Chemoresistance In Non-small Cell Lung Cancer Research Articles

New Paradigms to Assess Consequences of Long-Term, Low-Dose Curcumin Exposure in Lung Cancer Cells.

Curcumin has been investigated extensively for cancer prevention, but it has been proposed that long-term treatments may promote clonal evolution and gain of cellular resistance, potentially rendering cancer cells less sensitive to future therapeutic interventions. Here, we used long-term, low-dose treatments to determine the potential for adverse effects in non-small cell lung cancer (NSCLC) cells. IC50s for curcumin, cisplatin, and pemetrexed in A549, PC9, and PC9ER NSCLC cells were evaluated using growth curves. IC50s were subsequently re-assessed following long-term, low-dose curcumin treatment and a three-month treatment withdrawal period, with a concurrent assessment of oncology-related protein expression. Doublet cisplatin/pemetrexed-resistant cell lines were created and the IC50 for curcumin was determined. Organotypic NSCLC-fibroblast co-culture models were used to assess the effects of curcumin on invasive capacity. Following long-term treatment/treatment withdrawal, there was no significant change in IC50s for the chemotherapy drugs, with chemotherapy-resistant cell lines exhibiting similar sensitivity to curcumin as their non-resistant counterparts. Curcumin (0.25–0.5 µM) was able to inhibit the invasion of both native and chemo-resistant NSCLC cells in the organotypic co-culture model. In summary, long-term curcumin treatment in models of NSCLC neither resulted in the acquisition of pro-carcinogenic phenotypes nor caused resistance to chemotherapy agents.

The MyoD family inhibitor domain-containing protein enhances the chemoresistance of cancer stem cells in the epithelial state by increasing β-catenin activity

Cancer cells with mesenchymal attributes potentially display chemoresistance. Cancer stem cells (CSCs), which are intrinsically resistant to most chemotherapy agents, exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal states. However, the drug response of CSCs in the epithelial and mesenchymal states has not been completely investigated. In this study, we found that epithelial-type (E-cadherinhigh/CD133high) CSCs displayed a higher sphere formation ability and chemoresistance than mesenchymal-type (E-cadherinlowCD133high) CSCs. Gene expression profiling of the CSC and non-CSC subpopulations with distinct epithelial-to-mesenchymal transition (EMT) states showed that MyoD family inhibitor domain-containing (MDFIC) was selectively upregulated in epithelial-type CSCs. Knockdown of MDFIC sensitized epithelial-type CSCs to chemotherapy agents. Ectopic expression of MDFIC increased the chemoresistance of mesenchymal-type CSCs. In a tissue microarray, high MDFIC expression was associated with poor prognosis of non-small cell lung cancer (NSCLC) patients. A mechanistic study showed that the MDFIC p32 isoform, which is located in the cytoplasm, interacted with the destruction complex, Axin/GSK-3/β-catenin. This interaction stabilized β-catenin by inhibiting β-catenin phosphorylation at S33/37 and increased the nuclear translocation and transcriptional activity of β-catenin. Knockdown of β-catenin decreased MDFIC-enhanced chemoresistance. These results suggested that the upregulation of MDFIC enhanced the chemoresistance of epithelial-type CSCs by elevating β-catenin activity. Thus, targeting MDFIC-regulated β-catenin signaling of epithelial-type CSCs may be a potential strategy to overcome chemoresistance in NSCLC.

CircMTDH.4/miR-630/AEG-1 axis participates in the regulation of proliferation, migration, invasion, chemoresistance, and radioresistance of NSCLC.

