Non-small Cell Lung Cancer HCC827 Research Articles

Deoxybouvardin targets EGFR, MET, and AKT signaling to suppress non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) remains a significant challenge, as it is one of the leading causes of cancer-related deaths, and the development of resistance to anticancer therapy makes it difficult to treat. In this study, we investigated the anticancer mechanism of deoxybouvardin (DB), a cyclic hexapeptide, in gefitinib (GEF)-sensitive and -resistant NSCLC HCC827 cells. DB inhibited the viability and growth of HCC827 cells in a concentration- and time-dependent manner. In vitro kinase assay showed DB inhibited epidermal growth factor receptor (EGFR), mesenchymal–epithelial transition (MET), and AKT, and their phosphorylation was suppressed in HCC827 cells treated with DB. A molecular docking model suggested that DB interacts with these kinases in the ATP-binding pockets. DB induces ROS generation and cell cycle arrest. DB treatment of HCC827 cells leads to mitochondrial membrane depolarization. The induction of apoptosis through caspase activation was confirmed by Z-VAD-FMK treatment. Taken together, DB inhibited the growth of both GEF-sensitive and GEF-resistant NSCLC cells by targeting EGFR, MET, and AKT and inducing ROS generation and caspase activation. Further studies on DB can improve the treatment of chemotherapy-resistant NSCLC through the development of effective DB-based anticancer agents.

Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells.

Epithelial-mesenchymal-transition (EMT) is an epigenetic-based mechanism contributing to the acquired treatment resistance against receptor tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR)-mutations. Delineating the exact epigenetic and gene-expression alterations in EMT-associated EGFR TKI-resistance (EMT-E-TKI-R) is vital for improved diagnosis and treatment of NSCLC patients. We characterized genome-wide changes in mRNA-expression, DNA-methylation and the histone-modification H3K36me3 in EGFR-mutated NSCLC HCC827 cells in result of acquired EMT-E-TKI-R. CRISPR/Cas9 was used to functional examine key findings from the omics analyses. Acquired EMT-E-TKI-R was analyzed with three omics approaches. RNA-sequencing identified 2,233 and 1,972 up- and down-regulated genes, respectively, and among these were established EMT-markers. DNA-methylation EPIC array analyses identified 14,163 and 7,999 hyper- and hypo-methylated, respectively, differential methylated positions of which several were present in EMT-markers. Finally, H3K36me3 chromatin immunoprecipitation (ChIP)-sequencing detected 2,873 and 3,836 genes with enrichment and depletion, respectively, and among these were established EMT-markers. Correlation analyses showed that EMT-E-TKI-R mRNA-expression changes correlated better with H3K36me3 changes than with DNA-methylation changes. Moreover, the omics data supported the involvement of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 signaling axis for acquired EMT-E-TKI-R. CRISPR/Cas9-mediated analyses corroborated the importance of ZEB1 in acquired EMT-E-TKI-R, MIR200C and MIR141 to be in an EMT-E-TKI-R-associated auto-regulatory loop with ZEB1, and FGFR1 to mediate cell survival in EMT-E-TKI-R. The current study describes the synchronous genome-wide changes in mRNA-expression, DNA-methylation, and H3K36me3 in NSCLC EMT-E-TKI-R. The omics approaches revealed potential novel diagnostic markers and treatment targets. Besides, the study consolidates the functional impact of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1-signaling axis in NSCLC EMT-E-TKI-R.

CD47 blockade improves the therapeutic effect of osimertinib in non-small cell lung cancer.

