Cell Lines HCC827 Research Articles

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

79 Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827

79 Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827

Moving a Carbohydrate Mimetic Peptide into the clinic

Tumor-Associated Carbohydrate Antigens (TACAs) are broad-spectrum targets for immunotherapy. Immunization with Carbohydrate Mimetic Peptides (CMPs) is a strategy to induce broad-spectrum TACA-reactive antibodies hypothesized to interfere with cellular pathways involved in tumor cell survival. A Phase I study was conducted with a first-in-man CMP referred to as P10s, conjugated to the Pan T cell carrier PADRE, along with MONTANIDE™ ISA 51 VG as adjuvant over a course of 5 immunizations. While designed as a safety and tolerability study, the potential for therapeutic impact was observed in a subject with metastatic lesions as evaluated before and after vaccine treatment. The subject received Vinorelbine and Trastuzumab (VT) for two months prior to study eligibility. PET scans showed partial response in the lungs and complete resolution of a previously enlarged subpectoral lymph node. Immunization with P10s vaccine resulted in responses to P10s, with serum and plasma antibodies reactive with and cytotoxic to human breast cancer cells in vitro, including the Trastuzumab-resistant HCC1954 cell line. However, the patient developed cystic masses in the brain parenchyma with no apparent evidence of metastases. The subject was switched to Docetaxel, Pertuzumab and Trastuzumab a year later, and her last PET scan showed a complete response in the lungs and lymph nodes. Incubation of cancer cells with a combination of vaccine-induced serum and docetaxel suggests that the induced antibodies sensitize tumor cells for more efficient killing upon administration of docetaxel. The data suggest that P10s-PADRE induces anti-tumor antibody response that in combination with chemotherapy can affect metastatic lesions in breast cancer patients.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Abstract 113: A comparative preclinical PET imaging study between [11C]erlotinib and [18F]afatinib

Abstract Background: The epidermal growth factor receptor (EGFR) is an established target for the treatment of advanced non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib have been approved for treatment of NSCLC. The Efficacy of those TKIs depends on the presence of activating mutations of EGFR (exon 19 del. or L858R point mutation). Therefore, the EGFR mutational status needs to be ascertained by an invasive biopsy before treatment; however this is often compromised by sampling errors due to tumor heterogeneity or by lack of access to the tumor tissue. We hypothesize that PET-imaging with radiolabeled TKIs (TKI-PET) can provide a tool to determine and predict responsiveness to EGFR TKIs in vivo. Previously, this was demonstrated with the reversible EGFR inhibitor [11C]erlotinib that showed higher uptake in patients harboring exon 19 deletions of EGFR. Therefore, to validate the application of TKI-PET imaging, we developed irreversible second generation TKI afatinib as a PET tracer and extensively evaluated both [18F]afatinib and [11C]erlotinib in a comparative preclinical imaging study to determine the feasibility of TKI-PET as a potential personalized medicine tool. Methods: The synthesis of [11C]erlotinib was already established in our center. The synthesis of [18F]afatinib was developed using an innovative three step radiosynthesis. Both tracers were thoroughly evaluated in a PET imaging study in mice bearing a panel of three clinically relevant EGFR expressing human NSCLC xenografts. The three selected cell lines are: A549 with wild-type EGFR, H1975 with the sensitizing mutation L858R and the acquired resistance related mutation T790M and HCC827 cell line with the sensitizing exon 19 deletion. Other factors influencing uptake were also investigated, such as tracer metabolism and effects of drug efflux transporters. Results: [18F]afatinib was successfully synthesized in an overall decay corrected yield of 17 ± 2.3% with a radiochemical purity of >97% and a specific activity of >200 GBq/µmole at end of synthesis. In a panel of lung cancer xenograft models, [11C]erlotinib and [18F]afatinib both showed an increased uptake in the treatment sensitive cell line, while showing low to no uptake in wild type or acquired resistance cell lines. [18F]afatinib showed a higher tumor-to-background ratio than [11C]erlotinib in the sensitive cell line. In vivo blocking of the P-gp drug efflux transporter with tariquidar revealed an increased uptake of both tracers indicating that they are substrates for efflux pumps. Conclusions: The new TKI-PET tracer [18F]afatinib has been synthesized successfully. [11C]erlotinib and [18F]afatinib show similar uptake patterns across EGFR mutations, while uptake appears to correspond with treatment sensitivity. A treatment study in mice will be performed to directly correlate uptake to sensitivity. Citation Format: Paul Slobbe, Albert Windhorst, Marijke Stigter-van Walsum, Robert Schuit, Egbert Smit, Heiko Niessen, Guus van Dongen, Alex Poot. A comparative preclinical PET imaging study between [11C]erlotinib and [18F]afatinib. [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 113. doi:10.1158/1538-7445.AM2014-113