Astrocyte elevated gene-1 (AEG-1) plays a critical role in the development, progression, and metastasis of a variety of cancers, including non-small-cell lung cancer (NSCLC). The objective of the current study is to unravel the upstream signaling of AEG-1. A cohort of 28 NSCLC tissues and 30 normal tissues were collected. Quantitative reverse transcription-polymerase chain reaction and Western blotting were used to examine AEG-1, migration, and invasion related markers in NSCLC cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay coupled with colony formation assay were conducted to monitor cell growth. Transwell assay was performed to determine cell migration and invasion. Apoptotic cells were detected by costaining with Annexin-V-fluorescein isothiocyanate and propidium iodide. Immunofluorescent staining was used to observe the levels of migration and invasion related markers. Xenograft models were used to investigate tumor formation in vivo. Dual-luciferase reporter assay and RNA immunoprecipitation were carried out to determine the interaction between circMTDH.4 and miR-630, as well as the associated between miR-630 and AEG-1. AEG-1 was highly expressed in NSCLC tissues and cell lines. Silencing of AEG-1 inhibited cell proliferation, migration, invasion, and chemoresistance/radioresistance in NCI-H1650 and A549 cells. circMTDH.4 regulated AEG-1 expression via sponging miR-630. Knockdown of circMTDH.4 and/or overexpression of miR-630 inhibited chemoresistance and radioresistance in NSCLC cells, whereas overexpression of AEG-1 or knockdown of miR-630 exerted rescue effects. circMTDH.4/miR-630/AEG-1 axis is responsible for chemoresistance and radioresistance in NSCLC cells.

Upregulation of Long Noncoding RNA (lncRNA) X-Inactive Specific Transcript (XIST) is Associated with Cisplatin Resistance in Non-Small Cell Lung Cancer (NSCLC) by Downregulating MicroRNA-144-3p.

BackgroundPatients with advanced non-small cell lung cancer (NSCLC) treated with cisplatin, also termed cis-diamminedichloroplatinum (CDDP) or diamminedichloroplatinum (DDP), may develop chemoresistance. This study aimed to investigate the role of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) and multidrug resistance-1 (MDR1) in tumor tissue samples and the chemoresistant human NSCLC cell lines, H460/DDP and A549/DDP, and in a murine A549/DDP tumor xenograft.Material/MethodsTissue samples were from patients with NSCLC who responded cisplatin (DDP-sensitive) (n=24), patients with NSCLC unresponsive to cisplatin (DDP-resistant) (n=30), and normal lung tissue (n=25). In H460/DDP and A549/DDP cells, expression of XIST, microRNA (miR)-144-3p, MDR1, and multidrug resistance-associated protein 1 (MRP1) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The MTT assay measured cell survival and proliferation, a transwell assay evaluated cell migration, and flow cytometry measured apoptosis. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays examined the relationship between XIST and miR-144-3p. Tumor xenografts from A549/DDP cells were studied in BALB/c nude mice.ResultsIn tissue from patients with DDP-resistant NSCLC and the mouse A549/DDP tumor xenograft, lncRNA-XIST expression was upregulated and miR-144-3p expression was inhibited. In A549/DDP and H460/DDP cells, down-regulation of lncRNA-XIST and upregulation of miR-144-3p reduced cell survival, proliferation, migration, induced apoptosis and suppressed MDR1 and MRP1 expression.ConclusionsUpregulation of lncRNA-XIST was associated with cisplatin resistance in NSCLC by downregulating miRNA-144-3p in H460/DDP and A549/DDP cells, a murine A549/DDP tumor xenograft, and human tumor tissues from patients with cisplatin-resistant NSCLC.

PIDD interaction with KEAP1 as a new mutation-independent mechanism to promote NRF2 stabilization and chemoresistance in NSCLC

Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A major mechanism of chemoresistance involves stabilization of the NRF2 transcription factor. NRF2 levels are normally tightly regulated through interaction with KEAP1, an adaptor that targets NRF2 to the CUL3 E3 ubiquitin ligase for proteolysis. In NSCLC, aberrant NRF2 stabilization is best understood through mutations in NRF2, KEAP1, or CUL3 that disrupt their interaction. Biochemical studies, however, have revealed that NRF2 can also be stabilized through expression of KEAP1-interacting proteins that competitively sequester KEAP1 away from NRF2. Here, we have identified PIDD, as a novel KEAP1-interactor in NSCLC that regulates NRF2. We show that this interaction allows PIDD to reduce NRF2 ubiquitination and increase its stability. We also demonstrate that PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo, and that this effect is dependent on NRF2. Finally, we report that NRF2 protein expression in a NSCLC cohort exceeds the typical incidence of combined NRF2, KEAP1, and CUL3 mutations, and that NRF2 expression in this cohort is correlated with PIDD levels. Our data identify PIDD as a new NRF2 regulator, and suggest that variations in PIDD levels contribute to differential chemosensitivities among NSCLC patients.

