental A549 cells. For the p53-deficient (H1299) NSCLC cells, the parental and irradiated populations differed in the expression of 1628 genes and 1616 pathways. The expression of genes associated with radioresistance reflects the complex biological processes involved in clinical cancer cell eradication and might serve as a potential biomarker and therapeutic target for NSCLC treatment.

Oxymatrine inhibits the development of radioresistance in NSCLC cells by reversing EMT through the DcR3/AKT/GSK-3β pathway

IntroductionLung cancer is the leading cause of cancer-associated mortality globally. In particular, non-small cell lung cancer (NSCLC) constitutes the largest percentage of all cases of lung cancer. In clinical practice, radioresistance contributes to poor responses to radiotherapy. Therefore, the demand remains to explore potential novel and effective mechanism underlying radioresistance to improve the efficacy of radiotherapy for NSCLC.Material and methodsWestern blotting was conducted to quantify the protein expression of epithelial-mesenchymal transition markers E-cadherin and vimentin in the A549 cell line. The proliferation of A549 cells was measured using the Cell Counting Kit-8 and colony forming assays. In addition, the apoptosis of A549 cells was analyzed by flow cytometry. Invasion and migration by NSCLC cells was quantified using Transwell and wound healing assays. Plasmids were used to overexpress decoy receptor 3 (DcR3) in A549 cells. Xenograft models were established to measure the extent of NSCLC tumor growth in vivo.ResultsOur study clarified the activation of the DcR3/protein kinase B (AKT)/glycogen synthase kinase 3β (GSK-3β) pathway in radioresistant NSCLC cells. Oxymatrine (OMT) treatment restored radiosensitivity and inhibited irradiation-induced epithelial-mesenchymal transition (EMT), invasion and migration in NSCLC cells through the DcR3/AKT/GSK-3β pathway in vitro. By contrast, OMT treatment promoted the suppressive effects of radiation on the weight and volume of the xenograft tumors in animal models.ConclusionsIn conclusion, OMT suppressed the development of radioresistance in NSCLC cells by promoting radiosensitivity, through the reversal of EMT process by inhibiting the DcR3/AKT/GSK-3β pathway.

Tumor-suppressive E3 ubiquitin ligase CHIP inhibits the PBK/ERK axis to repress stem cell properties and radioresistance in non-small cell lung cancer.

Recently, radioresistant cancer cells surviving radiotherapy have been suggested to show more aggressive phenotypes than parental cells, and the underlying mechanisms may be associated with cancer stem cells. This study provided novel mechanistic insights for E3 ubiquitin ligase CHIP in stem cell properties and radioresistance of non-small cell lung cancer (NSCLC). After bioinformatic prediction for key genes involved, NSCLC tissues and cells were collected to measure the expression of CHIP and PBK. E3 ubiquitin ligase CHIP was poorly expressed, while PBK was highly expressed in NSCLC tissues and cells. CHIP reduced the protein stability of PBK through the ubiquitin-protease pathway to repress the activation of ERK pathway. Based on the gain- or loss-of-function experiments, it was noted that restoration of CHIP curtailed stem cell properties and radioresistance in NSCLC, as manifested by inhibited sphere formation and cell proliferation, decreased number of CD133+CD44+ cells and expression of OCT4, SOX2, and NANOG, as well as facilitated apoptosis of NSCLC cells. Besides, in vivo animal experiments further confirmed that CHIP restrained tumorigenic ability and improved radiosensitivity of NSCLC cells by inhibiting PBK/ERK axis. Collectively, CHIP suppressed stem cell properties and radioresistance of NSCLC cells by inhibiting PBK/ERK axis, therefore offering a potential therapeutic target for enhancing efficacy of radiotherapy.

MiR-145 modulates the radiosensitivity of non-small cell lung cancer cells by suppression of TMOD3.

