Cisplatin Resistant NSCLC Cell Research Articles

Evodiamine potentiates cisplatin-induced cell death and overcomes cisplatin resistance in non-small-cell lung cancer by targeting SOX9-β-catenin axis.

In recent decades, phytotherapy has remained as a key therapeutic option for the treatment of various cancers. Evodiamine, an excellent phytocompound from Evodia fructus, exerts anticancer activity in several cancers by modulating drug resistance. However, the role of evodiamine in cisplatin-resistant NSCLC cells is not clear till now. Therefore, we have used evodiamine as a chemosensitizer to overcome cisplatin resistance in NSCLC. Here, we looked into SOX9 expression and how it affects the cisplatin sensitivity of cisplatin-resistant NSCLC cells. MTT and clonogenic assays were performed to check the cell proliferation. AO/EtBr and DAPI staining, ROS measurement assay, transfection, Western blot analysis, RT-PCR, Scratch & invasion, and comet assay were done to check the role of evodiamine in cisplatin-resistant NSCLC cells. SOX9 levels were observed to be higher in cisplatin-resistant A549 (A549CR) and NCI-H522 (NCI-H522CR) compared to parental A549 and NCI-H522. It was found that SOX9 promotes cisplatin resistance by regulating β-catenin. Depletion of SOX9 restores cisplatin sensitivity by decreasing cell proliferation and cell migration and inducing apoptosis in A549CR and NCI-H522CR. After evodiamine treatment, it was revealed that evodiamine increases cisplatin-induced cytotoxicity in A549CR and NCI-H522CR cells through increasing intracellular ROS generation. The combination of both drugs also significantly inhibited cell migration by inhibiting epithelial to mesenchymal transition (EMT). Mechanistic investigation revealed that evodiamine resensitizes cisplatin-resistant cells toward cisplatin by decreasing the expression of SOX9 and β-catenin. The combination of evodiamine and cisplatin may be a novel strategy for combating cisplatin resistance in NSCLC.

Metformin Sensitizes Cisplatin-induced Apoptosis Through Regulating Nucleotide Excision Repair Pathway In Cisplatin-resistant Human Lung Cancer Cells

Background: Lung cancer is a leading cause of cancer death globally. Platinum-based chemotherapeutic medications are essential for treating advanced NSCLC, despite that drug resistance severely limits its effectiveness. Objective: In this study, we investigated the cytotoxic effect of metformin on cisplatin-resistant NSCLC cells (A549/DDP) and its potential mechanisms. Methods: Anti-lung cancer efficacy of metformin, cisplatin, and metformin combined with cisplatin was examined in A549 and A549/DDP cells. The cell counting kit-8 (CCK-8) assay was applied for measuring cell proliferation. CalcuSyn software was used to calculate the combination index and estimate the synergistic effect of metformin and cisplatin on cell proliferation. The cell apoptosis was analyzed by flow cytometry and the expression of apoptosis-related proteins, Bcl-2, Bax and caspase-3 were analyzed using Western blot. Futhermore, the expression of key nucleotide excision repair (NER) proteins, ERCC1, XPF, and XPA, was also analyzed using Western blot. Results: We found that metformin had dose-dependent antiproliferative effects on A549/DDP and A549 cells. The combination of metformin and cisplatin had higher effectiveness in inhibiting A549/DDP and A549 cell growth than either of the two drugs alone. Flow cytometry analysis indicated that the combined treatment could cause more cell apoptosis than the single-drug treatment. Consistently, the combined treatment decreased the expression of Bcl-2 protein and elevated the expression of Bax, and cleaved caspase-3 proteins. The expression level of ERCC1, XPF, and XPA proteins were lower in the combined treatment than in either of metformin and cisplatin treatment alone. Conclusions: Our study suggested that metformin and cisplatin had synergistic antitumorigenic effects in A549/DDP cells. The combination of cisplatin and metformin could be promising drug candidates to sensitize cisplatin-induced apoptosis through regulating nucleotide excision repair pathways in lung cancer.

