Pemetrexed Sensitivity Research Articles

A novel lncRNA-hidden polypeptide regulates malignant phenotypes and pemetrexed sensitivity in A549 pulmonary adenocarcinoma cells

The advance of high-throughput sequencing enhances the discovery of short ORFs embedded in long non-coding RNAs (lncRNAs). Here, we uncovered the production and biological activity of lncRNA-hidden polypeptides in lung adenocarcinoma (LUAD). In the present study, bioinformatics was used to screen the lncRNA-hidden polypeptides in LUAD. Analysis of protein expression was done by western blot or immunofluorescence assay. The functions of the polypeptide were determined by detecting its effects on cell viability, proliferation, migration, invasion, and pemetrexed (PEM) sensitivity. The protein interactors of the polypeptide were analyzed by mass spectrometry after Co-immunoprecipitation (Co-IP) assay. The results showed that the lncRNA LINC00954 was confirmed to encode a novel polypeptide LINC00954-ORF. The polypeptide had tumor-suppressor features in A549 cells by repressing cell growth, motility and invasion. Moreover, the polypeptide enhanced PEM sensitivity and suppressed growth in A549/PEM cells. The protein interactors of this polypeptide had close correlations with RNA processing, amide metabolic process, translation, RNA binding, RNA transport, and DNA replication. As a conclusion, the LINC00954-ORF polypeptide embedded in lncRNA LINC00954 possesses tumor-suppressor features in A549 and PEM-resistant A549 cells and sensitizes PEM-resistant A549 cells to PEM, providing evidence that the LINC00954-ORF polypeptide is a potential anti-cancer agent in LUAD.

Warburg effect enhanced by AKR1B10 promotes acquired resistance to pemetrexed in lung cancer-derived brain metastasis

BackgroundResistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown.MethodsPEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches.ResultsThe lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle.ConclusionsOur finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.

The Potential Mechanism of HDAC1-Catalyzed Histone Crotonylation of Caspase-1 in Nonsmall Cell Lung Cancer.

Nonsmall cell lung cancer (NSCLC) is a predominant subtype of lung cancer and accounts for over 80% of all lung cancer cases. The resistance to pemetrexed (PEM) is frequently occurred and severely affects the NSCLC therapy. Proteomic analysis of histones indicated that the histone deacetylase 1 (HDAC1) complex could hydrolyze lysine crotonylation on histone3 (H3K18cr), affecting epigenetic regulation in cancers. However, the effect of HDAC1-mediated H3K18cr on the PEM resistance of NSCLC is still unclear. Here, we aimed to explore the function of HDAC1-mediated H3K18cr in NSCLC PEM resistance. The expression of HDAC1 was upregulated in clinical NSCLC tissues and cell lines and correlated with the poor prognosis of NSCLC samples. We constructed the PEM-resistant NSCLC cell lines, and the depletion of HDAC1 remarkably reduced the viability of the cells. The proliferation of PEM-resistant NSCLC cells was decreased by HDAC1 knockdown, and the IC50 of PEM was repressed by the silencing of HDAC1 in the cells. Mechanically, we identified the enrichment of HDAC1 on the promoter of caspase-1 in PEM-resistant NSCLC cells. The depletion of HDAC1 inhibited the enrichment of histone H3K18cr and RNA polymerase II (RNA pol II) on the caspase-1 promoter in the cells. The expression of caspase-1 was suppressed by HDAC1 knockdown. The knockdown of HDAC1 reduced proliferation of PEM-resistant NSCLC cells, in which caspase-1 or GSDMD depletion reversed the effect. Clinically, the HDAC1 expression was negatively associated with caspase-1 and GSDMD in clinical NSCLC tissues, while caspase-1 and GSDMD expression was positively correlated in the samples. Therefore, we concluded that HDAC1-catalyzed histone crotonylation of caspase-1 modulates PEM sensitivity of NSCLC by targeting GSDMD.

BMI1-Mediated Pemetrexed Resistance in Non-Small Cell Lung Cancer Cells Is Associated with Increased SP1 Activation and Cancer Stemness.