Radioresistance is a major problem encountered in the treatment of non-small cell lung cancer (NSCLC). Aberrant microRNA (miRNA) expression contributes to multiple cancer-associated signaling pathways and profoundly influences effects of radiotherapy (RT) in cancers. MicroRNA-145-5p (miR-145) is recognized as a tumor suppresser in NSCLC. However, the roles of miR-145 during radiotherapy of NSCLC are largely unknown. The present study aimed to investigate the function and underlying mechanism of miR-145 in modulation of radiosensitivity in NSCLC. We generated radioresistant H460 and A549 subclones, named H460R and A549R, respectively, and found that irradiation (IR) could suppress the expression levels of miR-145 in radioresistant NSCLC cells. Furthermore, overexpression of miR-145 could sensitize radioresistant NSCLC cells to IR, whereas knockdown of miR-145 in NSCLC cells acted the converse manner. Mechanically, miR-145 was able to directly target 3'UTR of tropomodulin 3 (TMOD3) mRNA and decrease the expression of TMOD3 at the levels of mRNA and protein. Additionally, we confirmed that miR-145 could enhance the radiosensitivity of radioresistant NSCLC cells by targeting TMOD3 in vitro and in vivo, and could be used as a target in clinical treatment of NSCLC. Collectively, restoration of miR-145 expression increases the radiosensitivity of radioresistant NSCLC cells by suppression of TMOD3, and miR-145 can act as a new radiosensitizer for NSCLC therapy.

Silencing of the lncRNA H19 enhances sensitivity to X-ray and carbon-ions through the miR-130a-3p /WNK3 signaling axis in NSCLC cells

BackgroundThe lncRNA H19 is believed to act as an oncogene in various types of tumors and is considered to be a therapeutic target and diagnostic marker. However, the role of the lncRNA H19 in regulating the radiosensitivity of non-small cell lung cancer (NSCLC) cells is unknown.MethodsThe expression profiles of lncRNAs in NSCLC were explored via transcriptome sequencing. CCK-8, EdU incorporation and clonogenic survival assays were conducted to evaluate the proliferation and radiosensitivity of NSCLC cells. Flow cytometry and Western blotting were conducted to measure the level of apoptosis. The binding relationship between the lncRNA H19 and miR-130a-3p was determined by a dual-luciferase reporter assay. A binding relationship was also identified between miR-130a-3p and With-No-Lysine Kinase 3 (WNK3).ResultsExpression patterns of lncRNAs revealed that the lncRNA H19 was upregulated in radioresistant NSCLC (A549-R11) cells compared with A549 cells. Knockdown of the lncRNA H19 enhanced the sensitivity of NSCLC cell lines to X-ray and carbon ion irradiation. Mechanistically, the lncRNA H19 serves as a sponge of miR-130a-3p, which downregulates WNK3 expression. The lncRNA H19–miR-130a-3p–WNK3 axis modulates radiosensitivity by regulating apoptosis in NSCLC cell lines.ConclusionKnockdown of the lncRNA H19 promotes the sensitivity of NSCLC cells to X-ray and carbon ion irradiation. Hence, the lncRNA H19 might function as a potential therapeutic target that enhances the antitumor effects of radiotherapy in NSCLC.

Prognosis biomarker and potential therapeutic target CRIP2 associated with radiosensitivity in NSCLC cells

Radiotherapy plays a major role in non-small cell lung cancer (NSCLC) treatment. The curative efficacy of advanced NSCLC is unsatisfactory because of its radioresistance to conventional radiotherapy. The biomarkers which can be used to diagnose radiosensitivity or predict for prognosis are beneficial in promoting curative effects. In this study, NSCLC cell lines with acquired radioresistance to X-rays were obtained through fractionated irradiation. The differentially expressed proteins (DEPs) between the self-established radioresistant NSCLC cell line A549-R11 and control (A549-CK) were measured by proteomic analysis. Among the detected DEPs, CRIP2, ARHGDIB, and PADI3 were validated to be up-regulated in radioresistant cells, in mRNA and protein levels. Further analysis of bioinformatics deciphered that CRIP2, as a potential biomarker for diagnosis and a key biomarker for prediction of prognosis, may impact the X-ray radiosensitivity of NSCLC by regulating the occurrence of apoptosis and cell cycle arrest; as such, it may serve as a potent therapeutic target to facilitate NSCLC radiotherapy. CRIP2 and other DEPs may shed new light on the recognition of complex factors associated with radiation-responsiveness and finally be beneficial in the advancement of personalized therapies and precision medical treatment.