The third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib (OSI) has been approved as the first-line treatment for EGFR-mutant non-small cell lung cancer (NSCLC). This study aims to explore a rational combination strategy for enhancing the OSI efficacy. In this study, OSI induced higher CD47 expression, an important anti-phagocytic immune checkpoint, via the NF-κB pathway in EGFR-mutant NSCLC HCC827 and NCI-H1975 cells. The combination treatment of OSI and the anti-CD47 antibody exhibited dramatically increasing phagocytosis in HCC827 and NCI-H1975 cells, which highly relied on the antibody-dependent cellular phagocytosis effect. Consistently, the enhanced phagocytosis index from combination treatment was reversed in CD47 knockout HCC827 cells. Meanwhile, combining the anti-CD47 antibody significantly augmented the anticancer effect of OSI in HCC827 xenograft mice model. Notably, OSI induced the surface exposure of "eat me" signal calreticulin and reduced the expression of immune-inhibitory receptor PD-L1 in cancer cells, which might contribute to the increased phagocytosis on cancer cells pretreated with OSI. In summary, these findings suggest the multidimensional regulation by OSI and encourage the further exploration of combining anti-CD47 antibody with OSI as a new strategy to enhance the anticancer efficacy in EGFR-mutant NSCLC with CD47 activation induced by OSI.

TPD7 inhibits the non-small cell lung cancer HCC827 cell growth by regulating EGFR signalling pathway

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, and is characterized by more insensitivity to chemotherapy and poor prognosis. Epidermal growth factor receptor (EGFR) has been confirmed as a tumorigenic driving factor of NSCLC. Taspine has been proved effective in the inhibition of malignant tumours. Here, we found TPD7, a novel taspine derivative, exerted most inhibitory effect on EGFR-dependent HCC827 cells and investigated the underling mechanism. In addition, TPD7 could block cell cycle at G0/G1 phase of HCC827 cells by regulating the expression of cyclin D1 and cyclin E. Furthermore, TPD7 induced HCC827 cell apoptosis by regulating the expression of BCL-2 family proteins. Further study revealed that TPD7 could down-regulate the phosphorylation of EGFR and downstream members. TPD7 might present a potential EGFR inhibitor in the treatment of NSCLC.

Abstract 1099: Resistance to anti-EGFR therapy can spread horizontally among cancer cells

Abstract Targeted therapies almost universally fail due to the development of resistance. EGFR-mutant non-small cell lung cancer (NSCLC) is a well characterized model of such mechanism. In this model, treatment with anti-EGFR therapy leads to resistance driven by the presence of the EGFR T790M mutation in about 50% of the cases. However, clinical data shows the EGFR resistance mutation is present only in a subpopulation of the relapsing cells. How cancer cells devoid of the EGFR T790M mutation become resistant is not known. The aim of this study is to explore if T790M-mediated resistance to anti-EGFR therapy can spread horizontally, through a mitosis-independent mechanism, among NSCLC cells. Immunodeficient mice (Rag2-/-Il2rg-/-) were engrafted with NSCLC HCC827 cells, harboring the Erlotinib-sensitive mutation EGFR E746-A750del, and H1975 cells, carrying the EGFR T790M Erlotinib-resistant mutation. These animals received a single inoculation of HCC827 cells in a single flank (n=10), or dual inoculation of HCC827 and H1975 cells in opposite flanks (n=20). Erlotinib was orally administered three times a week (25 mg/Kg) and tumors were collected at humane endpoint for genetic and transcriptomic analysis by next generation sequencing and digital PCR. T790M-driven resistance to Erlotinib was transferred in vivo from resistant to sensitive cells in dual inoculated animals, resulting in a significant decrease of relapse-free survival. Short tandem repeat profiling of the tumors showed absence of H1975 cells in sensitive tumors in dual inoculated animals, ruling out cell migration as the mechanism of resistance. This result can also not be explained by transfer of mutated DNA from resistant to sensitive cells because high-end sequencing detected the presence of the T790M mutation only in few sensitive tumors from dual inoculated mice and with an extremely low allele frequency (<0,1%). These results support our contention that anti-EGFR therapy resistance does not imply the presence of the T790M mutation in every resistant cell. RNA sequencing revealed differences in transcriptomic profiles from sensitive tumors between single and dual inoculated mice. Gene ontology analysis identified several pathways, such as endocytosis, lysosome biogenesis and RNA transport, as segregators of sensitive tumors that relapsed in the presence or absence of resistant tumors. Our findings suggest that mutation-driven resistance to anti-EGFR can spread horizontally among lung cancer cells and results in alteration of the transcriptional landscape of sensitive cells. Our results question the established concept that acquisition of resistance to targeted therapies relies solely on the selection of tumor cells carrying a specific resistance mutation, which is then passed on to its progeny. PhD Scholarship: SFRH/BD/115099/2016; FEDER funds: COMPETE 2020—(POCI), Portugal 2020; FCT: PTDC/DTP-PIC/2500/2014, NORTE 2020: NORTE- 01-0145-FEDER-000029. Citation Format: Susana Junqueira-Neto, Ana Rita Oliveira, Joana Marques, Sónia Melo, José Luís Costa, José Carlos Machado. Resistance to anti-EGFR therapy can spread horizontally among cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1099.