Lung cancer is the leading cause of cancer death worldwide and the therapeutic strategies include surgery, chemotherapy and radiation therapy. Non-small cell lung cancers (NSCLCs) account for around 85% of cases of lung cancers. Pemetrexed is an antifolate agent that is currently used as the second line chemotherapy drug in the treatment of advanced NSCLC patients with a response rate of 20–40%. The search for any combination therapy to improve the efficacy of pemetrexed is required. The existence of cancer stem cells (CSCs) is considered as the main reason for drug resistance of cancers. In this study, we first found that pemetrexed-resistant NSCLC cells derived from A549 cells displayed higher CSC activity in comparison to the parental cells. The expression of CSC related proteins, such as BMI1 or CD44, and the epithelial–mesenchymal transition (EMT) signature was elevated in pemetrexed-resistant NSCLC cells. We next discovered that the overexpression of BMI1 in A549 cells caused the pemetrexed resistance and inhibition of BMI1 by a small molecule inhibitor, PTC-209, or transducing of BMI1-specific shRNAs suppressed cell growth and the expression of thymidylate synthase (TS) in pemetrexed-resistant A549 cells. We further identified that BMI1 positively regulated SP1 expression and treatment of mithramycin A, a SP1 inhibitor, inhibited cell proliferation, as well as TS expression, of pemetrexed-resistant A549 cells. Furthermore, overexpression of BMI1 in A549 cells also caused the activation of EMT in and the enhancement of CSC activity. Finally, we demonstrated that pretreatment of PTC-209 in mice bearing pemetrexed-resistant A549 tumors sensitized them to pemetrexed treatment and the expression of Ki-67, BMI1, and SP1 expression in tumor tissues was observed to be reduced. In conclusion, BMI1 expression level mediates pemetrexed sensitivity of NSCLC cells and the inhibition of BMI1 will be an effective strategy in NSCLC patients when pemetrexed resistance has developed.

Circular RNA CDR1-AS contributes to pemetrexed and cisplatin chemoresistance through EGFR/PI3K signaling pathway in lung adenocarcinoma

Recent studies found circRNAs were involved in tumorigenesis and became new tumor biomarkers and therapeutic targets in lung adenocarcinoma (LUAD), which played a critical role in various biological processes and had been implicated in resistance to chemotherapeutic drugs. However, the role of CDR1-AS in chemoresistance of LUAD to pemetrexed (PTX) and cisplatin (CDDP) is poorly understood. Here, we found that CDR1-AS was up-regulated in LUAD tissues and cell lines, its high-expression was relevant to smoking history, T stage and neoadjuvant chemotherapy (PTX and CDDP) of LUAD patients. CDR1-AS was an independent prognostic biomarker for LUAD patients. CDR1-AS was highly expressed in PTX and CDDP resistant LUAD tissues and cell line (A549/CR). Silence of CDR1-AS re-sensitized A549/CR cells to PTX and CDDP. CDR1-AS was closely related with EGFR/PI3K signaling pathway in A549/CR cells. The activating of EGFR/PI3K pathway mostly restored the effecting of CDR1-AS silence on PTX and CDDP sensitivity in A549/CR cells, CDR1-AS contributed to PTX and CDDP chemoresistance through EGFR/PI3K signaling pathway in LUAD. In conclusion, the CDR1-AS is high-expressed in LUAD and is an independent prognostic biomarker for LUAD patients. The high-expression of CDR1-AS is related with the PTX and CDDP insensitivity of LUAD patients, CDR1-AS promotes PTX and CDDP chemoresistance through EGFR/PI3K signaling pathway in LUAD.

Blood mRNA expression of REV3L and TYMS as potential predictive biomarkers from platinum-based chemotherapy plus pemetrexed in non-small cell lung cancer patients.

Therapeutic options for cancer patients have increased in the last years, although drugs resistance problem remains unresolved. Genetic background in individual susceptibility to cancer treatment could influence the therapy responses. The aim of this study was to explore the feasibility of using blood 4 genes (AEG-1, BRCA-1, REV3L and TYMS) expression levels as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer. Sixteen patients from the Medical Oncology Department at "12 de Octubre" Hospital, were included in the study. Total mRNA was isolated from blood samples, and gene expression was analyzed by RT-qPCR. A panel of lung tumor cell lines were used in cell proliferation tests and siRNA-mediated silencing assays. Similarity between blood gene expression levels and protein expression in matched tumor tissue was observed in 54.54% (REV3L) and 81.81% (TYMS) of cases. Gene expression of REV3L and TYMS in blood correlated directly and inversely, respectively, with progression-free survival and overall survival in the patients from our cohort. In tumor cell lines, the knockdown of REV3L conferred resistance to pemetrexed treatment, and the TYMS silencing increased the pemetrexed sensitivity of tumor cells. The use of peripheral blood samples for expression quantification of interest genes is an affordable method with promising results in the evaluation of response to pemetrexed treatment. Therefore, expression levels of REV3L and TYMS genes might be used as predictive biomarkers in advanced NSCLC patients.