LINC01224/ZNF91 Promote Stem Cell-Like Properties and Drive Radioresistance in Non-Small Cell Lung Cancer

BackgroundRadioresistance is the main reason for the failure of radiotherapy in non-small-cell lung cancer (NSCLC); however, the molecular mechanism of radioresistance is still unclear.MethodsAn RNA-Seq assay was used to screen differentially expressed long non-coding RNAs (lncRNAs) and genes in irradiation-resistant NSCLC cells. RT-PCR and Western blotting assays were performed to analyze the expressions of lncRNAs and genes. The chromosome conformation capture (3C) assay was performed to measure chromatin interactions. Cell cytotoxicity, cell apoptosis, sphere formation and Transwell assays were performed to assess cellular function.ResultsIn this study, it was found that LINC01224 increased during the induction of radioresistance in NSCLC cells. LINC01224 was located within the enhancer of ZNF91, and LINC01224 could affect the transcription of ZNF91 by regulating the long-range interactions between the ZNF91 enhancer and promoter. Moreover, upregulation of LINC01224 and ZNF91 could promote irradiation resistance by regulating the stem cell-like properties of NSCLC cells. In addition, high expression levels of LINC01224 and ZNF91 in tissue samples were associated with radioresistance in NSCLC patients.ConclusionOur findings demonstrated that LINC01224/ZNF91 drove radioresistance regulation by promoting the stem cell-like properties in NSCLC.

CircZNF208 enhances the sensitivity to X-rays instead of carbon-ions through the miR-7-5p /SNCA signal axis in non-small-cell lung cancer cells

BackgroundMounting evidence suggests that circular RNAs (circRNAs) are closely related to the regulation of gene expression during tumour development. However, the role of circRNAs in modulating the radiosensitivity of non-small cell lung cancer (NSCLC) cells has not been explored. MethodsTranscriptome sequencing was used to explore the expression profiles of circRNAs in NSCLC. The expression level of circRNAs was changed by inducing instantaneous knockdown or overexpression. Changes in proliferation and radiosensitivity of NSCLC cells were investigated using CCK-8, EDU, and clonal survivals. ResultsBy analysing the circRNA expression profile of NSCLC cells, we found that circRNA ZNF208 (circZNF208) was significantly upregulated in a radioresistant NSCLC cell line (A549-R11), which was acquired from the parental NSCLC cell line A549. Knockout experiments indicated that circZNF208 enhanced the radiosensitivity of A549 and A549-R11 cells to X-rays. Mechanistically, circZNF208 upregulated SNCA expression by acting as a sponge of miR-7-5p and subsequently promoted the resistance of NSCLC cells to low linear energy transfer (LET) X-rays. However, this effect was not observed in NSCLC cells exposed to high-LET carbon ions. ConclusionsKnockdown of circZNF208 altered the radiosensitivity of patients with NSCLC to X-rays but did not significantly change the sensitivity to carbon ions. Therefore, circZNF208 might serve as a potential biomarker and therapeutic target for NSCLC treatment with radiotherapy of different modalities.

MiR-129-5p Promotes Radio-sensitivity of NSCLC Cells by Targeting SOX4 and RUNX1

Dysregulation of microRNAs (miRNAs) figures prominently in the radio- sensitivity of non-small cell lung cancer (NSCLC). MiR-129-5p can block the development of a variety of tumors. However, whether miR-129-5p modulates radio-sensitivity of NSCLC cells remains unknown. This study was aimed to explore the role and the underlying mechanism of miR-129-5p in the radiosensitivity of NSCLC. Radio-resistant NSCLC cell lines (A549-R and H1299-R) were constructed using A549 and H1299 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to quantify miR-129-5p, SRY-box transcription factor 4 (SOX4) mRNA, and RUNX family transcription factor 1 (RUNX1) mRNA expression levels. Cell apoptosis and cell cycle were detected by flow cytometry. Cell counting kit-8 (CCK-8) assay and colony formation experiments were used to measure cell proliferation. γ-H2AX was examined by Western blot to confirm DNA injury. Dual- luciferase reporter experiments were applied to analyze the interactions among miR-129-5p, RUNX1, and SOX4. In A549-R and H1299-R cells, compared with the wild-type cell lines, miR-129-5p expression was remarkably reduced while SOX4 and RUNX1 expressions were increased. The transfection of miR-129-5p into NSCLC cell lines markedly induced cell apoptosis, DNA injury, cell cycle arrest, and inhibited cell proliferation and colony formation. RUNX1 and SOX4 were validated as target genes of miR-129-5p, and the restoration of RUNX1 or SOX4 could counteract the influence of miR-129-5p on A549-R cells. MiR-129-5p sensitizes A549-R and H1299-R cells to radiation by targeting RUNX1 and SOX4.