Abstract 4875: In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations

Abstract Background: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (TKIs) e.g. Erlotinib (Tarceva®) and Gefitinib (Iressa®). Most NSCLC patients with activating EGFR mutations will respond to EGFR-TKIs; however, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of c-MET kinase, Her-2 and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel NSCLC patient-derived xenograft (PDX) model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. Methods: LU6422 is a Caucasian NSCLC adenocarcinoma PDX model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. HCC827 is NSCLC adenocarcinoma cell line with an activating EGFR mutation (exon 19 del E746-A750). Resistant models of LU6422 and HCC827 were generated in vivo and in vitro (respectively) through repeated dosing or exposure to EGFR-TKIs. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression and genomic amplification by quantitative PCR. Combination efficacy treatment (in vitro and in vivo) was carried out to verify documented resistance mechanisms. Results: Naïve LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p<0.0001). Following successive cycles of EGFR-TKI treatment several resistant subtypes were generated, cloned and characterized. Combination treatment of resistant HCC827 variants restored efficacy to naïve treatment levels. Conclusions: EGFR-TKI resistant subtypes were generated in vivo and in vitro from a proprietary patient-derived xenograft model (LU6422) and the HCC827 cell line and were characterised for their resistance mechanisms. Pre-clinical modes of acquired resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR-TKIs. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Martin Page, Henry Li, Rajendra Kumari, Jane Wrigley. In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations. [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 4875. doi:10.1158/1538-7445.AM2014-4875

Abstract 1686: Synergistic combination of Trabectedin and Olaparib in breast cancer tumor cell lines

Abstract Trabectedin (Yondelis, ET-743) is a marine-derived antitumor agent that is currently used for the treatment of sarcoma and, in combination with pegylated-liposomal doxorubicin, of platinum-sensitive ovarian cancer patients. After binding to the DNA minor groove, trabectedin induces a potent transcription inhibition on tumor cells and tumor-associated macrophages. The resolution of functional DNA adducts generated by the drug is known to occur through the coordinated action of multiple DNA repair pathways, including homologous recombination (HR), a process that gives rise to double-strand breaks (DSBs). Trabectedin treatment at nanomolar concentrations does indeed produce a high proportion of DSB-positive tumor cells with abundant γ-H2AX foci and an S-phase delay/arrest by activation of the DNA replication checkpoints. We had previously shown the synergism (CI < 1) of the combination of Trabectedin with Olaparib (AZD-2281) in the breast cancer cell lines HCC-1937, MCF-7, MDA-MB-231 and MDA-MB-436. In this work we have investigated the mechanism of this synergistic effect at the molecular level. While the treatment with either compound alone (0.05 nM Trabectedin or 5 μM Olaparib for 72 hours) induced only a mild antiproliferative effect, the combination of both compounds at the mentioned concentrations induced strong apoptosis in all the breast cancer cell lines. Exposure to 0.5 and 2.5 nM Trabectedin for 72 hours induced a clear accumulation of γ-H2AX foci. Exposure to Olaparib alone induced only a mild DNA damage at 72 hours with all the concentrations tested. Olaparib induced a clear inhibition of PARylation at concentrations from 1-2.5 μM of the compound. The combination of both drugs, keeping Olaparib at 5 μM and varying the concentrations of Trabectedin from 0.05 to 2.5 nM, demonstrated a synergistic effect on the accumulation of histone H2AX phosphorylation, as analyzed both by western blotting and immunofluorescence microscopy. When comparing all these results, it was clearly seen that, although Trabectedin or Olaparib generated a considerable amount of DSBs, the combination of both drugs induced a higher proportion of DNA damage that remained at very high levels even after 72 hours of treatment. Thus, the observed synergistic effect seems to be the result of higher accumulation of DSBs after the administration of the combination of Trabectedin with Olaparib. Citation Format: Sonia Avila, Marta Martínez, Victoria Moneo, Juan F. Martínez-Leal, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galamarini. Synergistic combination of Trabectedin and Olaparib in breast cancer tumor cell lines. [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 1686. doi:10.1158/1538-7445.AM2014-1686