AMPK activation induced in pemetrexed-treated cells is associated with development of drug resistance independently of target enzyme expression.

Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first‐line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP‐activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM‐resistant mesothelioma cell lines in which expression of the PEM‐target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM‐resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.

FGF2-FGFR1 pathway activation together with thymidylate synthase upregulation is induced in pemetrexed-resistant lung cancer cells.

Pemetrexed (MTA) is a folate antimetabolite used for treating non-small cell lung cancer. To elucidate the mechanisms of pemetrexed resistance in lung cancer, we established pemetrexed-resistant sublines in PC9 (mutant EGFR) and H1993 (wild-type EGFR) lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Gene expression profile comparison by microarray analyses revealed enhanced fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1) expression, confirmed by Western blotting, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. ERK phosphorylation was increased in PC9-MTA but decreased in H1993-MTA along with decreased downstream signaling molecule phosphorylation. Cellular morphological change from epithelial to spindle-shape together with increased mesenchymal marker protein expression was observed in H1993-MTA. SiRNA-mediated FGF2 knockdown partially restored pemetrexed sensitivity in both lines, whereas anti-FGFR1 inhibitor PD173074 restored pemetrexed sensitivity in PC9-MTA. FGF2 or FGFR1 inhibition decreased pERK levels in PC9-MTA but increased pEGFR levels together with downstream signaling molecule activation and reversed epithelial-mesenchymal transition marker protein expression in H1993-MTA. Although thymidylate synthase strongly facilitates the development of pemetrexed resistance, our results reveal involvement of the FGF2-FGFR1 pathway in pemetrexed resistance in lung cancer cells and suggest that cellular function alterations induced by FGF2-FGFR1 pathway activation depend on the innate feature of cancer cells.

Dexamethasone alleviates pemetrexed-induced senescence in Non-Small-Cell Lung Cancer

Pemetrexed (PEM) is a novel and multi-targeted antifolate used as an antineoplastic agent for non-small cell lung cancer (NSCLC) and pleural mesothelioma. Although glucocorticoid was often used with PEM to reduce toxicity during the chemotherapy, it is not clear yet whether glucocorticoid co-administration could affect PEM efficacy in NSCLC. Here we established NSCLC cell lines and examined the effects of dexamethasone (DEX) on PEM sensitivity in vitro and in xenograft models. DEX co-administration reduced chemotherapy sensitivity to PEM in xenograft models. DEX co-administration promoted cell growth and weakened senescence growth arrest, such as altered secretions of proinflammatory and mitogenic cytokines, reminiscent of a senescence associate secretory phenotype (SASP). CSCs in DEX co-administration group were subsequently found to be less sensitive towards PEM treatment as measured by cell proliferation and generation of tumor spheres in the presence of PEM. Survival molecule B-cell lymphoma-2 (Bcl-2) may involve in this process and blockage of Bcl-2 could reverse altered senescence and CSCs abilities, thus alleviated PEM insensitivity. As such, DEX might suppress the antitumor activity of PEM through altered SASP level that had induced traits similar to CSCs.

High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase.

The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53-null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild-type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.

Dasatinib modulates sensitivity to pemetrexed in malignant pleural mesothelioma cell lines.

BackgroundThymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated.ResultsMPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling.Cell lines and MethodsIn three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed.ConclusionsThese data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.

The ERK\u2013ZEB1 pathway mediates epithelial\u2013mesenchymal transition in pemetrexed resistant lung cancer cells with suppression by vinca alkaloids