Circular RNA circ_0001287 inhibits the proliferation, metastasis, and radiosensitivity of non-small cell lung cancer cells by sponging microRNA miR-21 and up-regulating phosphatase and tensin homolog expression

ABSTRACT As a type of non-coding RNA, circular RNA (circRNA) figures prominently in human cancer progression. Nonetheless, the expression, function, and regulatory mechanism of circ_0001287 in non-small cell lung cancer (NSCLC) remain obscure. In this work, quantitative real-time polymerase chain reaction (qRT-PCR) was implemented to quantify circ_0001287 and miR-21 expressions in NSCLC tissues and cells. The relationship between circ_0001287 expression and the clinicopathological parameters of NSCLC patients was examined. Cell counting kit-8 (CCK-8), 5-bromo-2©-deoxyuridine (BrdU), and Transwell experiments were conducted to detect the multiplication, migration, and invasion of NSCLC cells after circ_0001287 was overexpressed or knocked down. The survival of NSCLC cells was studied using colony formation experiment under different doses of radiation. RNA immunoprecipitation (RIP) experiment and luciferase reporter gene experiment verified the binding relationship between circ_0001287 and miR-21. Western blot was employed to examine the regulatory effects of circ_0001287 and miR-21 on phosphatase and tensin homolog (PTEN) expression. We reported that circ_0001287 expression was down-modulated in NSCLC tissues and cell lines. Besides, circ_0001287 low expression was associated with low differentiation and positive lymph node invasion of NSCLC. Circ_0001287 overexpression suppressed the multiplication, migration, invasion, and radioresistance of NSCLC cells, whereas circ_0001287 knockdown promoted the above phenotypes. Circ_0001287 could adsorb miR-21 and repress its expression, and indirectly up-modulate PTEN expression in NSCLC cells. Taken together, circ_0001287/miR-21/PTEN axis is probably involved in regulating NSCLC cell multiplication, metastasis, and radioresistance.

Integrated analysis of circRNA-miRNA-mRNA network reveals potential prognostic biomarkers for radiotherapies with X-rays and carbon ions in non-small cell lung cancer.

BackgroundThis work was aimed at exploring the regulatory network of non-coding RNA (ncRNA) especially circular RNA (circRNA) and microRNA (miRNA), in the sensitivity of non-small cell lung cancer (NSCLC) cells to low linear energy transfer (LET) X-ray and high-LET carbon ion irradiations.MethodsThe radioresistant NSCLC cell line A549-R11 was obtained from its parental cell line A549 through irradiation with X-rays of 2.0 Gy per fraction for 30 times. The sensitivities of A549, A549-R11 and H1299 cells exposed to X-rays and carbon ions were verified using the colony formation assay. A comprehensive circRNA-miRNA-mRNA network was constructed through the sequencing data in parental A549, acquired radioresistant A549-R11 and intrinsic radioresistant H1299 cells, and the network was further optimized according to the prognostic results from the TCGA and GEO databases.ResultsBased on high-throughput sequencing of circRNAs, we found that 40 circRNAs were up-regulated while 184 circRNAs were down-regulated in the intersection of the sets of A549-R11 and H1299 cells. Subsequently, a circRNA- miRNA-mRNA network, including 14 interactive pairs and 8 circRNAs, 4 overall survival-associated miRNAs, and 4 mRNAs, was constructed through the high-throughput data screening and bioinformatics methods.ConclusionsOur results provide a complete understanding to the regulatory mechanism of the sensitivities to low-LET X-ray and high-LET carbon ion irradiations, and might be helpful to screen potential biomarkers for predicting the Carbon-ion radiotherapy (CIRT) and X-ray radiotherapy responses in NSCLC.

A novel PPAR\u0263 ligand, PPZ023, overcomes radioresistance via ER stress and cell death in human non-small-cell lung cancer cells