LncRNA FOXD3-AS1 promoted chemo-resistance of NSCLC cells via directly acting on miR-127-3p/MDM2 axis

BackgroundThis study aims to investigate the mechanism underlying the high level of long non-coding RNA FOXD3-AS1 in cisplatin-resistant NSCLC cells.MethodsCisplatin-resistant cells were generated from A549 cells. CCK-8 were used to evaluate cell proliferation. The FOXD3-AS1, miR-127-3p, MDM2 and MRP1 mRNA expression levels were confirmed by qRT-PCR. Protein levels of MDM2 and MRP1 were determined by western blot assay. Luciferase reporter and RNA pull-down assays were evaluated the relationship between miR-127-3p and FOXD3-AS1/MDM2. In vivo tumor growth was evaluated in a xenograft nude mice model.ResultsFOXD3-AS1 was up-regulated in cisplatin-resistant NSCLC cells (A549/DDP and H1299/DDP cells) in comparison with their parental cell lines. Overexpression of FOXD3-AS1 promoted cisplatin-resistance in A549 and H1299 cells; while FOXD3-AS1 knockdown sensitized A549/DDP and H1299/DDP cells to cisplatin treatment. FOXD3-AS1 regulated miR-127-3p expression by acting as a competing endogenous RNA, and miR-127-3p repressed MDM2 expression via targeting the 3′UTR. MiR-127-3p overexpression and MDM2 knockdown both increased the chemo-sensitivity in A549/DDP cells; while miR-127-3p knockdown and MDM2 overexpression both promoted chemoresistance in A549 cells. Further rescue experiments revealed that miR-127-3p knockdown or MDM2 overexpression counteracted the suppressive effects of FOXD3-AS1 knockdown on chemo-resistance and MRP1 expression in A549/DDP cells. In vivo studies showed that FOXD3-AS1 knockdown potentiated the antitumor effects of cisplatin treatment. Inspection of clinical samples showed the upregulation of FOXD3-AS1 and MDM2, and down-regulation of miR-127-3p in NSCLC tissues compared to normal adjacent tissues.ConclusionIn conclusion, our results suggest that LncRNA FOXD3-AS1 promotes chemo-resistance of NSCLC cells via directly acting on miR-127-3p/MDM2 axis. Our findings may provide novel perspectives for the treatment of NSCLC in patients resistant to chemotherapy.

The miR 495-UBE2C-ABCG2/ERCC1 Axis Reverses DDP Resistance via Downregulation of Anti-Drug Genes in Cisplatin Resistant NSCLC Cells

Resistance to DDP has become the leading cause of mortality in NSCLC. The microRNA dysregulation plays a vital role in tumor progression. In our study, we found that miR-495 was significantly downregulated in lung cancer tissue specimens. Our objective was to elucidate the functions, direct target genes, and molecular mechanisms of miR-495 in lung cancer. We found that miR-495 downregulated the expression of its substrate UBE2C by directly binds to the 3′-UTR of UBE2C. UBE2C is a proto-oncogene that is activated in lung cancer; however, its role in drug resistance is unclear. We found here that UBE2C expression was higher in cisplatin resistant NSCLC cells; this was associated with induced cisplatin resistant NSCLC cells proliferation, invasion and cisplatin resistant. EMT was contributed to cisplatin resistance. We also found that UBE2C knockdown downregulated the expression of the vimentin. In contrast, E-cadherin was upregulated. Importantly we found that miR-495 and UBE2C were associated with cisplatin resistance. We performed to evaluate their effects on cell proliferation and cisplatin resistant. We also performed EMT, cell migration and invasion assays in cisplatin resistant NSCLC cells overexpressing miR-495 and under expressing UBE2C. Furthermore, Bioinformatics assays coupled with WB and luciferase assays revealed that UBE2C directly binds to the 5′-UTR of the drug resistance genes, ABCG2 and ERCC1. We also found that miR-495 reduced the expression of ABCG2 and ERCC1 via regulation of UBE2C. Collectively, our results indicated that miR495-UBE2C-ABCG2/ERCC1 axis reverses DDP resistance via downregulation of anti-drug genes and reducing EMT in cisplatin resistant NSCLC cells. Funding Statement: This work was supported by the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017PH028), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14). Competing Interests: The authors declare no competing financial interests. Ethics Approval Statement: The experimental protocol was approved by the Research Ethics Committee of Binzhou Medical University, China (No. 2017-016-01 for human lung cancer specimen and No. 2017-009-09 for mouse experiments in vivo) and the written informed consent was obtained from all subjects. Informed consent was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th Edition of the AJCC Cancer Staging Manual: Stomach (2010).