Abstract 2455: BRCA1 and BARD1 expression and localization in breast cell lines

Abstract One of the principal functions of BRCA1 is to keep the integrity of DNA by homologous recombination which repairs DNA double strand breaks, thus its localization is mainly nuclear in a normal cell. BRCA1 nuclear function is enhanced by its interaction with BARD1, which promotes the import and nuclear retention of BRCA1 by masking its nuclear export signal. Therefore, the interaction of these two proteins is relevant in BRCA1 nuclear function. On the other hand, in tumor cell lines, splicing variants of BARD1 lacking the BRCA1 interaction domain have been described. The expression of these splicing variants of BARD1 could contribute to the loss of nuclear localization of BRCA1. Recently, our group found that a high percentage of triple negative breast cancer tumors showed cytoplasmic or no expression of BRCA1 (unpublished data). Our interest is to study the molecular mechanisms that explain the cytoplasmic localization of BRCA1 in breast cancer cells. We studied mRNA expression of BRCA1, BARD1 and BARD1β through RT-PCR and expression of both proteins by western blot, immunocytochemistry and immunofluorescence in two breast cancer cell lines (HCC1937: ER-, mutated in BRCA1, and T47D: ER+) and one non-tumor breast cell line (MCF10A). BRCA1 and BARD1 expression was coherent between immunocytochemistry and immunofluorescence assay. The HCC1937 cell line presented a weak BRCA1 protein expression mainly in the cytoplasm. The other cell lines had a high nuclear and weak cytoplasmic expression. BARD1 protein is expressed in the nucleus and cytoplasm in all cell lines. BARD1 full mRNA had an equal expression in the three cell lines, and its isoform BARD1β, which do not interact with BRCA1, was expressed at higher levels in tumor cell lines than in the non-tumor cell lines. However, T47D cell line showed nuclear localization of BRCA1 despite high levels of BARD1β mRNA. This suggests that BARD1β either does not express at protein level, or that full-length BARD1 levels are enough to keep BRCA1 in the nucleus. The study of molecular causes of BRCA1 localization in cell lines will allow us to better understand subcellular localization of BRCA1 in tumors and its interaction with BARD1. FONDECYT 1120200. Citation Format: Cristobal Herrera, Elisa Pérez, Elías Blanco, Patricia Gajardo, Pilar Carvallo. BRCA1 and BARD1 expression and localization in breast cell lines. [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 2455. doi:10.1158/1538-7445.AM2014-2455