Combination of Huanglian Jiedu Decoction and erlotinib delays growth and improves sensitivity of EGFR‑mutated NSCLC cells invitro and invivo via STAT3/Bcl‑2 signaling.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is widely used in the treatment of non-small cell lung cancer (NSCLC). However, erlotinib resistance leads to high mortality in patients with NSCLC, while the activation of STAT3 is closely related to erlotinib resistance. Studies have shown that the main components of Huanglian Jiedu Decoction (HJD) have antitumor effects. Therefore, the anticancer effect of HJD combined with erlotinib on NSCLC cells was investigated. The NSCLC HCC827, HCC827ER, and H1975 cell lines as well as xenograft nude mice were selected as models to study the effects of HJD. The proapoptotic effects of HJD were examined by CCK-8 and apoptosis assays. ELISA, immunostaining, and western blot analysis were also performed. HJD considerably enhanced the anticancer effect of erlotinib in both EGFR-TKI-resistant and -sensitive NSCLC cells. HJD promoted erlotinib-induced apoptosis and caspase 3 activity. The co-treatment also inhibited the expression of Bcl-XL, Bcl-2, and p-STAT3. In addition, siSTAT3 had similar functions with HJD. In particular, the apoptotic rates of erlotinib-stimulated HCC827, HCC827ER, and H1975 cells were enhanced by transfecting siSTAT3. Furthermore, overexpression of STAT3 significantly inhibited HJD-mediated erlotinib sensitization. The combined use of HJD with erlotinib significantly reduced tumor growth in erlotinib-resistant HCC827ER and H1975 ×enografts, induced caspase 3, and inhibited Ki67, STAT3, and Bcl-2 expression. HJD significantly alleviated erlotinib resistance by regulating the STAT3/Bcl-2 signaling pathway, which is a promising method to overcome the EGFR-TKI resistance of NSCLC.

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5–100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 μM) with cross-resistance to gefitinib (IC50 > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes — such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) — are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12–123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.