The Role of Prostaglandin-Endoperoxide Synthase-2 in Chemoresistance of Non-Small Cell Lung Cancer.

The prostaglandin-endoperoxide synthase-2 (PTGS2) plays essential roles in diverse pathological process. Although recent studies implied that PTGS2 was closely related with chemoresistance, the precise roles and the underlying mechanisms of PTGS2 in the developing process of chemoresistance in non-small cell lung cancer (NSCLC) remained elusive. In the present study, we revealed a novel molecular mechanism of PTGS2 implicated in the chemoresistance of NSCLC and proposed a model for the positive feedback regulation of PTGS2 in the process of developing resistance phenotype in NSCLC cells. Our results demonstrated that cisplatin induced PTGS2 expression through the ROS-ERK1/2-NF-κB signaling axis. The prostaglandin E2 (PGE2) derived from PTGS2 catalyzation further strengthened PTGS2 expression via the PGE2-EPs-ERK1/2 positive feedback loop, which induced multidrug resistance of NSCLC cells through up-regulation of BCL2 expression and the subsequent attenuation of cell apoptosis. Consistently, high levels of both PTGS2 and BCL2 were closely associated with poor survival in NSCLC patients. Inhibition of PTGS2 significantly reversed the chemoresistance in the resistant NSCLC in vitro and in vivo. Our results suggested that PTGS2 might be employed as an adjunctive therapeutic target for improving the response to the therapeutic agents in a subset of resistant NSCLC.

P53 sensitizes chemoresistant non-small cell lung cancer via elevation of reactive oxygen species and suppression of EGFR/PI3K/AKT signaling

BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer deaths primarily due to chemoresistance. Somatic mutation of TP53 (36%) and epidermal growth factor receptor (EGFR; > 30%) are major contributors to cisplatin (CDDP) resistance. Substantial evidence suggests the elevated levels of reactive oxygen species (ROS) is a key determinant in cancer. The elevated ROS can affect the cellular responses to chemotherapeutic treatments. Although the role of EGFR in PI3K/Akt signaling cascade in NSCLC is extensively studied, the molecular link between EGFR and p53 and the role of ROS in pathogenesis of NSCLC are limitedly addressed. In this study, we investigated the role of p53 in regulation of ROS production and EGFR signaling, and the chemosensitivity of NSCLC.MethodsIn multiple NSCLC cell lines with varied p53 and EGFR status, we compared and examined the protein contents involved in EGFR-Akt-P53 signaling loop (EGFR, P-EGFR, Akt, P-Akt, p53, P-p53) by Western blot. Apoptosis was determined based on nuclear morphological assessment using Hoechst 33258 staining. Cellular ROS levels were measured by dichlorofluorescin diacetate (DCFDA) staining followed by flow cytometry analysis.ResultsWe have demonstrated for the first time that activation of p53 sensitizes chemoresistant NSCLC cells to CDDP by down-regulating EGFR signaling pathway and promoting intracellular ROS production. Likewise, blocking EGFR/PI3K/AKT signaling with PI3K inhibitor elicited a similar response. Our findings suggest that CDDP-induced apoptosis in chemosensitive NSCLC cells involves p53 activation, leading to suppressed EGFR signaling and ROS production. In contrast, in chemoresistant NSCLC, activated Akt promotes EGFR signaling by the positive feedback loop and suppresses CDDP-induced ROS production and apoptosis.ConclusionCollectively, our study reveals that the interaction of the p53 and Akt feedback loops determine the fate of NSCLC cells and their CDDP sensitivity.