Abstract 4513: IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors

Abstract EGFR is one of the oncogenic drivers in non-small-cell lung adenocarcinoma (NSCLC AC). EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib and afatinib, show remarkable single agent activity in NSCLC AC with EGFR mutations. However, a majority of the tumors develop resistance within twelve months. T790M secondary EGFR mutation and activation of alternative signaling pathways, such as MET, are the two most common resistance mechanisms. To address this clinical challenge, we developed IMGN289, an EGFR antibody-drug conjugate consisting of the novel J2898A antibody (Ab), which selectively binds to and inhibits EGFR-driven tumor cell growth, conjugated to the maytansinoid DM1, a potent anti-microtubule agent, via the SMCC thioether linker. IMGN289 showed significantly enhanced cytotoxic activity, relative to cetuximab or unconjugated J2898A, against tumor cell line models that are dependent on EGFR signaling (Chittenden et al., AACR 2013). To evaluate the ability of IMGN289 to overcome mechanisms of EGFR TKI resistance, IMGN289 activity was tested in erlotinib-resistant EGFR-mutant cell lines which overexpressed MET (HCC827-ER) or carry the T790M EGFR mutation (HCC827-EPR) (Suda et al., Clin Cancer Res 2010). Despite variable levels of EGFR expression in these cell lines, (193,000, 32,500 and 422,000 antigens per cell in HCC827, HCC827-ER and HCC827-EPR, respectively), IMGN289 showed comparable cytotoxic activities against these cell lines in vitro. In addition to MET overexpression, the MET pathway can also be activated by HGF, the MET ligand. While addition of HGF made the HCC827 cell line resistant to EGFR TKIs (Yano et al., Cancer Res 2008) and decreased growth inhibition by cetuximab, IMGN289 activity was unaffected. Additionally, we generated an erlotinib-resistant HCC827-ER-E4 cell line by exposing the EGFR-mutant HCC827 cells to increasing concentrations of erlotinib for ∼6 months, followed by subcloning. The HCC827-ER-E4 cell line expressed mesenchymal markers and acquired a mesenchymal phenotype. It expressed lower levels of EGFR, HER3 and MET compared to the parental cell line and showed no loss of PTEN. In the presence of erlotinib, HCC827-ER-E4 cells retained high levels of pAKT and pERK, suggesting that activation of alternative signaling pathways may be responsible for the erlotinib resistance. In vitro cytotoxic assays showed that IMGN289 was equally cytotoxic to the parental and the HCC827-ER-E4 cell line. In conclusion, IMGN289 demonstrated EGFR-targeted cytotoxicity against NSCLC cell lines that harbor some of the most common mechanisms of resistance to EGFR TKIs. IMGN289 represents a promising novel candidate for treatment of EGFR-expressing NSCLC AC that has acquired resistance to EGFR TKIs. Citation Format: Yulius Y. Setiady, Ling Dong, Anna Skaletskaya, Jan Pinkas, Robert J. Lutz, John M. Lambert, Thomas Chittenden. IMGN289, an EGFR-targeting antibody-drug conjugate, is effective against tumor cells that are resistant to EGFR tyrosine kinase inhibitors. [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 4513. doi:10.1158/1538-7445.AM2014-4513

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

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

Overcoming EMT-associated resistance to anti-cancer drugs via Src/FAK pathway inhibition.

Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, "EGFR-addicted" cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro. To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.

EGFR-targeted plasmonic magnetic nanoparticles suppress lung tumor growth by abrogating G2/M cell-cycle arrest and inducing DNA damage.

BackgroundWe have previously demonstrated the epidermal growth factor receptor (EGFR)-targeted hybrid plasmonic magnetic nanoparticles (225-NP) produce a therapeutic effect in human lung cancer cell lines in vitro. In the present study, we investigated the molecular mechanism of 225-NP-mediated antitumor activity both in vitro and in vivo using the EGFR-mutant HCC827 cell line.MethodsThe growth inhibitory effect of 225-NP on lung tumor cells was determined by cell viability and cell-cycle analysis. Protein expression related to autophagy, apoptosis, and DNA-damage were determined by Western blotting and immunofluorescence. An in vivo efficacy study was conducted using a human lung tumor xenograft mouse model.ResultsThe 225-NP treatment markedly reduced tumor cell viability at 72 hours compared with the cell viability in control treatment groups. Cell-cycle analysis showed the percentage of cells in the G2/M phase was reduced when treated with 225-NP, with a concomitant increase in the number of cells in Sub-G1 phase, indicative of cell death. Western blotting showed LC3B and PARP cleavage, indicating 225-NP-treatment activated both autophagy- and apoptosis-mediated cell death. The 225-NP strongly induced γH2AX and phosphorylated histone H3, markers indicative of DNA damage and mitosis, respectively. Additionally, significant γH2AX foci formation was observed in 225-NP-treated cells compared with control treatment groups, suggesting 225-NP induced cell death by triggering DNA damage. The 225-NP-mediated DNA damage involved abrogation of the G2/M checkpoint by inhibiting BRCA1, Chk1, and phospho-Cdc2/CDK1 protein expression. In vivo therapy studies showed 225-NP treatment reduced EGFR phosphorylation, increased γH2AX foci, and induced tumor cell apoptosis, resulting in suppression of tumor growth.ConclusionThe 225-NP treatment induces DNA damage and abrogates G2/M phase of the cell cycle, leading to cellular apoptosis and suppression of lung tumor growth both in vitro and in vivo. Our findings provide a rationale for combining 225-NP with other DNA-damaging agents for achieving enhanced anticancer activity.