PO-510 XRCC6BP1: a novel role in the DNA repair of platinum resistant NSCLC cells

IntroductionAlterations in the DNA repair capacity of damaged cells are now recognised as an important factor in mediating resistance to chemotherapeutic agents.Material and methodsDNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level. The translational relevance of differentially expressed genes was examined in a cohort of chemo-naïve matched normal and tumour lung tissues from NSCLC patients. Loss of function studies were carried out using siRNA technology. The effect of XRCC6BP1 gene knockdown on apoptosis was assessed by FACS. Cellular expression and localization of XRCC6BP1 protein and gH2AX foci in response to cisplatin were examined by immunofluorescence (Cytell). To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like subpopulations within chemoresistant cells. XRCC6BP1 mRNA analysis was also examined in ALDH1+ and ALDH1- subpopulations. Immunohistochemistry analysis was carried out in resected lung tumour tissues and XRCC6BP1 expression was correlated with survival and other clinicopathological parameters.Results and discussionsWe identified a number of critical DNA repair genes that were differentially expressed between PT and CisR NSCLC cells. XRCC6BP1 mRNA and protein expression was significantly increased in H460 CisR cells relative to their PT counterparts. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in lung adenocarcinoma patients. Gene silencing of XRCC6BP1 induced significant apoptosis of chemoresistant cells and reduced their DNA repair capacity. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and gH2AX foci in CisR cells. SP analysis revealed a significantly higher stem cell population in resistant cells, while XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1+ CisR cells compared to ALDH1- cells. IHC scoring of XRCC6BP1 showed that lung cancer patients with high expression had worse survival outcomes.ConclusionOur data highlight the potential of targeting components of the DNA repair pathway in chemoresistant lung cancer, in particular XRCC6BP1, either alone or in combination with conventional cytotoxic therapies such as cisplatin.

P2.01-086 Luteolin is a Novel Target of Axl Receptor Tyrosine Kinase to Inhibit Cell Proliferation and Circumvent Chemoresistance in Lung Cancer Cells

Axl receptor tyrosine kinase (RTK) plays a critical role in cell growth, proliferation, and anti-apoptosis. In this study, we demonstrated the effect of luteolin, a non-toxic flavonoid widely found in various plants, on expression and activation of Axl RTK in NSCLC, H460, and its cisplatin-resistant cell, H460/CisR. 1. Cell viability measurement & Clonogenic assay. 2. Western blot analysis. 3. Promoter activity test. 4. Ectopic expression of Axl. 5. siRNA trasnfection for Axl knockdown. Luteolin treatment of H460 and H460/CisR cells was found to cause a dose-dependent decline of Axl protein as well as mRNA levels. Axl promoter activity was also reduced by luteolin, suggesting the transcriptional down-regulation of Axl expression by luteolin. Axl phosphorylation upon its ligand, Gas6, was inhibited by luteolin, indicating that luteolin also abrogates Gas6-induced Axl phosphorylation. Next, it was found that treatment of both H460 and H460/CisR cells with luteolin decreased the cell viability and clonogenic ability in dose-dependent manner. We further observed the synergistic anti-proliferative effect of luteolin in cells transfected with Axl specific siRNA, while the reduction of its cytotoxic effect in Axl RTK overexpressing cells, confirming that luteolin exerts its anticancer potential via interference of Axl expression. In addition, luteolin was found to result in the increase of p21, a cyclin-dependent kinase inhibitor, in H460 and H460/CisR cells. In summary, our data demonstrate that luteolin inhibits Axl expression and the activation which are associated with its anti-proliferative activity in both parental and cisplatin-resistant NSCLC cells. Thus, Axl seems to be a potent therapeutic target of luteolin to inhibit cell proliferation and to overcome chemoresistance of NSCLC cells.