Abstract 3367: Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies

Abstract Cancer immunotherapy with immune checkpoint inhibitors which enhances T-cell regulatory pathways has provided unprecedented benefit to cancer patients. Programmed death 1 ligand (PD-L1) being expressed by cancer cells binds to programmed death-1 (PD-1) to avoid anti-tumor activity of immune cells. Antibodies to disrupt the interaction have been developed for cancer therapeutics. YBL-006 is a new anti-PD-1 human IgG4 monoclonal antibody. Here, we described in vitro & in vivo studies to evaluate pharmacological effect of YBL-006 along with non-clinical studies of pharmacokinetics, toxicokinetics & tissue cross reactivity (TCR). YBL-006 was bound specifically to human PD-1 among receptors of B7 family by ELISA. When affinity to recombinant PD-1 of various species was measured using surface plasmon response system, KD of YBL-006 was 0.372 nM to human PD-1 & 0.070 nM to cynomolgus monkey PD-1 which is higher affinity than nivolumab (1.37 nM & 2.50 nM) & pembrolizumab (1.44 nM & 0.817 nM). It did bind to mouse PD-1 with less affinity than to human PD1, while nivolumab & pembrolizumab did not bind to mouse PD-1. YBL-006 was able to inhibit interaction between PD-1 & both PD-L1 & PD-L2 which was confirmed in competitive binding assay. Inhibition of PD-1 by YBL-006 increased the level of IFN-γ in a mixed lymphocyte reaction model. In a syngeneic mouse model of MC38 colon cancer, weight of tumors in mice treated with YBL-006 was less than that in mice with human IgG control by 25%. In a humanized mouse model (miXeno mouse) of HCC827 non-small cell lung cancer, volume of tumors in YBL-006-treated mice was smaller (35 % less comparing to control mice) than that in nivolumab-treated mice (24 % less). Pharmacokinetic analysis in monkey showed that exposures to YBL-006 increased dose-dependently & in a dose-proportional manner. Maximum mean serum concentrations (Cmax) of YBL-006 were reached (Tmax) between 0.5- & 1.0-hour post onset of infusion & YBL-006 mean serum concentrations slowly declined at a mean estimated t1/2 value of 70.8~110 hours. Clearance & mean volume of distribution suggest that YBL-006 was mainly distributed throughout the blood. In 1-month IV infusion toxicity study in monkey, there was no toxicity when they were infused up to 100 mg/kg/dose of YBL-006, but anti-drug antibody was observed (7 out of 22 monkeys). There was no potential toxicity in TCR study. YBL-006 is an anti-PD-1 antibody with high affinity, promising anti-tumor activity in animal models, & favorable safety profile. First-in-human phase I trial to investigate the safety & efficacy of YBL-006 in advanced solid cancer will be held in 2020. Citation Format: Jaebong Yoon, Hyunmi Lee, Eunyoung Jeon, Jinsil Lee, Eunyoung Shim, Hyunju Lee, Eunjin Lee, Younggyu Cho, Bumchan Park, Hanseung Lee, Youngwoo Park. Preclinical characterization of YBL-006, a fully human anti-PD-1 antibody being ready for clinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3367.

Mechanism of PLOD2 induced osimertinib resistance in non-small cell lung cancer HCC827 cells

Objective: To investigate the effects of osimertinib on proliferation, migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance. Methods: We transfected HCC827 cells with LV-vector and LV-over/PLOD2. The expression of PLOD2 was detected by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays. The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence (IF). The expressions of epithelial-mesenchymal transition (EMT), FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting. Results: The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib. The 50% inhibitory concentration (IC(50)) and resistance index of osimertinib for HCC827-PLOD2 cells was over 1 000 nmol/L and over 100, respectively. The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about (2.13±0.21) fold changes as that of HCC827-vector cells. The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were (212.78±10.43), significantly higher than (101.32±12.52) of HCC827-vector cells (P<0.01). The result of IF showed that compared with HCC827-vector cells, the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells. Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells. The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level. Osimertinib inhibited the expression of p-EGFR, but did not affect the expressions of PLOD2, p-FAK, p-AKT, p-ERK, vimentin and E-cadherin in HCC827-PLOD2 cells. Conclusion: PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT, FAK-PI3K/AKT and MAPK signal pathways.

Regulation of breast cancer resistance protein and P-glycoprotein by ezrin, radixin and moesin in lung, intestinal and renal cancer cell lines.