Triple Inhibition of EGFR, Met, and VEGF Suppresses Regrowth of HGF-Triggered, Erlotinib-Resistant Lung Cancer Harboring an EGFR Mutation

Introduction:Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer.Methods:Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene–transfected PC-9 (PC-9/HGF) cells were examined.Results:Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment.Conclusion:These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis.

AKT inhibition synergistically enhances growth-inhibitory effects of gefitinib and increases apoptosis in non-small cell lung cancer cell lines

ObjectivesEGFR inhibitors are ineffective against most EGFR wild-type non-small cell lung cancer, for which novel treatment strategies are needed. AKT signalling is essential for mediating EGFR survival signals in NSCLC. We evaluated the combination of gefitinib and two different AKT inhibitors, the allosteric inhibitor AKTi-1/2 and the ATP-competitive pan-AKT inhibitor AZD5363, in EGFR-mutant (HCC-827 and PC-9) and -wild-type (NCI-H522, NCI-H1651), non-small cell lung cancer cell lines. Materials and methodsDrug interaction was studied in two EGFR mutant and two EGFR wild-type non-small cell lung cancer cell lines by calculating combination index (CI) using median effect analysis. The effects on p-EGFR, p-ERK, p-AKT, p-S6 and apoptosis were studied by Western blot analysis. ResultsThe combination of gefitinib and AKTi-1/2 or AZD5363 showed synergistic growth inhibition in all cell lines. CI values for the combination of gefitinib and AKTi-1/2 were 0.35 (p=0.0048), 0.56 (p=0.036), 0.75 (p=0.13) and 0.64 (p=0.0003) in NCI-H522, NCI-H1651, HCC-827 and PC-9 cell lines, respectively; CI values of 0.45 (p=0.0087) and 0.22 (p<0.0001) were observed in NCI-H522 and PC-9 cells, respectively, when gefitinib was combined with AZD5363. Additive inhibition of signalling output through AKT and key downstream proteins (S6) and increased apoptosis were demonstrated. ConclusionDual inhibition of EGFR and AKT may be a useful up-front strategy for patients with EGFR-mutant and -wild-type non-small cell lung cancer.

Performance of EGFR mutant-specific antibodies in different cytological preparations: a validation study.

Molecular testing for epidermal growth factor receptor (EGFR) mutations is required to select the most appropriate treatment for advanced-stage non-small cell lung cancer (NSCLC). In routine practice, cytological samples are often the only specimens available for testing. When the number of neoplastic cells is large, DNA-based assays are the gold standard. When cytological samples contain only a few neoplastic cells, immunocytochemistry (ICC) using anti-EGFR mutant-specific antibodies may be more effective. We aim to assess the specificity and sensitivity of IHC staining in cytological specimens using mutated cell lines subjected to different cytopreparations and staining methods. HCC827 (exon 19 p.E746-A750 del) and H3255 (exon 21 p.L858R) cell lines were subjected to different fixation (air dried, alcohol or CytoLyt(®)), staining (Diff-Quik(®) or Papanicolaou) and preparation (smears or cell blocks) methods before ICC. In a second set of experiments, mutated cells were mixed with EGFR wild-type cells to obtain low-level (10%) mutated cytological samples. The intensity and percentage of cells stained were evaluated against validated molecular techniques. Moreover, the cell lines were subjected to poor growing conditions to simulate routine specimens that are less optimal than in vitro samples. The cytological preparations showing the most intense staining were formalin-fixed cell blocks and samples fixed with CytoLyt or alcohol, including Papanicolaou-destained samples. Conversely, air-dried slides showed the least intense staining. Mutant antibodies allowed the detection of mutated cells, even when representing only 10% of the total population. Although, in necrotic specimens, an aspecific background signal appeared, the viable cells still retained anti-mutant EGFR positivity. All cytological preparations are suitable for ICC using anti-EGFR mutant-specific antibodies, in particular formalin-fixed cell blocks and alcohol- or CitoLyt-fixed samples. the method is also validated to detect even a few mutant cells in less than optimal samples.