Knockdown of TRIM65 inhibits autophagy and cisplatin resistance in A549/DDP cells by regulating miR-138-5p/ATG7

Cisplatin resistance is the main cause of treatment failure in patients with non-small-cell lung cancer (NSCLC). Autophagy is a key mechanism of resistance to chemotherapy. Given that tripartite motif (TRIM)-containing proteins are involved in the regulation of autophagy and chemoresistance, we aimed to study the functions of TRIM protein members in autophagy-mediated chemoresistance of NSCLC. We found that TRIM65 was significantly increased in cisplatin-resistant NSCLC cell line (A549/DDP) as compared to the parental cell line (A549). Knockdown of TRIM65 can enhance cisplatin-induced apoptosis and inhibit autophagy in A549/DDP cells, as indicated by Annexin V/PI staining, caspase3 activity test, and LC3-II immunofluorescence staining. Additionally, knockdown of TRIM65 significantly decreased the expression of an important autophagy mediator, ATG7, which was a potential target of miR-138-5p. miR-138-5p inhibitor significantly abolished the effects of TRIM65 knockdown on autophagy and cisplatin-induced apoptosis. Moreover, TRIM65 induced the ubiquitination and degradation of TNRC6A, resulting in the suppressed expression of miR-138-5p. TRIM65 knockdown inhibited the growth of tumors derived from A549/DDP cells. Furthermore, cisplatin-resistant NSCLC tissues displayed higher expression of TRIM65 mRNA and lower expression of miR-138-5p as compared to cisplatin non-resistant ones. miR-138-5p expression was negatively correlated with TRIM65 mRNA in NSCLC tissues. Collectively, the present study indicates that TRIM65 knockdown attenuates autophagy and cisplatin resistance in A549/DDP cells via regulating miR-138-5p.

Hypoxic non-small-cell lung cancer cell-derived exosomal miR-21 promotes resistance of normoxic cell to cisplatin.

PurposeTo explore the effects of hypoxic non-small-cell lung cancer (NSCLC)-derived exosomes on NSCLC resistance to cisplatin.Materials and methodsExosomes were isolated by differential centrifugation and characterized by transmission electron microscope and Western blotting. Quantitative real-time PCR was used to measure miR-21 levels. MTT assays and colony formation assays were performed to investigate the effects of hypoxia-induced exosomes on cisplatin resistance.ResultsHypoxic NSCLC cell-derived exosomes facilitate normoxic cell resistance to cisplatin. In addition, hypoxia enhanced the miR-21 expression in NSCLC cells and cell-derived exosomes. Interestingly, changes in miR-21 levels in the hypoxia-induced exosomes affected the sensitivity of recipient cells to cisplatin. Mechanically, exosomal miR-21 promoted cisplatin resistance by downregulating phosphatase and tensin homolog (PTEN). The expression of miR-21 in tumor cell lines and clinical NSCLC tumor samples was positively correlated with hypoxia-inducible factor-1α and negatively correlated with PTEN. Moreover, high miR-21 expression was associated with shorter median survival period in patients undergoing pharmacotherapy, but no association was observed in patients who were not under pharmacotherapy.ConclusionExosomal miR-21 derived from hypoxic NSCLC cells may promote cisplatin resistance, which indicates that exosomal miR-21 might be a potential biomarker and therapeutic target to address NSCLC chemoresistance.

CHD1L contributes to cisplatin resistance by upregulating the ABCB1\u2013NF-\u03baB axis in human non-small-cell lung cancer

Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is a recently identified gene associated with malignant tumor progression and patient chemotherapy resistance in human hepatocellular carcinoma (HCC). Previously, we found an association between CHD1L overexpression and poor patient survival in non-small-cell lung cancer (NSCLC). However, little is known about the relationship between CHD1L expression and chemotherapy resistance of NSCLC. By employing immunohistochemistry, we analyzed the expression of CHD1L in NSCLC samples and elucidated the roles and mechanism of CHD1L in NSCLC chemoresistance. We found that the increased expression of CHD1L is positively correlated with a shorter survival time of patients who had received chemotherapy after surgery. We also found that the expression of CHD1L was increased after cisplatin treatment in A549 cells. Conversely, the depletion of CHD1L in cisplatin-resistance cells increased the cell sensitivity to cisplatin, indicating that CHD1L plays a critical role in cisplatin resistance of NSCLC cells. Importantly, we identified the ATP-Binding Cassette Sub-Family B Member (ABCB1) gene as a potential downstream target of CHD1L in NSCLC cells. Knocking down ABCB1 coupled with ectopic expression of CHD1L enhanced the effect of cisplatin on NSCLC cells apoptosis. In addition, overexpressed CHD1L increase the transcription of c-Jun which targeted directly to the promoter of ABCB1. Our data demonstrate that CHD1L could induce cisplatin resistance in NSCLC via c-Jun-ABCB1–NF-κB axis, and may serve as a novel predictive marker and the potential therapeutic target for cisplatin resistance in NSCLC.