MA02.02 A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC

MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumor biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumors, including lung cancer. MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7th generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumor tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x103 cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumor volume was measured over time and harvested once the tumor mass measured 500mm3 and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumors and cisplatin resistance was investigated relative to cisplatin sensitive controls. Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT. A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.

P2.03a-063 Small Molecule Cancer Stemness Inhibitor, BBI608, Restores Cisplatin Sensitivity in Resistant NSCLC: Topic: Clinical Trials

The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesizes that there is a robustly resistant stem-like population of cells that survive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumor via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. To date, preclinical studies investigating BBI608 in in vitro and in vivo models of pancreatic and prostate cancer have shown promise. Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as a key CSC subset across cisplatin resistant NSCLC cell lines. PT and CisR cell lines were treated with BBI608 (1μM) and stemness factors investigated, the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin (0-100μM) in the presence and absence of 1μM BBI608. The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity. To date, BBI608 has not been investigated in terms of a cisplatin resistant CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials recruiting patients to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.

P2.03a-064 Inhibition and Exploitation of Aldehyde Dehydrogenase 1 as a Cancer Stem Cell Marker to Overcome Cisplatin Resistant NSCLC: Topic: Clinical Trials

The root of therapeutic resistance is hypothesized to be the presence a rare CSC population within the tumor population which survives chemotherapeutic treatment and has the potential to recapitulate a heterogenic tumor. Aldehyde dehydrogenase 1 (ALDH1) is involved the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified ALDH1 as a promising CSC-marker associated with cisplatin resistance across NSCLC histologies. The H460 CisR subline was separated by FACS into ALDH1-positive and negative subpopulations and subcutaneously injected into NOD/SCID mice to assess tumor initiation and growth. ALDH1 was inhibited in vitro within the cell lines using two pharmacological ALDH1 inhibitors, DEAB and disulfiram, alone and in combination with cisplatin. Cell lines were treated in vitro with retinol and all-trans retinoic acid (ATRA) to exploit the vitamin A/retinoic acid axis in which ALDH1 is involved. The CisR sublines showed significantly greater ALDH1 activity relative to their PT counterparts. In vivo subcutaneous injection of ALDH1-positive and negative subpopulations revealed no significant difference in tumor initiation or growth rate. ALDH1 inhibition in combination with cisplatin significantly decreased clonogenic and proliferative competencies and increased apoptosis cell death compared to cisplatin alone. Vitamin A supplementation and ATRA treatment in combination with cisplatin showed similar re-sensitizing effects. This pharmacological CSC depletion in conjunction with cisplatin treatment resulted in re-sensitization of cisplatin resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of ATRA or an ALDH1 inhibitor to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumor recurrence and cisplatin resistance.

75P Inhibition and exploitation of aldehyde dehydrogenase 1 (ALDH1) as a cancer stem cell marker in cisplatin resistant NSCLC