Peroxisome proliferator-activated receptor gamma (PPARɣ) agonists exert powerful anticancer effects by suppressing tumor growth. In this study, we developed PPZ023 (1-(2-(ethylthio)benzyl)-4-(2-methoxyphenyl)piperazine), a novel PPAR ligand candidate, and investigated the underlying signaling pathways in both non-small-cell lung cancer (NSCLC) and radio-resistant NSCLC cells. To identify whether PPZ023 has anticancer effects in NSCLC and radioresistant NSCLC cells, we performed WST-1, LDH, Western blot, and caspase-3 and -9 activity assays. Furthermore, we isolated exosomes from PPZ023-treated NSCLC cells and studied cell death signaling. PPZ023 reduces cell viability and increases LDH cytotoxicity and caspase-3 activity in NSCLC cells. PPZ023 induces cell death by generating reactive oxygen species (ROS) and triggering mitochondrial cytochrome c release. PPZ023 treatment causes cell death via the PERK–eIF2α–CHOP axis in both NSCLC cell lysates and exosomes, and PERK and CHOP knockdown significantly blocks ER stress-mediated apoptosis by reducing cleaved caspase-3. Interestingly, diphenyleneiodonium (DPI, a Nox inhibitor) inhibits PPZ023-induced cell death via ER stress, and PPARɣ knockdown inhibits PPZ023-induced ROS, ER stress, and cell death. Moreover, PPZ023, in combination with radiation, causes synergic cell death via exosomal ER stress in radioresistant NSCLC cells, indicating that PPZ023/radiation overcomes radioresistance. Taken together, our results suggest that PPZ023 is a powerful anticancer reagent for overcoming radioresistance.

Y Chromosome LncRNA Are Involved in Radiation Response of Male Non-Small Cell Lung Cancer Cells.

Numerous studies have implicated changes in the Y chromosome in male cancers, yet few have investigated the biological importance of Y chromosome noncoding RNA. Here we identify a group of Y chromosome-expressed long noncoding RNA (lncRNA) that are involved in male non-small cell lung cancer (NSCLC) radiation sensitivity. Radiosensitive male NSCLC cell lines demonstrated a dose-dependent induction of linc-SPRY3-2/3/4 following irradiation, which was not observed in radioresistant male NSCLC cell lines. Cytogenetics revealed the loss of chromosome Y (LOY) in the radioresistant male NSCLC cell lines. Gain- and loss-of-function experiments indicated that linc-SPRY3-2/3/4 transcripts affect cell viability and apoptosis. Computational prediction of RNA binding proteins (RBP) motifs and UV-cross-linking and immunoprecipitation (CLIP) assays identified IGF2BP3, an RBP involved in mRNA stability, as a binding partner for linc-SPRY3-2/3/4 RNA. The presence of linc-SPRY3-2/3/4 reduced the half-life of known IGF2BP3 binding mRNA, such as the antiapoptotic HMGA2 mRNA, as well as the oncogenic c-MYC mRNA. Assessment of Y chromosome in NSCLC tissue microarrays and expression of linc-SPRY3-2/3/4 in NSCLC RNA-seq and microarray data revealed a negative correlation between the loss of the Y chromosome or linc-SPRY3-2/3/4 and overall survival. Thus, linc-SPRY3-2/3/4 expression and LOY could represent an important marker of radiotherapy in NSCLC. SIGNIFICANCE: This study describes previously unknown Y chromosome-expressed lncRNA regulators of radiation response in male NSCLC and show a correlation between loss of chromosome Y and radioresistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4046/F1.large.jpg.

CB13, a novel PPAR\u03b3 ligand, overcomes radio-resistance via ROS generation and ER stress in human non-small cell lung cancer

Peroxisome proliferator-activated receptor gamma (PPARγ) is a well-known therapeutic target for type 2 diabetes as well as is a potential target for effective anti-cancer drug, since PPARγ ligands such as ciglitazone (Cig) frequently cause cell death in many types of cancer cells and suppress tumor growth. However, many cancer patients acquire chemo-resistance or radio-resistance after chemo or radiotherapy, and it is still unclear. In the difficulty of well-known anti-cancer drugs, we developed a novel PPARγ agonist CB13 (1-benzyl-5-(4-methylphenyl) pyrido [2,3-d]pyrimidine-2,4(1H,3H)-dione) and investigated the anti-cancer effect and cell death mechanism on human non-small cell lung cancer (NSCLC) cells. With anti-cancer effect of Cig, CB13 also causes inhibition of cell growth by decreasing cell viability, increasing the release of LDH, and increasing caspase-3, and caspase-9 activities. CB13 generates reactive oxygen species (ROS) and causes cell death via ER stress in NSCLC and radio-resistant NSCLC cells (A549R and H460R), and a combination of CB13 and radiation induces greater ER stress and cell death when compared to CB13 alone. Taken together, our results suggest that a combination of CB13 and radiation may overcome radio-resistance caused by radiotherapy.