Daurinoline suppressed the migration and invasion of chemo-resistant human non-small cell lung cancer cells by reversing EMT and Notch-1 and sensitized the cells to Taxol

Non-small cell lung cancer (NSCLC) is one of the most common malignancies, and Taxol is a cornerstone in the treatment. However, taxol-resistance eventually limits the clinical effects and applications. Daurinoline could restore the sensitivity of resistant MCF-7/adr and KBv200 cells. Whereas, the effect of daurinoline on the chemo-resistant NSCLC cells and the mechanism has not been elucidated. In this study, daurinoline was firstly demonstrated that inhibited the proliferation, migration, invasion and EMT phenotype of chemo-resistant NSCLC cells. And these effects were associated with EMT and Notch-1 reversal. Moreover, daurinoline could significantly enhance the anti-tumor effect of Taxol rather than epirubicin, adriamycin and cisplatin. And the reverse fold (RF) value of daurinoline was greater than terfenadine reported before. There are little cytotoxic effects of daurinoline and its derivatives reported by L.W. Fu, et al. (2001). Therefore, daurinoline may be a potential anti-tumor agent or chemosensitizer for chemo-resistant NSCLC patients.

Self-targeted knockdown of CD44 improves cisplatin sensitivity of chemoresistant non-small cell lung cancer cells.

Chemoresistance remains a major challenge for effective chemotherapy of non-small-cell lung carcinoma (NSCLC). CD44 expression is related to the susceptibility of various cancer cell types to anticancer drugs. Here, we systematically investigated the CD44-dependent chemoresistance of NSCLC cells and developed a liposomal siRNA delivery system to overcome this chemoresistance by the self-targeted downregulation of CD44. We confirmed the relationship between the expression of CD44 and the chemosensitivity of NSCLC cells using flow cytometry and MTT assay. We then generated and characterized cisplatin-resistant cell lines and compared the expression of CD44 in resistant cells to that in parental cells using western blotting. To evaluate whether the chemosensitivity of resistant cells depends on CD44 expression, we performed CD44 knockdown using CD44 siRNA and detected the chemosensitivity of these cells. Additionally, we prepared hyaluronic acid (HA)-coated liposomes as a targeted delivery system to selectively deliver CD44-specific siRNA to chemoresistant NSCLC cells and observed whether the chemosensitivity of these cells was improved. We found that CD44 expression is inversely proportional to the degree of cellular response to cisplatin chemotherapy and that CD44 is overexpressed in chemoresistant NSCLC cells. By performing CD44 knockdown using siRNA, we reconfirmed that the chemosensitivity of resistant cells depends on CD44 expression. We also observed that HA-liposome-mediated siRNA delivery prior to cisplatin chemotherapy significantly reduced CD44 expression and enhanced cisplatin sensitivity in chemoresistant NSCLC cells. These results suggest that self-targeted downregulation of chemoresistance-associated cell surface proteins during chemotherapy is an effective therapeutic strategy for overcoming the chemoresistance of NSCLC cells.

LncRNA BLACAT1 is involved in chemoresistance of non‑small cell lung cancer cells by regulating autophagy.