Background The root of therapeutic resistance is hypothesized to be due to the presence of a rare CSC population within the tumour population. When challenged with chemotherapy, this tumour cell subset survives the insult and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved in the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. Methods FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines of different histologies was used to examine ALDH1 activity. The H460 CisR subline was FACS-sorted (MoFlo®) into ALDH1-positive and ALDH1-negative fractions and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. The effects of inhibiting ALDH1 on cell proliferation (BrdU), apoptosis (Annexin-V/propidium iodide) and colony formation (crystal violet) in PT and CisR cell lines was assessed in vitro using two ALDH1 inhibitors, DEAB (N,N-diethylaminobenzaldehyde) and disulfiram (Antabuse), alone and in combination with cisplatin. Lung cancer cells were treated with retinol and All-trans retinoic acid (ATRA) to exploit the role of ALDH1 in the vitamin A/retinoic acid axis in NSCLC. Results ALDH1 activity was significantly increased in all CisR cell lines relative to their parental counterparts in which little or no ALDH1 expression was detected. Xenograft studies using ALDH1+ and ALDH1– fractions from H460 CisR cells showed no significant difference in tumour growth in mice. ALDH1 inhibition, in combination with cisplatin, significantly decreased cell viability, proliferation and clonogenic survival of CisR sublines only and increased apoptotic cell death compared to cells treated with cisplatin alone. Similarly, treatment of cells with retinol or ATRA, in combination with cisplatin, showed similar re-sensitizing effects. Conclusions In this study, we show that inhibition of the CSC marker, ALDH1, re-sensitizes cisplatin resistant NSCLC cells to the cytotoxic effects of cisplatin. Furthermore, our data suggest a role for ALDH1 as a key enzyme in the vitamin A/retinoic acid axis.

80P XRCC6BP1: A key DNA repair gene in platinum-resistant NSCLC

Background In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to these agents, in particular, cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. Methods DNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes in H460 cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines previously generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated using RT-PCR and western blot analysis, respectively. Loss of function studies were carried out using siRNA technology. The effect of gene knockdown on apoptosis was assessed by Annexin V/propidium iodide (PI) staining using the Cytell® Imaging System. DNA damage and repair in response to cisplatin following gene knockdown was investigated using the γH2AX foci formation assay. The translational relevance of these genes was examined in a cohort of chemo-naive matched normal and tumour lung tissues (n = 20). Results We identified a number of important DNA repair genes differentially regulated between H460 PT and CisR NSCLC cells. These included XRCC6BP1, TOP3A, XPA, PMS1 and hSSB1. XRCC6BP1 mRNA was significantly increased (19.4-fold) in H460 CisR cells relative to their PT counterparts. Gene silencing of XRCC6BP1 re-sensitized CisR lung cancer cells to the pro-apoptotic effects of cisplatin and significantly reduced the DNA repair capacity of these cells. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in adenocarcinoma tissues (AD), while hSSB1 mRNA was significantly increased in both AD and squamous cell carcinoma (SCC) tissues. Conclusions We have identified XRCC6BP1, in addition to hSSB1, as key DNA repair genes implicated in cisplatin resistant NSCLC. Our data highlights the potential of targeting XRCC6BP1, at least in part, in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin chemotherapy.

70P Identification of a novel microRNA signature: Potential diagnostic biomarkers and predictors of cisplatin response?

Background In addition to their involvement in mechanisms underlying cancer initiation and progression, emerging evidence has highlighted a key role for microRNAs in resistance to a number of anti-cancer therapies. Methods MicroRNA profiling and validation was carried out on a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines using 7th generation miRCURY LNA™ arrays (Exiqon). Transient transfections were used to either inhibit or over-express these microRNAs using antagomirs or pre-miRs, respectively. The effects of modulating these miRNAs on cell proliferation, apoptosis and clonogenic survival were examined in response to cisplatin. The translational relevance of these miRNAs in lung cancer was confirmed in a cohort of chemo-naive matched normal and tumour lung tissues and sera from NSCLC patients. MicroRNAs were also assessed in mouse xenograft FFPE tissues from H460 CisR-derived ALDH1+ and ALDH1– cancer stem cell subpopulations. Results A 5-miRNA signature consisting of members of the miR-30 family, miR-34a and miR-4286 was identified and validated. MicroRNAs were significantly up-regulated in all CisR cell lines relative to their parental counterparts, in contrast to miR-4286 which was significantly down-regulated. Inhibition of the miR-30 family and miR-34a and forced over-expression of miR-4286 significantly reduced the clonogenic survival ability of CisR cells in response to cisplatin. The miR-30 family was significantly decreased in AD and SCC tissues. MiR-34a expression was significantly lower in SCC tissues while miR-4286 levels were significantly increased in AD tissues only. A significant-fold increase in miR-4286 was found in sera from SCC lung cancer patients. Similar to that found at the in vitro level, the miR-30 family and miR-34a were up-regulated in mouse xenograft FFPE tissues derived from CisR and PT cell lines in vivo. Conclusions In this study, a 5-microRNA signature was identified using an in vitro model of cisplatin resistance. Differential expression of these microRNAs was found between matched normal and tumour lung tissues from NSCLC patients. These may offer potential as diagnostic markers in NSCLC and in predicting response to cisplatin chemotherapy.