High thymidylate synthase (TS) level in cancer tissue is considered to result in resistance to pemetrexed therapy for advanced stages of nonsquamous non-small cell lung cancers. To further investigate the mechanism of pemetrexed resistance and potential prognostic outcomes in lung cancer, we established pemetrexed-resistant lung adenocarcinoma cell sublines from CL1 harboring a mutated TP53 gene (R248W) and A549 harboring wild-type TP53. We found the TS expression is upregulated in both pemetrexed-resistant sublines and the reduced TS level achieved through shRNA inhibition resulted in higher pemetrexed sensitivity. We also demonstrated that the acquisitions of pemetrexed resistance enhances epithelial–mesenchymal transition (EMT) in vivo with a mice animal model and in vitro with CL1 and A549 sublines, which was associated with upregulation of ZEB1 which, in turn, downregulates E-cadherin and upregulates fibronectin. When ERK1/2 phosphorylation was reduced by an inhibitor (U0126) or siRNA inhibition, both pemetrexed-resistant sublines reduced their migration and invasion abilities. Therefore, the ERK-mediated pathways induce apoptosis with pemetrexed treatment, and may in turn mediate EMT when cancer cells are resistant to pemetrexed. We further demonstrated that the growth of pemetrexed-resistant tumors could be inhibited by vinblastine in vivo and vincristine in vitro. Our data indicate that pemetrexed resistance could be relieved by non-cross-resistant chemotherapeutic drugs such as vinca alkaloids and might be independent to TP53 status. Furthermore, the phosphorylation of ERK was reduced by vincristine. This finding provides a new insight for overcoming pemetrexed resistance and metastasis by application of vinca alkaloids.

Inhibition of MEK/ERK activation attenuates autophagy and potentiates pemetrexed-induced activity against HepG2 hepatocellular carcinoma cells

Identification of efficient chemo-therapeutic/chemo-preventive agents for treatment of hepatocellular carcinoma (HCC) is important. In this study, we examined the activity of pemetrexed, an anti-folate chemotherapy drug, against HepG2 human HCC cells. Pemetrexed treatment in vitro exerted weak but significant cytotoxic activity against HepG2 cells. When analyzing the possible pemetrexed-resistance factors, we indentified that pemetrexed treatment in HepG2 cells induced cyto-protective autophagy activation, evidenced by GFP–light chain 3B (LC3B) puncta formation, p62 downregulation and Beclin-1/LC3B-II upregulation. Correspondingly, autophagy inhibitors, including bafliomycin A1, 3-methyladenine and chloroquine, enhanced pemetrexed-induced cytotoxicity against HepG2 cells. Further, RNAi-mediated knockdown of Beclin-1 in HepG2 cells also increased pemetrexed sensitivity. Pemetrexed activated MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular-signal-regulated kinase) signaling in HepG2 cells, which was required for autophagy induction. Pharmacological inhibition of MEK/ERK activation attenuated pemetrexed-induced autophagy, enhanced HepG2 cell death and apoptosis. In summary, pemetrexed activates MEK/ERK-dependent cyto-protective autophagy, and inhibition of this pathway potentiates pemetrexed’s activity in HepG2 cells.

Abstract 784: Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed

Abstract Background: Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear. Materials and Methods: We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis. Results: Three cell lines were sensitive to PEM. Eighteen transcripts and fourteen genes identified by Gene-chips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative RT-PCR analysis and Western blotting. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy. Conclusion: PEM has anti-tumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM. Citation Format: Susumu Takeuchi, Masahiro Seike, Rintaro Noro, Chie Soeno, Teppei Sugano, Fenfei Zou, Masaru Matsumoto, Akihiko Miyanaga, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma. Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 784. doi:10.1158/1538-7445.AM2014-784

Significance of osteopontin in the sensitivity of malignant pleural mesothelioma to pemetrexed

Pemetrexed (PEM) is currently recommended as one of the standard anticancer drugs for malignant pleural mesothelioma (MPM). However, the mechanism of the sensitivity of MPM to PEM remains unclear. We analyzed the antitumor effects of PEM in six MPM cell lines by MTS assay. To identify genes associated with drug sensitivity, we conducted gene expression profiling on the same set of cell lines using GeneChips and pathway analysis. Three cell lines were sensitive to PEM. A total fo 18 transcripts and 14 genes identified by GeneChips were significantly correlated with sensitivity to PEM. Pathway analysis revealed that osteopontin (SPP1/OPN) was an important target in PEM sensitivity. Overexpression of SPP1/OPN was observed in the sensitive cells by quantitative PCR and western blot analysis. Introduction of SPP1/OPN by lentiviral vector significantly enhanced the invasion activities of MPM cells. PEM treatment with SPP1/OPN knockdown inhibited the PEM-induced cell growth-inhibitory effect in PEM-sensitive cells. Expression of SPP1/OPN and AKT phosphorylation significantly decreased after PEM treatment of the PEM-sensitive cells. High immunohistochemical expression of SPP1/OPN was observed in two of three MPM patients who had a partial response to PEM-based chemotherapy. PEM has antitumor effects in MPM cells dependent on SPP1/OPN overexpression resulting in AKT activation. Our results suggest that SPP1 may be used as a single predictive biomarker of the effectiveness of PEM treatment in MPM.