The aim of the present study was to determine the effect of the long non‑coding RNA (lncRNA) bladder cancer‑associated transcript1 (BLACAT1) in chemoresistance of non‑small cell lung cancer (NSCLC) cells. Expression of lncRNA BLACAT1, microRNA (miR)‑17, autophagy‑related protein7 (ATG7), multidrug‑resistance protein1 (MRP1), and the autophagy‑associated proteins light chain3 (LC3)‑II/LC3‑I and Beclin1 were detected using the reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Cell viability was determined using an MTT assay. The interaction between BLACAT1 and miR‑17 was determined using RNA immunoprecipitation and RNA pull‑down assays. A cisplatin (DDP)‑resistant NSCLC cell A549/DDP xenograft model in nude mice was established to investigate the effect of BLACAT1 on the chemoresistance of NSCLC cells. Compared with in DDP‑sensitive NSCLC cells, expression of BLACAT1, ATG7, MRP1, LC3‑II/LC3‑I and Beclin1 was significantly upregulated in DDP‑resistant NSCLC cells, whereas miR‑17 was downregulated in DDP‑resistant NSCLC cells. Short interfering RNA against BLACAT1 decreased the viability of DDP‑resistant NSCLC cells. In addition, BLACAT1 interacted with miR‑17, and negatively regulated miR‑17. BLACAT1 promoted ATG7 expression through miR‑17, and facilitated autophagy and promoted chemoresistance of NSCLC cells through miR‑17/ATG7. Finally, in vivo experiments indicated that inhibition of BLACAT1 ameliorated the chemoresistance of NSCLC. BLACAT1 was upregulated in DDP‑resistant NSCLC cells, and promoted autophagy and chemoresistance of NSCLC cells through the miR‑17/ATG7 signaling pathway.

NEAT1 contributes to the CSC-like traits of A549/CDDP cells via activating Wnt signaling pathway

Long non-coding RNAs (lncRNAs) have been identified to exert crucial roles in tumorigenesis and can serve as novel biomarkers for cancer therapy including lung cancer. Cisplatin is a first-line chemotherapeutic agent in non-small cell lung cancer (NSCLC), but the therapeutic effect is unsatisfactory, partly due to drug resistance. Emerging evidence showed that chemo-resistance is associated with acquisition of cancer stem cell (CSC)-like properties. Cisplatin resistance remains a major obstacle in the treatment of lung cancer, and its mechanism is still not fully elucidated. Meanwhile, CSCs have been involved in tumor metastasis, tumor recurrence and chemotherapy resistance. So far, the mechanism of nuclear enriched abundant transcript 1 (NEAT1) in modulating CSCs in lung cancer remains barely known. Therefore, we aimed to explore the correlation between NEAT1 and cancer stem cells in lung cancer. In our current study, we observed that CSC-like traits were much more enriched in cisplatin-resistant A549/CDDP cells. In addition, NEAT1 was obviously up-regulated in A549/CDDP cells compared with parental A549 cells. Knockdown of NEAT1 decreased the CSC-like properties of A549/CDDP cells through inhibiting tumor cell sphere volume, repressing CSC-like biomarkers levels and restraining CD44 positive cell ratios. Oppositely, overexpression of NEAT1 enhanced the stemness respectively. Moreover, it has been reported that Wnt pathway is implicated in many vital cellular functions including cancer stem cells. Here, it was exhibited that Wnt signal pathway was inactivated by knockdown of NEAT1 whereas activated by NEAT1 overexpression in A549/CDDP cells. Taken these together, it was indicated that NEAT1 could exert a novel biological role in NSCLC chemo-resistance.

Knockdown of miR‑935 increases paclitaxel sensitivity via regulation of SOX7 in non‑small‑cell lung cancer.

Sex determining region Y‑box (SOX)7 is a member of the SOX family and is responsible for various developmental processes. As a tumor suppressor, decreased expression of SOX7 has been observed in several cancer types, including non‑small‑cell lung cancer (NSCLC). However, the mechanism underlying SOX7 downregulation and its role in chemoresistance in NSCLC remains poorly understood. In the present study, the inhibition of microRNA (miR)‑935 increased the expression of SOX7 at the mRNA and protein levels in A549 cells. The luciferase reporter assay verified that miR‑935 could directly bind to the 3'untranslated region of SOX7 mRNA to suppress its expression in A549 cells. In addition, the inhibition of miR‑935 enhanced the anticancer effect of paclitaxel, i.e., induced cell growth arrest and apoptosis in A549 cells. It was further observed that the inhibition of miR‑935 decreased the B cell lymphoma (Bcl)‑2 and phosphorylated‑RAC‑α serine/threonine‑protein kinase (AKT) protein levels and increased the Bcl‑2 associated X, apoptosis regulator protein levels, without affecting the AKT levels in the presence of paclitaxel within A549 cells. The findings of the present study validate miR‑935 as a predictor of paclitaxel sensitivity in NSCLC.