Immediate early response gene X-1 promotes the apoptosis of the breast cancer cell line HCC1937 induced by tumor necrosis factor-related apoptosis inducing ligand

Objective To observe the effect of immediate early response gene X-1 (IER3) on the apoptosis of the breast cancer cell line HCC1937 induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL).Methods The expression of gene IRAIL and IER3 was detected before and after treatment by real-time quantitative polymerase chain reaction (Real-time PCR) at 48 h,The proliferation of breast cancer cells HCC1937 was mearsured by using Methylthiazol tetrazolium (MTT) assay at 24,48,72,96,and 120 h,The clone formation ability was mearsuerd by clone formation assay after 10-14 d,breast cancer cells HCC1937 were examined by Hoechst 33258 stain to detect the apoptpsis after 24 h.Results Compared with the negative control group,the relative expression of TRAIL in the TRAIL and the IER3 + TRAIL groups were 42.69 ± 3.88 and 46.36 ± 3.55 times respectively.And the relative expression of the IER3 in the IER3 and the IER3 + TRAIL groups were 5.08 ± 1.40 and 9.66 ± 2.25 times respectively.The inhibition rate (％) of cells in TRAIL,IER3 and TRAIL + IER3 groups were (50.95 ± 7.45),(25.39 ± 4.49) and (72.01 ± 2.95) respectively.The cell clone formation ability in IER3 +TRAIL group was obviously reduced.Compared with the negative control group,the apoptosis rate of cells in IER3 + TRAIL group was also increased significantly.Conclusion IER3 could promote the apoptosis of the breast cancer cell line HCC1937 induced by TRAIL. Key words: Immediate early response gene X-1 ; Tumor necrosis factor-related apoptosis inducing ligand ; Breast carcinoma

A novel cytotoxic terpenoid from the flowers of Kaunia lasiophthalma Griseb

A phytochemical study of the flowers of Kaunia lasiophthalma G. (Asteraceae) yielded a novel triterpene (1) together with several known sesquiterpenoids. The structure of the new compound was elucidated by analysis of the spectroscopic data. The biosynthetic origin of 1 is proposed to be a dimerization of an oxidized derivative (3) of the germacrane sesquiterpene costunolide (2), also present in the flowers. The anticancer activity of 1 in the five breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7 and SK-BR-3 was compared with the cytotoxicity in the normal-like breast epithelial cell line MCF-10A. 1 exhibited high cytotoxicity in all investigated cancer cell lines with IC50 values ranging from 0.67 to 7.0μM, although it is lacking selectivity as the MCF-10A cells were almost as sensitive.

MiR-342 Regulates BRCA1 Expression through Modulation of ID4 in Breast Cancer

A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its “in silico” predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.

Abstract A24: Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples.