CLPTM1L induces estrogen receptor \u03b2 signaling-mediated radioresistance in non-small cell lung cancer cells

IntroductionRadioresistance is a major challenge in lung cancer radiotherapy, and new radiosensitizers are urgently needed. Estrogen receptor β (ERβ) is involved in the progression of non-small cell lung cancer (NSCLC), however, the role of ERβ in the response to radiotherapy in lung cancer remains elusive. In the present study, we investigated the mechanism underlying ERβ-mediated transcriptional activation and radioresistance of NSCLC cells.MethodsQuantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of CLPTM1L, ERβ and other target genes. The mechanism of CLPTM1L in modulation of radiosensitivity was investigated by chromatin immunoprecipitation assay, luciferase reporter gene assay, immunofluorescence staining, confocal microscopy, coimmunoprecipitation and GST pull-down assays. The functional role of CLPTM1L was detected by function assays in vitro and in vivo.ResultsCLPTM1L expression was negatively correlated with the radiosensitivity of NSCLC cell lines, and irradiation upregulated CLPTM1L in radioresistant (A549) but not in radiosensitive (H460) NSCLC cells. Meanwhile, IR induced the translocation of CLPTM1L from the cytoplasm into the nucleus in NSCLC cells. Moreover, CLPTM1L induced radioresistance in NSCLC cells. iTRAQ-based analysis and cDNA microarray identified irradiation-related genes commonly targeted by CLPTM1L and ERβ, and CLPTM1L upregulated ERβ-induced genes CDC25A, c-Jun, and BCL2. Mechanistically, CLPTM1L coactivated ERβ by directly interacting with ERβ through the LXXLL NR (nuclear receptor)-binding motif. Functionally, ERβ silencing was sufficient to block CLPTM1L-enhanced radioresistance of NSCLC cells in vitro. CLPTM1L shRNA treatment in combination with irradiation significantly inhibited cancer cell growth in NSCLC xenograft tumors in vivo.ConclusionsThe present results indicate that CLPTM1L acts as a critical coactivator of ERβ to promote the transcription of its target genes and induce radioresistance of NSCLC cells, suggesting a new target for radiosensitization in NSCLC therapy.39qrTZQYVJS_ThcYeHjY54Video

Radiotranscriptomics signature‐based predictive nomograms for radiotherapy response in patients with nonsmall cell lung cancer: Combination and association of CT features and serum miRNAs levels

PurposeWe aimed to establish radiotranscriptomics signatures based on serum miRNA levels and computed tomography (CT) texture features and develop nomogram models for predicting radiotherapy response in patients with nonsmall cell lung cancer (NSCLC).MethodsWe first used established radioresistant NSCLC cell lines for miRNA selection. At the same time, patients (103 for training set and 71 for validation set) with NSCLC were enrolled. Their pretreatment contrast‐enhanced CT texture features were extracted and their serum miRNA levels were obtained. Then, radiotranscriptomics feature selection was implemented with the least absolute shrinkage and selection operator (LASSO), and signatures were generated by logistic or Cox regression for objective response rate (ORR), overall survival (OS), and progression‐free survival (PFS). Afterward, radiotranscriptomics signature‐based nomograms were constructed and assessed for clinical use.ResultsFour miRNAs and 22 reproducible contrast‐enhanced CT features were used for radiotranscriptomics feature selection and we generated ORR‐, OS‐, and PFS‐ related radiotranscriptomics signatures. In patients with NSCLC who received radiotherapy, the radiotranscriptomics signatures were independently associated with ORR, OS, and PFS in both the training (OR: 2.94, P < .001; HR: 2.90, P < .001; HR: 3.58, P = .001) and validation set (OR: 2.94, P = .026; HR: 2.14, P = .004; HR: 2.64, P = .016). We also obtained a satisfactory nomogram for ORR. The C‐index values for the ORR nomogram were 0.86 [95% confidence interval (CI), 0.75 to 0.92] in the training set and 0.81 (95% CI, 0.69 to 0.89) in the validation set. The calibration‐in‐the‐large and calibration slope performed well. Decision curve analysis indicated a satisfactory net benefit.ConclusionsThe radiotranscriptomics signature could be an independent biomarker for evaluating radiotherapeutic responses in patients with NSCLC. The radiotranscriptomics signature‐based nomogram could be used to predict patients’ ORR, which would represent progress in individualized medicine.