Ezrin (Ezr), radixin (Rdx) and moesin (Msn) (ERM) proteins anchor other proteins to the cell membrane, serving to regulate their localization and function. Here, we examined whether ERM proteins functionally regulate breast cancer resistance protein (BCRP) and P-glycoprotein in cell lines derived from lung, intestinal and renal cancers. ERM proteins were each silenced with appropriate siRNA. BCRP and P-gp functions were evaluated by means of efflux and uptake assays using 7-ethyl-10-hydroxycamptothecin (SN-38) and rhodamine123 (Rho123) as specific substrates, respectively, in non-small cell lung cancer HCC827 cells, intestinal cancer Caco-2 cells and renal cancer Caki-1 cells. In HCC827 cells, the efflux rates of SN-38 and Rho123 were significantly decreased by knockdown of Ezr or Msn, but not Rdx. However, BCRP function was unaffected by Ezr or Rdx knockdown in Caco-2 cells, which do not express Msn. In Caki-1 cells, Rdx knockdown increased the intracellular SN-38 concentration, while knockdown of Ezr or Msn had no effect. Our findings indicate that regulation of BCRP and P-gp functions by ERM proteins is organ-specific. Thus, if the appropriate ERM protein(s) are functionally suppressed, accumulation of BCRP or P-gp substrates in lung, intestine or kidney cancer tissue might be specifically increased.

IL-22 Confers EGFR-TKI Resistance in NSCLC via the AKT and ERK Signaling Pathways

Background: The efficacy of an EGFR-targeted treatment strategy for non-small cell lung cancer (NSCLC) is reduced by drug resistance. IL-22 enhances tumor growth and induces chemotherapy resistance in human lung cancer cells. The present study elucidated the IL-22-induced mechanism underlying EGFR-tyrosine kinase inhibitor (TKI) resistance in NSCLC.Methods: The plasma and tissues of patients who received EGFR-TKIs were utilized to determine the association between IL-22 expression and gefitinib efficacy. The IL-22 effect on the EGFR/ERK/AKT pathways in NSCLC HCC827 and PC-9 cells was determined using the CCK-8 assay, western blot, and flow cytometric analysis. A PC-9 xenograft model of IL-22 exposure was established. Gefitinib was administered to mice in combination with IL-22 or vehicle.Results: We showed that IL-22 expression was higher in the EGFR-TKI-resistant group compared to EGFR-TKI-sensitive group. IL-22 expression was associated with EGFR-TKI efficacy in plasma. Additional treatment of IL-22 induced gefitinib resistance and reduced apoptosis in PC-9 and HCC827 cell lines. Furthermore, we showed that the effects of IL-22 attributed to p-ERK, p-EGFR, and p-AKT up-regulation. IL-22 neutralizing antibody completely abrogated the effects of IL-22 on apoptosis and AKT/EGFR/ERK signaling. Finally, we showed that IL-22 enhanced tumor growth and induced gefitinib resistance in the PC-9 xenograft model. Moreover, compared with gefitinib alone, the combination of IL-22 and gefitinib led to an increase in Ki67-positive staining and a reduction in TUNEL staining.Conclusions: Our findings indicate that IL-22 plays a role in tumor progression and EGFR-TKI resistance in NSCLC. Thus, IL-22 might serve as a novel biomarker to overcome resistance of EGFR-TKI.

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.