8 Exploitation of the vitamin A/retinoic acid axis in the re-sensitisation of cisplatin resistant NSCLC cells

8 Exploitation of the vitamin A/retinoic acid axis in the re-sensitisation of cisplatin resistant NSCLC cells

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

Role of IGF-1/IGFBP3 in non-small cell lung cancer sensitivity to therapy.

e19123 Background: Molecular mechanisms of drug-resistance remain still unclear; it is believed multifactorial, involving host factors, numerous molecular events as well as genetic and epigenetic changes. We were aimed to use massive expression assays before and after epigenetic treatment to explore predictors for platinum and radiation therapy in NSCLC. Methods: By using an expression microarray analysis, we looked for those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Then, to investigate the importance of methylation in radiosensitivity, we have developed radiation clonogenic cell survival assays with three paired Sensitive/CDDP-Resistant cell lines that present a different gene expression and promoter methylation profile. Finally, we have explored in vitro data into three cohorts of patients from different institutions that were treated with radio, chemoradiation or chemotherapy alone. Results: IGF-1/IGFBP3 axis was significantly associated with response to therapy and promoter methylation of IGFBP3 was critical to predict biological behavior. In this regard, hypermethylated and semimethylated cell lines presented a higher sensitivity to radiotherapy and cisplatin than cell lines completely unmethylated for IGFBP3 promoter. However, when resistance to cisplatin treatment was not mediated by IGFBP3 promoter methylation there were also not significant changes regarding sensitivity to radiotherapy treatment. Finally, in vitro data for IGF-1/IGFBP3 axis were confirmed in three cohorts of patients. Conclusions: IGF-1/IGFBP3 axis plays a central role in resistance to chemo- and radiation therapy in NSCLC patients.

2 MicroRNA profiling and cisplatin–DNA interactions in an isogenic model of cisplatin resistant NSCLC cell lines

2 MicroRNA profiling and cisplatin–DNA interactions in an isogenic model of cisplatin resistant NSCLC cell lines

149P - Microrna Profiling and CDDP-GPG DNA Adduct Formation Analysis in a Panel of Cisplatin Resistant Non-Small Cell Lung Cancer Cell Lines

