EGFR Wild-type Cell Lines Research Articles

Targeting the miR-200c/LIN28B axis in acquired EGFR-TKI resistance non-small cell lung cancer cells harboring EMT features

MicroRNA (miR)-200 family members (miR-200s) are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT). We previously reported that miR-200s were silenced through promoter methylation in acquired EGFR-tyrosine kinase inhibitor (TKI) resistant non-small cell lung cancer (NSCLC) cells harboring EMT features. In this study, we examined the functional role of miR-200s in NSCLC cells and investigated a novel approach to overcoming acquired EGFR-TKI resistance. In the analysis of NSCLC cell lines, each of the miR-200s expression-silenced cell lines showed promoter methylation. Significant correlations between miR-200c silencing and several oncogenic pathway alterations, including EMT-changes and LIN28B overexpression, were observed in the database analysis. In addition, EGFR-wild type cell lines had lower miR-200s expression levels than EGFR-mutant cell lines. The introduction of miR-200c using pre-miR-200c caused LIN28B suppression in cells with acquired EGFR-TKI resistance that harbored EMT features. Interestingly, both the introduction of miR-200c and the knockdown of LIN28B produced an antitumor effect in acquired EGFR-TKI resistance cells, whereas these manipulations were not effective in parental cells. The miR-200c/LIN28B axis plays an important role in cells with acquired resistance to EGFR-TKI that harbor EMT features and might be a useful therapeutic target for overcoming resistance.

Effects of anti-PD-L1 monoclonal antibody and EGFR-TKI on the expression of PD-L1 and function of T lymphocytes in EGFR-mutated lung cancer cells

Objective: To investigate the effects of anti-PD-L1 monoclonal antibody and EGFR-TKI on expression of soluble PD-L1 and function of T lymphocytes in EGFR-mutated lung cancer cells. Methods: Flow cytometry was used to analyze the expression of membrane PD-LI. ELISA was performed to detect the level of sPD-L1 in the supernatant of cultured EGFR-mutated and wild type lung cancer cells before and after erlotinib treatment.After treated with anti-PD-L1 monoclonal antibody alone and in combination with erlotinib, the proliferation of T lymphocytes in co-culture system was measured using Cell Counting Kit-8 (CCK-8) assay. The expression levels of PD-LI and IFN-γ in tumor cells and T lymphocytes treated with erlotinib in co-culture system were analyzed by flow cytometry and ELISA, respectively. Results: PD-L1 was highly expressed in EGFR-mutated lung cancer PC9 cells (78.7±3.1)% and HCC827 cells (82.7±2.6)%.After treated with erlotinib, the expression rates of membrane PD-L1 in PC9 and HCC827 cells were down-regulated (64.7%±3.1% and 73.0%±2.6%, respectively), significantly lower than that in the two cell lines without erlotinib treatment (P<0.05), and the expression levels of sPD-L1 in the supernatant of PC9 and HCC827 cells were also down-regulated (0.680±0.120)ng/ml and (0.903±0.047)ng/ml, respectively, significantly lower than that in the two cell lines without erlotinib treatment (P<0.01). However, no significant changes of membrane PD-L1 and sPD-L1 expression were found in EGFR wild type lung cancer cells (H1299 and A549) before and after erlotinib treatment. In the co-culture system composed of T cells and EGFR-mutated lung cancer cells, treatment with erlotinib alone promoted the proliferation of T lymphocytes (P<0.05), and combined treatment of anti-PD-L1 monoclonal antibody with erlotinib had a stronger effect (P<0.05). In the co-culture system composed of T cells and EGFR wild type cell lines, the proliferation of T cells was not changed after using erlotinib alone or combination of erlotinib and anti-PD-L1 monoclonal antibody (P>0.05). Before and after treatment with erlotinib, the secretion levels of IFN-γ were (856.0±70.3)pg/ml and (1 697.3±161.0)pg/ml, respectively, showing a significant difference (P<0.001). The expression rates of membrane PD-L1 were (76.2±0.5)% and (50.9±0.9)%, respectively, also showing a significant difference (P<0.001). However, no significant changes in the expression of IFN-γ and membrane PD-L1 were found in the co-culture system composed of T cells and A549 cells. Conclusions: Anti-PD-L1 monoclonal antibody combined with EGFR-TKI can effectively promote the proliferation and secretion function of T lymphocytes in the microenvironment of EGFR-mutated lung cancer cells.