PO-409 Signal peptide of PD-1 inhibits cancer cell growth

IntroductionCancer cells escape immune system surveillance by activating molecules called ‘immune checkpoints’, of which programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) are known representative molecules. Immune cells do not become active when the PD-1 present on their cell surface recognises the PD-L1 present on a cancer cell’s surface, allowing cancer cells to evade the immune cell’s attack. Immune checkpoint-blocking antibodies developed to target PD-1/PD-L1 have remarkable anti-cancer activity. However, the biological reasons why cancer cells activate PD-L1 remain unclear. In addition, there is an issue with cells, such as myocardial cells, with PD-L1 present on their cell surfaces also being affected by immune checkpoint-blocking antibodies. We focused on a signal peptide of PD-1 expressed on the immune cell surface. Our hypothesis was that the signal peptide produced as part of the production process that leads to mature PD-1 has an anti-tumour function. We evaluated whether an artificial peptide designed based on this PD-1 signal peptide had cell growth inhibition activity against human melanoma A2058 and non-small cell lung cancer (NSCLC) HCC-827.Material and methodsWe chemically synthesised a conjugation peptide of the human PD-1 signal peptide (MQIPQAPWPVVWAVLQLGWR) and a cell penetrating peptide TAT (RRKKRRQRRR) and named it ‘PD1SP-TAT’ (MQIPQAPWPVVWAVLQLGWRRRKKRRQRRR). PD1SP-TAT was evaluated for growth inhibitory activity against A2058 and HCC-827 by WST assay.Results and discussionsThe results were 95% of human melanoma A2058 growth and NSCLC HCC-827 growth were inhibited in cells treated with a concentration of 25 µM PD1SP-TAT for 48 hours.ConclusionFrom these surprising results, PD1SP-TAT, based on the signal peptide of PD-1, shows remarkable anti-cancer activity against human melanoma and NSCLC, and suggests that PD1SP-TAT could be a candidate for a novel anti-cancer agent.

MicroRNAs expression profile of gefitinib resistant non-small cell lung cancer HCC827 cells

MicroRNAs expression profile of gefitinib resistant non-small cell lung cancer HCC827 cells

Abstract 2688: The HSP90 inhibitor, onalespib (AT13387), delays the emergence of resistance to erlotinib in an EGFR-driven xenograft model

Abstract Background: Kinase inhibitors have been used successfully in the clinic, but relapse is common due to the emergence of resistance. Inhibition of HSP90 leads to the depletion of oncogenic ‘client’ proteins and the simultaneous inhibition of many signalling pathways. As such, the use of HSP90 inhibitors to overcome resistance has been widely investigated. Onalespib (AT13387) is a potent, second generation HSP90 inhibitor, which has been studied in many preclinical models of kinase inhibitor resistance. Recent findings indicate that an upfront combination of onalespib and either vemurafenib or crizotinib, in models of mutant BRAF melanoma or ALK-translocated non-small cell lung cancer (NSCLC), can delay the emergence of resistance to these therapies. Here we have extended this work to a combination of onalespib and erlotinib in an EGFR-driven NSCLC model. Methods: Mice bearing HCC827 tumor xenografts were dosed with either 12.5 mg/kg erlotinib po qd, or a combination of 12.5 mg/kg erlotinib and 55 mg/kg onalespib ip qw for 52 weeks. Results: Initially both the erlotinib single-agent and onalespib/erlotinib combination treatments significantly inhibited tumor growth of the EGFR-driven NSCLC HCC827 xenograft model; all tumors in the monotherapy and combination groups regressed rapidly and achieved complete response (CR) (median times to CR were 79 and 56 days for monotherapy and combination treatments respectively). However, after 20 weeks of continuous treatment, tumor relapse was observed in one of the xenografts dosed with erlotinib only and this was followed by regrowth of a further 2 tumors in this group over the 52-week study period, with these tumors reaching 50% of the original tumor volumes by 26 and 46 weeks. Of the remaining tumors, 5 out of 6 were detectable at the end of the study. In comparison, tumor growth remained inhibited in the group treated with the erlotinib/onalespib combination; no tumors were observed to regrow over the course of the study and none (of 9) were palpable when treatments were terminated. This suggests that while treatment with monotherapy erlotinib in this model leads to relapse, as seen in the clinic, combination treatment with onalespib can delay the emergence of resistance. Conclusions: These data demonstrate that an upfront combination of onalespib and erlotinib can delay the emergence of resistance in an EGFR-driven xenograft model, suggesting there is therapeutic potential for an upfront erlotinib/onalespib combination in the clinic. Onalespib is currently being tested in a Phase 2 clinical trial in combination with crizotinib in ALK-positive NSCLC. The preclinical data presented here, alongside our previous data on combinations of onalespib with vemurafenib or crizotinib, suggest that the concept of using an HSP90 inhibitor combination upfront in the clinic to delay resistance could be extended to further targeted therapies. Citation Format: Aurélie Courtin, Tomoko Smyth, Keisha Hearn, John Lyons, Neil Thompson, Nicola G. Wallis. The HSP90 inhibitor, onalespib (AT13387), delays the emergence of resistance to erlotinib in an EGFR-driven xenograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2688. doi:10.1158/1538-7445.AM2015-2688