Downregulation of both EGFR and ErbB3 improves the cellular response to pemetrexed in an established pemetrexed-resistant lung adenocarcinoma A549 cell line

Epidermal growth factor receptor (EGFR) and ErbB3 (HER3) play important roles in the regulation of cell proliferation, differentiation, anti-apoptosis and chemoresistance; however, their dysregulation in pemetrexed (PEM) resistance remains unclear. The aim of the present study was to clarify the relationship between PEM resistance and gene expression of EGFR and ErbB3, by establishing the PEM-resistant lung adenocarcinoma A549 cell line, A549/PEM. Compared with A549 cells, the A549/PEM cells were significantly more resistant to PEM (P=0.0024). The downregulation of S phase and arrest at G1 stage were detected in the A549/PEM cell line when compared to the A549 cells (P<0.05). The apoptosis rate of A549/PEM cells was much lower than that of the A549 cells after a 24 h continuous exposure to PEM (P<0.001). Real-time PCR and western blotting demonstrated the overexpression of EGFR and ErbB3 in A549/PEM cells. However, downregulation of EGFR or ErbB3 by lentiviral delivered shRNAs in A549/PEM cells showed no significant correlation with PEM sensitivity while silencing both EGFR and ErbB3 increased the cellular response to PEM in the A549/PEM cells and significantly decreased phosphorylation of STAT3, AKT and ERK. Together, these data suggest that either high expression of EGFR or ErbB3 plays a critical role in the cellular response to PEM in human lung adenocarcinoma cells though EGFR/ErbB3-dependent pathways.

Significance of thymidylate synthase expression for resistance to pemetrexed in pulmonary adenocarcinoma

Pemetrexed (PEM), a multi-targeted antifolate, has promising clinical activity in non-squamous non-small cell lung cancer. However, the majority of patients eventually acquire resistance to PEM. To evaluate the resistant mechanisms, the A549 lung adenocarcinoma cell line was exposed to stepwise increasing PEM concentrations of 1.6, 6.4 and 16 μM to establish three PEM-resistant lung cancer cell lines, A549/PEM-1.6, -6.4 and -16. Growth inhibition was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), reduced folate carrier (RFC) and folypoly-γ-glutamate synthetase (FPGS) were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The three A549 cell lines showed more resistance to PEM (3.7-, 17.3− and 38.0-fold, respectively) compared with that of the parental cell line, which also showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine and 5-Fluorouracil (5-FU). TS gene expression was significantly increased in three PEM-resistant cells, relative to that of the parental cells, in a PEM dose-dependent manner. Knockdown of TS expression with siRNA enhanced the cytotoxicity of PEM in A549/PEM-16 cells. By contrast, the levels of RFC and FPGS gene expression in A549/PEM-1.6 and -6.4 cells were significantly decreased, whereas the levels of the two genes were restored in A549/PEM-16 cells. In summary, PEM-resistant A549 cells remained sensitive to docetaxel, vinorelbine and 5-FU. TS expression appeared to be associated with resistance to PEM, which may be a predictive marker for PEM sensitivity in lung adenocarcinoma.

Effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells

The study investigated the effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells. Six human colorectal cancer cells were exposed to pemetrexed, gefitinib, and their combination. Antitumor effects were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Thymidylate synthase (TS) mRNA expression and EGFR mutation were studied by real-time RT-PCR and DNA sequence determination. Pharmacological interaction was studied using the combination index method. Cell cycle distribution and apoptosis were determined by flow cytometry. Activity assay was performed to assess the effects of drugs on TS activity, and Western blot was performed to assess the protein expression of pEGFR, pAKT, and pERK 1/2. Six colorectal cancer cells are all sensitive to pemetrexed, and TS gene expression of cells was negatively related to pemetrexed sensitivity. The cytotoxic synergism was observed in concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib. The combination of pemetrexed and gefitinib modulated cell cycle and induced apoptosis. Pemetrexed combined with gefitinib decreased TS mRNA expression and in situ activity. Pemetrexed induced an EGFR-mediated activation of the phosphatidylinositol 3-kinase/AKT and ERK pathway, which was inhibited by gefitinib. Pemetrexed is a promising agent, and pemetrexed combined with gefitinib has a significantly synergistic effect on colorectal cancer cells, which seems to present a strategy of pemetrexed combined with EGFR-TKIs in colorectal cancer treatment.