Abstract 4814: miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and next generation EGFR tyrosine kinase inhibitors (TKIs) in NSCLC

Abstract Background: miRNAs play a critical role in regulating key biological processes by modulating the expression of up to several hundred genes across multiple cellular pathways. miR-34a, one of the most widely studied miRNAs, is lost or expressed at reduced levels in many tumors, and normally functions as a natural tumor suppressor by down-regulating expression of &amp;gt;30 different oncogenes, as well as genes involved in tumor immune evasion, including PD-L1. MRX34 is a potential first-in-class liposome-encapsulated miR-34a mimic in Phase 1 study (NCT01829971) as monotherapy in patients with advanced malignancies. The ability of miR-34a to regulate the expression of key oncogenes across multiple oncogenic pathways makes MRX34 a rational candidate to combine with other anticancer therapies which are frequently subject to primary and acquired resistance in the clinic. Previous studies showed that miR-34a greatly sensitizes both EGFR wild-type and mutant NSCLC cell lines, as well as hepatocellular carcinoma cell lines, to the first generation EGFR TKI erlotinib. Here we report research combining miR-34a and the next generation EGFR TKIs afatinib (Gilotrif®) and rociletinib (CO-1686) in NSCLC cell lines. Methods: Combination studies using single-drug ratios (∼IC50 ratio of miR-34a and TKI) and multiple ratios above and below were performed in a panel of EGFR wild-type (A549, H460, H1299, H226) and EGFR mutant (H1975, HCC827 parent and HCC827 erl res) NSCLC cell lines. Cells were transfected with miR-34a and incubated 24 hrs later with afatinib or rociletinib for 72 hrs, with cellular proliferation then determined by AlamarBlue. Synergistic, additive, or antagonistic effects were determined by combination index (CI) values (based on Loewe's concept of additivity), isobolograms, and curve-shift analyses. Results: Strong synergy was observed between miR-34a and both TKIs in all EGFR-mutant cell lines tested (CI &amp;lt;0.5 at effect levels ≥50%). Synergy was also observed for miR-34a + rociletinib in most EGFR wild-type cell lines (CI &amp;lt;0.6 at effect levels of ∼50-80%), but not for miR-34a + afatinib. Best synergies overall were observed in EGFR-mutant cells with acquired erlotinib resistance. The effects were observed across a range of different dose levels and drug ratios, with maximal synergy providing a high level of inhibition (80%) at doses likely achievable in the clinic and well below those projected to be required for similar inhibition by the single agents alone. Conclusions: Complementing previous results with miR-34a + erlotinib, the data demonstrate strongly synergistic anticancer effects between miR-34a and next generation EGFR TKIs in combination against a range of EGFR wild-type and mutant NSCLC cell lines. The results support clinical study of MRX34 + EGFR TKI combinations in patients with advanced NSCLC, including those with EGFR-mutant NSCLC that has progressed on EGFR TKI monotherapy. Citation Format: Jane Zhao, Adriana Guerrero, Kevin Kelnar, Heidi J. Peltier, Andreas G. Bader. miRNA combination therapy: In vitro anticancer synergy between miR-34a mimic and next generation EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4814.

Abstract 3502: Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity.