Abstract A66: Ablative radiotherapy increases invasion potential in EGFR-wildtype non-small cell lung cancer cells compared to fractionated radiotherapy.

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for over 80% of all lung cancer cases. Nearly 70% of lung cancer patients will undergo radiotherapy (RT). Recent trends in RT have shown an increase in the use of ablative radiotherapy (ART) in inoperable small tumors with response rates of 80-90% in NSCLC patients. However, despite the high rate of local control, early clinical data shows no advantage in survival due to distant metastasis after ART compared to conventional fractionated radiotherapy (FRT). In addition, patient selection for ART is independent of the status of the epidermal growth factor receptor (EGFR) which has been shown to play a role in radiation response. We investigated the effect of ART compared to FRT on cell invasion, proliferation, senescence and clonogenic survival of NSCLC cell lines harboring wild type and mutated EGFR. Methods: The cell lines used were A549, H1975 and HCC827. Cell irradiation was performed using a Faxitron X-Ray machine. Radiation doses of 8Gy and12Gy were delivered in fractionated or single sessions. The effect of radiation on cells was investigated using the clonogenic assay, MTT proliferation assay, matrigel invasion assay and senescence-associated beta-galactosidase. Results: ART significantly suppressed the proliferative and clonogenic potential of wild-type EGFR A549 cells compared to FRT. In addition, a significant increase in the number of senescent as well as large, polynucleated cells was observed in the ART-treated group compared to the FRT-treated group. Analysis of the invasive potential of the cells revealed a 2-fold increase in invasion 5 days after exposure to ART of 12Gy compared to control. The FRT group showed a 1.4-fold increase in invasion compared to control. Contrary to A549 cells, EGFR-mutated NSCLC HCC827 and H1975 cells, showed no significant difference after exposure to ART compared to FRT. In addition, both ART and FRT-treated groups showed a similar increase in the number of senescent, large and polynucleated cells. A significant reduction in the number of invading cells after exposure to either FRT or ART was observed compared to the control. Conclusion: ART significantly reduces cell proliferation and clonogenic survival compared to FRT in wild-type EGFR A549 cells. This differential response between the type of treatment (ART vs FRT) was not seen in NSCLC cell lines harboring EGFR mutations. In contrast to reduced cell proliferation, there was a significant increase in the invasive capacity of cells after ART compared to FRT in A549 cells only. These results can have significant consequences in the selection and treatment of NSCLC patients for ART. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A66. Citation Format: Ayman J. Oweida, Jack Xu, Siham Sabri, Bassam Abdulkarim. Ablative radiotherapy increases invasion potential in EGFR-wildtype non-small cell lung cancer cells compared to fractionated radiotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A66.

Design, synthesis and biological evaluation of new molecules inhibiting epidermal growth factor receptor threonine790→ methionine790 mutant

A series of 5,8-dioxo-pyrimido[4,5-e][1,4]diazepine derivatives were designed and synthesized as new inhibitors against wild-type EGFR and a panel of mutants, including the clinical resistance related T790M mutants. One of the most potent compounds 2l inhibited all forms of EGFR evaluated with low nM IC50 values. It also strongly suppressed the proliferation of H1975 and HCC827 non-small cell lung cancer cells with IC50 values of 69 and 71 nM, respectively.

Malpas joins SpecialChem's board of advisers

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5–100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 μM) with cross-resistance to gefitinib (IC50 > 5 μM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes — such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) — are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12–123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.