PO-233 Chemoresistant NSCLC cells are hypersensitive to metabolic drugs due to mTOR-mediated inhibition of autophagy

IntroductionIn non-small cell lung cancer (NSCLC) cisplatin is the first line chemotherapy and triggers a strong apoptotic response mainly by inducing DNA interstrand crosslinks. However, the effectiveness of cytotoxic cancer therapy is often limited by the emergence of drug resistant tumour cells. Here, we identify strategies for overcoming resistance to DNA interstrand crosslinkers such as cisplatin.Material and methodsWe treated the NSCLC cell line H460 continuously with DNA crosslinking agents resulting in resistant subclones that displayed cross-resistance to multiple DNA-damaging cancer drugs, which are commonly used in the clinic.Results and discussionsDrug resistance was found to be mediated by mTOR-dependent upregulation of DNA repair via the Fanconi anaemia pathway. Strikingly, despite being multidrug-resistant, resistant cells were highly sensitive to treatment with the metabolic drugs 2-deoxyglucose (2-DG) and dichloroacetate (DCA), whereas parental H460 cells failed to respond. Moreover, xenograft studies in mice revealed, that DCA preferentially reduces growth of resistant tumour cells. Interestingly, mTOR inhibition by RNAi or pharmacological inhibitors not only reversed resistance to crosslinking drugs but in parallel mitigated the apoptotic response to metabolic drugs, thereby linking sensitivity to metabolic drugs with mTOR signalling in chemoresistant cells. Mechanistically, inhibition of autophagy by mTOR was found to be decisive. We observed elevated levels of the mTOR-induced inhibitory S757 phosphorylation of the central autophagy-initiating kinase Ulk1 in chemoresistant tumour cells. Matching these observations, autophagosome formation was detected only in parental, but was abrogated in resistant tumour cells upon DCA treatment. In line, knockout of ATG7 using the CRISPR/Cas9 system or direct inhibition of different steps of autophagy with pharmacological autophagy inhibitors was sufficient to render parental cells sensitive to metabolic drugs. Failure to initiate autophagy resulted in lethal energy stress indicating that autophagy is absolutely required to survive metabolic drug treatment.ConclusionTogether these results demonstrate that mTOR-triggered resistance to DNA-damaging cancer drugs generates a therapeutic vulnerability to metabolic drugs due to mTOR-mediated inhibition of autophagy.

Exosome-mediated transfer of lncRNA RP11‑838N2.4 promotes erlotinib resistance in non-small cell lung cancer.

Currently, resistance to tyrosine kinase inhibitors, such as erlotinib, has become a major obstacle for improving the clinical outcome of patients with metastatic and advanced-stage non-small cell lung cancer (NSCLC). While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the roles of lncRNAs within extracellular vesicles (exosomes) are largely unknown. To this end, in this study, the involvement and regulatory functions of potential lncRNAs wrapped by exosomes during the development of chemoresistance in human NSCLC were investigated. Erlotinib-resistant cell lines were established by grafting HCC827 and HCC4006 cells into mice and which were treated with erlotinib. After one treatment course, xenografted NSCLC cells were isolated and transplanted into nude mice again followed by erlotinib treatment. This process was repeated until 4th generation xenografts were isolated and confirmed to be erlotinib-resistant NSCLC cells. lncRNA microarray assays followed by RT-qPCR were then performed which identified that lncRNA RP11-838N2.4 was upregulated in erlotinib-resistant cells when compared to normal NSCLC cells. Furthermore, bioinformatics analysis and chromatin immunoprecipitation revealed that forkhead box protein O1 (FOXO1) could bind to the promoter region of lncRNA RP11-838N2.4, resulting in its silencing through the recruitment of histone deacetylase. Functional experiments demonstrated that the knockdown of lncRNA RP11-838N2.4 potently promoted erlotinib-induced cytotoxicity. Furthermore, extracellular lncRNA RP11-838N2.4 could be incorporated into exosomes and transmitted to sensitive cells, thus disseminating erlotinib resistance. Treatment-sensitive cells with exosomes containing lncRNA RP11-838N2.4 induced erlotinib resistance, while the knockdown of lncRNA RP11-838N2.4 abrogated this effect. In addition, the serum expression levels of exosomal lncRNA RP11-838N2.4 were upregulated in patients exhibiting resistance to erlotinib treatment. On the whole, exosomal lncRNA RP11-838N2.4 may serve as a therapeutic target for patients with NSCLC.