Abstract Background: Binding of PD-L1 expressed on tumor cells to the PD1 on T lymphocytes transduces immuno-inhibitory signals which cripples the T cell's ability to combat the tumor. Several anti-PD-L1 and anti-PD1 agents are in clinical trials and both regimens have reported promising preliminary results in NSCLC patients. (Brahmer et al., 2012 and Inman, 2013).These studies suggest that tumor expression of PD-L1 is associated with a response to either anti-PD-L1 and anti-PD1 treatment. Immunohistochemistry (IHC) is the current method to assess PD-L1 expression in FFPE tissue; however, PD-L1 IHC has yielded mixed results; some studies showed high false positive by IHC while another study showed that 13% of the PD-L1 negative patients responded to treatment. Moreover, IHC is low throughput and assessing multiple druggable targets by IHC is tissue consuming. As such, there is an urgent need to develop quantitative and highly multiplexed tests to assess biomarker expression. We have developed and clinically validated a quantitative mass spectrometric assay to measure PD-L1 protein expression in FFPE tissue biopsies. Method: We used trypsin digestion mapping of recombinant PD-L1 to identify optimal quantitative peptides. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in pyrococcus complex matrix to determine LOD, LLOQ, accuracy, precision and linearity of the assay. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was then run on archived FFPE sections from in 9 normal tissues, 21 early staged (stage 1 and 2) and 4 advanced staged (stage 3) NSCLC patients. We also used Lung OncoPlex assay to sub-classify NSCLC samples to adenocarcinoma and squamous cell carcinoma. All of the samples were screened in replicates and multiple machines were used to check technical variability. Results: A 10 point calibration curve using five replicates was used to determine the LOD (75 amol) and LOQ (100 amol) for the PD-L1 assay. Fourteen (14) cell lines were assayed for PD-L1 expression by LT-SRM. PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis (Broad Institute) demonstrated excellent correlation (R2=0.8894). The NSCLC cell line HCC827 and breast cancer cell line MDA-MB-231 had the highest levels of PD-L1, 374.78 and 298.27 amol/μg protein, respectively. Our initial clinical analysis of NSCLC tissue shows that while no normal lung tissue expresses detectable levels of PD-L1, ~24 % of early stage NSCLC (5/21) and 50 % of advanced stage NSCLC (2/4) express measurable PD-L1 protein. Interestingly, in this initial cohort, all of the PD-L1 positive early staged NSCLC were squamous cell carcinoma while in a small set of advanced staged NSCLC, PD-L1 expression was seen in both squamous cell carcinoma (1/3) and adenocarcinoma (1/1). Characterization of larger cohorts of NSCLC tissue is currently underway and will be presented. Discussion: The need to characterize expression levels of druggable targets in small NSCLC biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Here we describe the development and initial clinical validation of a quantitative proteomic PD-L1 assay which accurately measures PD-L1 expression levels in FFPE tumor tissue. Initial PD-L1 screening using clinical NSCLC samples suggests that more advanced NSCLC patients are more likely to be PD-L1 positive compared to early stage NSCLC patients. Additionally, patients with squamous cell carcinoma are very likely to express PD-L1. Interestingly, Soria et al. (2013) has recently shown a high response rate to PD-L1 therapy in smokers with squamous cell carcinoma. We are currently expanding this initial clinical validation to assess PD-L1 expression levels in larger cohorts, including both adeno and squamous carcinoma. Additional quantitative assays for both lymphocyte (CD3, CD8, CD68) and immunotargets (PD1, B7-H3) are under development. This proteomic assay promises to be a critical component of our multiplexed biomarker analysis, and will allow more accurate identification of potential candidates for PD-L1 or PD1 targeted therapies. Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Jaime Rodriguez, Ravi Salgia, Ignacio I. Wistuba, Jon Burrows, Todd Hembrough. Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A24.

Interaction of standardized mistletoe (Viscum album) extracts with chemotherapeutic drugs regarding cytostatic and cytotoxic effects in vitro.

BackgroundGiven the importance of complementary and alternative medicine (CAM) to cancer patients, there is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Mistletoe (Viscum album L.) belongs to the medicinal herbs that are used as supportive care during chemotherapy. In the in vitro study presented here the effect of standardized mistletoe preparations on the cytostatic and cytotoxic activity of several common conventional chemotherapeutic drugs was investigated using different cancer cell lines.MethodsHuman breast carcinoma cell lines HCC1937 and HCC1143 were treated with doxorubicin hydrochloride, pancreas adenocarcinoma cell line PA-TU-8902 with gemcitabine hydrochloride, prostate carcinoma cell line DU145 with docetaxel and mitoxantrone hydrochloride and lung carcinoma cell line NCI-H460 was treated with docetaxel and cisplatin. Each dose of the respective chemotherapeutic drug was combined with Viscum album extract (VAE) in clinically relevant concentrations and proliferation and apoptosis were measured.ResultsVAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of our experimental settings. At higher concentrations VAE showed an additive inhibitory effect.ConclusionsOur in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE simultaneously.