ABSTRACT Introduction The outcome of cisplatin therapy in NSCLC has reached a plateau, with the development of resistance being a major obstacle in the use of this drug. Understanding the molecular mechanisms underlying this resistance phenotype may aid in the development of novel agents that enhance the sensitivity of cisplatin. In this study, we examined the microRNA profile and levels of cisplatin-DNA adduct formation in a panel of cisplatin resistant NSCLC cell lines. Methods We have previously generated a panel of cisplatin resistant (CisR) cell lines (MOR, H460, A549, SKMES-1, H1299) from original, age-matched parent (PT) cells and extensively characterised these based on a number of functional assays including proliferation (MTT), apoptosis (FACS), cell cycle arrest (PI staining), survival ability (clonogenic assay), DNA copy number gains and losses (CGH arrays) and cancer stem cell marker expression (CD44, CD133, ALDH1, Nanog, Sox-2 and Oct-4) by FACS and Western blot analysis. MicroRNA profiling was carried out using the nCounter miRNA Expression Assay consisting of 749 target miRNA's. Immunofluorescence staining and measurement of specific DNA platination products (CDDP-GpG DNA adducts) was performed at 0, 4, 12 and 24h using immunofluorescence microscopy and quantitative digital image analysis using the ACAS 6.0 CytometryAnalysis System, respectively. Results In a panel of five NSCLC cell lines exhibiting a cisplatin resistant phenotype, a 3- and 13-miRNA signature was deduced, based on differential expression of 3 miRNA's (5/5 cell lines) and 13 miRNA's (4/5 cell lines) in the CisR cell lines relative to their parent counterparts. Levels of CDDP-GpG adducts were greatly reduced in resistant cells up to 24h in response to cisplatin compared to that observed in corresponding parent cells. Conclusion We have identified a distinct miRNA fingerprint in a clinically relevant, isogenic model of cisplatin resistance that may predict resistance to cisplatin in lung cancer. Furthermore, as cisplatin-DNA adducts correlate with cisplatin resistance in vitro, these may be used as a potential predictive marker in patient response to platinum therapy in NSCLC. Disclosure All authors have declared no conflicts of interest.

The effect of PARP inhibition on BAX/BAK independent synthetic lethality of BRCA1-deficient non-small cell lung cancer.

e13519 Background: Evasion of apoptosis contributes to both tumorigenesis and drug resistance. The proapoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. In contrast, prosurvival BCL-2 family proteins eg. MCL-1 and BCLX are among the most frequently amplified in human cancers, consistent with negative regulation of this pathway. New strategies for targeting cancer in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. Mutation of BRCA1 confers a defect in homologous recombination mediated DNA repair that can be exploited by synthetic lethality through inhibition of PARP. It is not known whether BAX and BAK play an essential role in mediating synthetic lethality. Methods: The effect of BRCA1 loss on response to PARP inhibitor therapy in non small cell lung cancer was studied in vitro using clonigenic assays in a panel of NSCLC cells, cells where both BAX and BAK expression was silenced (rendering them mitochondrial apoptosis defective), and cisplatin resistant NSCLC cells. BRCA1 immunohistochemistry was performed on a total of 302 primary NSCLC tumours from two independent cohorts to determine the frequency of BRCA1 immunonegativity in the patient population. Results: We show that BRCA1 silencing enhances sensitivity to PARP inhibitor therapy in a panel of NSCLC cell lines and in cells defective for mitochondrial apoptosis. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance but can still sensitize to PARP inhibition in the platinum resistant setting. BRCA1 immunohistochemistry showed an immunodeficiency (<10% cells stained positive) rate of 11% and 19% in two independent cohorts. Conclusions: In summary, targeting BRCA1 negative NSCLC by PARP inhibitors results in bypass of mitochondrial apoptosis block and can override platinum resistance. The existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic.

Differential expression of DNAmethyltransferases in sensitive versus cisplatin resistant NSCLC cell lines

Differential expression of DNAmethyltransferases in sensitive versus cisplatin resistant NSCLC cell lines

Identification of drug-sensitive prediction genes by an epigenetic reactivation screen of cisplatin-resistant NSCLC cell lines

In eukaryotes, a crucial step of translation initiation is the binding of the multifactor complex eIF4F to the 5′ end of the mRNA, a prerequisite to recruitment of the activated small ribosomal 43S particle. Histone H4 mRNAs have short 5′UTRs, which do not conform to the conventional scanning-initiation model. Here we show that the ORF of histone mRNA contains two structural elements critical for translation initiation. One of the two structures binds eIF4E without the need of the cap. Ribosomal 43S particles become tethered to this site and directly loaded in the vicinity of the AUG. The other structure, 19 nucleotides downstream of the initiation codon, forms a three-way helix junction, which sequesters the m7G cap. This element facilitates direct positioning of the ribosome on the cognate start codon. This unusual translation initiation mode might be considered as a hybrid mechanism between the canonical and the IRES-driven translation initiation process.