High expression of Aurora-B is correlated with poor prognosis and drug resistance in non-small cell lung cancer.

Aurora kinase B (Aurora-B) is a crucial regulator of accurate mitosis. Abnormal Aurora-B expression is associated with aneuploidy and has been implicated in the pathogenesis and drug resistance in a variety of human cancers. However, little evidence is available regarding the role of Aurora-B in regulating drug response in non-small cell lung cancer (NSCLC), which is the most common type of lung cancer, and is characterized with poor prognosis and high mortality. In the current study, we investigated the association of Aurora-B with the prognosis of NSCLC patients, and we also used the latest CRISPR/Cas9 system to explore the regulatory role of Aurora-B in NSCLC cells developing resistance to cisplatin (CDDP) and paclitaxel. We found that Aurora-B was correlated with significantly reduced overall survival and disease-free survival in NSCLC patients. Aurora-B overexpression was also observed in NSCLC cells developing impaired response to both CDDP and paclitaxel. Moreover, we found, for the first time, that Aurora-B may impair NSCLC drug response by disturbing cell proliferation and inhibiting p53-related DNA damage response and apoptotic pathway, while the knockout of Aurora-B resensitized NSCLC cells to chemo drugs by ensuring correct chromosome segregation and restoring p53 expression. Our results demonstrated the association of Aurora-B with chemoresistance in NSCLC, which may finally contribute to the poor prognosis of NSCLC patients. We also suggested Aurora-B as a promising therapeutic target in NSCLC treatment.

High Kpnβ1 expression promotes non-small cell lung cancer proliferation and chemoresistance via the PI3-kinase/AKT pathway.

Karyopherin β1 (Kpnβ1), also known as importin-β, is part of the karyopherin superfamily of nuclear transport proteins. Kpnβ1 is an oncogene that is overexpressed in various human cancers. Recent studies have showed that Kpnβ1 is one of the leading causes of cancer-related deaths in the world. However, the role of Kpnβ1 in non-small cell lung cancer (NSCLC) remains uncertain. In this study, we used western blotting to show that Kpnβ1 expression is higher in lung-cancer tissues and cells, and immunohistochemistry analysis revealed that Kpnβ1 was significantly associated with the clinicopathological features of NSCLC. Kaplan-Meier analysis showed that elevated Kpnβ1 expression correlated with a poor prognosis in NSCLC patients. Serum starvation-refeeding experiments and Kpnβ1-shRNA transfection assays revealed that elevated Kpnβ1 expression promoted cell proliferation and reduced sensitivity to cis-diamminedichloroplatinum. Immunoprecipitation assays showed that Kpnβ1 interacts with PI3 K to activate the PI3-kinase/AKT pathway, leading to enhanced cell survival and drug resistance in NSCLC cells. Collectively, our findings suggest that Kpnβ1 plays a significant role in NSCLC progression and chemoresistance. Our study provides new insights for targeted therapy to treat NSCLC.

The Roles of microRNAs in Regulating Chemotherapy Resistance of Non-Small Cell Lung Cancer.

Lung cancer is considered the leading cause of cancer death in the word. Among all lung cancer cases, non-small cell lung cancer (NSCLC) accounts for 85%. Currently, chemotherapy is still the major therapeutic method of lung cancer. However, chemoresistance is a serious obstacle in treating NSCLC. Up to now, many reports have suggested that microRNAs(miRNAs) could contribute to lung cancer cell chemosensitivity. MicroRNAs, a class of endogenous non-coding small RNAs, regulate gene expression by binding to their complementary target messenger RNAs, leading to mRNA degradation or translational suppression. Numerous studies indicate that changes of specific miRNA expression may contribute to chemoresistance or chemosensitivity. Consequently, targeting specific miRNAs could become an effective approach to predict the therapeutic effect of different anticancer agents, achieve more effective personalised treatments and enhance chemosensitivity. This review summarizes the several major drug resistance mechanisms and focuses on the roles of miRNAs in regulating chemoresistance in NSCLC.