Components of homologous recombination and translesion synthesis (TLS) in pemetrexed/cisplatin-treated non-small-cell lung cancer (NSCLC) patients (p).

11028 Background: REV3, the catalytic subunit of the TLS polymerase ξ, can continue replication past DNA adducts. Depletion of REV3 sensitizes A549 lung cancer cells to cisplatin. REV3 expression is part of a gene signature that predicted pemetrexed sensitivity in 17 NSCLC cell lines. Homologous recombination and TLS pathways have non-redundant functions in response to cisplatin. We hypothesized that low REV3 mRNA expression – alone or in combination with low expression levels of genes involved in homologous recombination – could correlate with better outcome to cisplatin/pemetrexed in NSCLC. Methods: REV3, BRCA1, RAP80, TS and AEG1 mRNA was examined by quantitative RT-PCR and categorized by terciles. Expression of each gene was correlated with outcome in 47 cisplatin/pemetrexed-treated NSCLC p. Results: 63.8% male; 47% smokers; 80.9% ECOG PS 1; 80.8% adenocarcinoma. Overall response rate was 51%, with no differences according to expression levels of any of the genes. Progression-free survival (PFS) for p with low, intermediate and high BRCA1 levels was 13.4, 5.5 and 3.9 months (m), respectively (P=0.005). Similar differences in PFS were observed according to TS (P=0.003) and AEG1 (P&lt;0.001) expression. Hazard ratio (HR) for PFS for p with high BRCA1 levels was 4 (P=0.002). Overall survival (OS) for p with low, intermediate and high BRCA1 levels was 29.7, 7.4 and 6.3 m, respectively (P=0.05). Similar differences in OS were observed according to TS (P=0.005) and AEG1 (P=0.001) expression.HR for OS for p with high BRCA1 levels was 3.6 (P=0.004). There were no differences in PFS or OS according to REV3 or RAP80 levels. However, the joint effect of BRCA1 and REV3 was significant for predictive modeling. PFS for p with low, intermediate and high levels of both genes was 14.9, 7.2 and 2.8 m, respectively (P=0.001). OS for p with low, intermediate and high levels of both genes was 29.7, 7.8 and 6.3 m, respectively (P=0.04). Conclusions: Low BRCA1 expression predicts longer PFS and OS in pemetrexed/cisplatin-treated NSCLC p. Low TS and AEG1 levels have similar predictive value. Analysis of these genes could be useful for customizing pemetrexed/platinum chemotherapy.

Abstract 4484: Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed.

Abstract Uracil misincorporation into DNA is a consequence of pemetrexed inhibition of thymidylate synthetase (TS). The DNA base excision repair (BER) enzyme, uracil DNA glycosylase (UNG) is the major glycosylase responsible for removal of misincorporated uracil. We previously illustrated hypersensitivity to pemetrexed in UNG-/- human colon cancer cells (DLD1) and proposed that cytotoxic uracil accumulation leads to collapsed replication forks, double strand breaks and cell death. Here, we examined the relationship between cellular capability to remove uracil-DNA and pemetrexed sensitivity in human lung cancer cells. Viral siRNA and shRNA knockdown of UNG enhanced pemetrexed sensitivity in A549 and H1975 cells, respectively. We further evaluated induction of UNG as a mechanism of pemetrexed resistance. Exposure of H1299 cells to step-wise increasing concentrations of pemetrexed established drug resistant sublines: H1299PR-1 and H1299PR-2. Significant induction of UNG protein was observed in both sublines. Importantly, co-treatment with the BER inhibitor, methoxyamine (MX), overcame pemetrexed resistance in H1299PR-1/2 underscoring the utility of BER directed therapeutics to offset acquired pemetrexed resistance. Lastly, differential expression of UNG in cell lines and primary lung cancer tissues was associated with pemetrexed sensitivity. Cell lines derived from squamous and small cell carcinoma, known to be clinically resistant to pemetrexed, had the highest levels of UNG expression in a panel of eight lung cancer cell lines. Analysis of cDNA microarrays derived from primary human lung cancer tissues also indicated high UNG expression in pemetrexed-resistant histological subtypes. Thus, we propose that UNG is a major determinant of pemetrexed sensitivity in human lung cancer. Citation Format: Lachelle D. Weeks, Pingfu Fu, Lili Liu, Stanton L. Gerson. Uracil DNA glycosylase expression determines human lung cancer cell sensitivity to pemetrexed. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4484. doi:10.1158/1538-7445.AM2013-4484