Abstract Predictive biomarkers are critical to optimize the use of receptor tyrosine kinase (RTK) inhibitors for cancer therapy. Because multi-protein complexes are critical to maintain oncogenic signaling, we hypothesized that assays that measure protein-protein interactions could identify patients with RTK activity and predict RTK inhibitor response. We developed in situ proximity ligation assays (PLA) to assess the degree of active EGFR signaling in both non-small cell lung cancer (NSCLC) cell lines and formalin-fixed paraffin-embedded (FFPE) patient specimens with the goal of establishing PLA assays as biomarkers of erlotinib sensitivity. Using in situ PLA, we evaluated EGFR association with its signaling adaptors, GRB2 and SHC1. By confocal microscopy we observed increased PLA signals in EGFR mutant NSCLC cell lines (PC9, H1650, HCC4006, HCC827) relative to EGFR wildtype (H23, H1299, H322, A549), indicative of constitutively active signaling complexes in mutant cell lines. PLA signal intensity was independent of EGFR, GRB2 and SHC1 protein expression levels, but did correlate with co-IP and pEGFR immunoblot results. Erlotinib dose-dependently reduced PLA signals in PC9 cells. H1650 xenografts exhibited high PLA signals that were reduced by erlotinib, corresponding with tumor shrinkage. Conversely, low PLA signals were observed in xenografts from EGFR wildtype cell lines (H322, H157). In human xenografts, PLA signals were significantly higher in EGFR mutant xenografts and reduced by erlotinib. In human FFPE specimens, PLA signals were absent in normal non-epithelial tissues and low in epithelial portions. FFPE lung tumor specimens (N = 221) revealed a wide range of PLA signal intensities (scored as 0+ to 3+), which localized to cytokeratin-positive regions. High PLA signals were found in approximately 25% of specimens across multiple histological classifications, including adenocarcinoma, squamous and large cell carcinoma. Furthermore, specimens from metastatic brain lesions were highly enriched for high PLA signal (p&amp;lt;.0001). Automated Quantitative Analysis (AQUA) of EGFR expression revealed multiple examples of EGFR expression-PLA signal discordance - we observed both high PLA signal (&amp;gt;2+) in specimens with relatively low EGFR protein expression and low PLA signal (&amp;lt;1+) in specimens with relatively high EGFR protein expression. Additionally, PLA signal was observed in both EGFR mutant and wildtype specimens, indicating that PLA provides additional information beyond immunohistochemistry and mutational analysis. Our results establish PLA as a powerful technique to detect signaling activity by monitoring protein complexes in clinical specimens. PLA-based approaches will likely have utility in biomarker studies for other receptor tyrosine kinase systems and their respective inhibitors. Citation Format: Matthew A. Smith, Richard Hall, Scott Haake, Jiannong Li, Eric B. Haura. Proximity ligation assays reveal protein-protein interactions associated with oncogenic signaling and drug sensitivity. [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 3502. doi:10.1158/1538-7445.AM2013-3502

The anti-proliferative effect of heat shock protein 90 inhibitor, 17-DMAG, on non-small-cell lung cancers being resistant to EGFR tyrosine kinase inhibitor

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC 50 values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 μM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 μM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.

Abstract 1616: The anti-tumor effect of TAE226, dual inhibitor of FAK and IGF-IR, on non-small-cell lung cancer with EGFR mutation

Abstract Background: TAE226 is the small molecule inhibitor that has been originally considered to inhibit focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). FAK and IGF-IR have been reported to be up-regulated in non-small cell lung cancer (NSCLC), suggesting their important roles on pathogenesis of NSCLC. In this study, we examined the anti-tumor effect of TAE226 on NSCLC cells in vitro and in vivo. Methods: We used NSCLC cell lines consisting of 4 EGFR-mutant cell lines (PC9, H3255, HCC827, H1975) and 3 EGFR-wild type cell lines (H1819, H1299, A549). We also used PC9-derived gefitinib-resistant cell line (RPC9). Anti-proliferative effect of TAE226 was examined with MTS assay. The status of EGFR and its downstream molecules was analyzed by Western blotting. The binding affinity of TAE226 to EGFR protein in the presence of ATP was compared between mutant and wild type EGFR proteins. The effect of TAE226 on xenograft mouse models was examined. Results: The value of IC50 (µmol/L) for EGFR-mutant cells (PC-9, H3255, HCC827, H1975 [T790M], and RPC-9 [T790M]) ranged from 0.086 to 0.31 and that of EGFR wild type cells (H1819, H1299, and A549) ranged from 1.4 to 4.7, indicating that EGFR-mutant cells are sensitive to TAE226. Western blotting assay showed that TAE226 preferentially inhibited phospho-EGFR and phospho-AKT. The binding affinity of TAE226 to mutant EGFR protein was 1000 times higher compared with wild-type EGFR protein. TAE226 via oral administration with dose of 30 mg/kg and 60 mg/kg exhibited significant inhibition of tumor growth in EGFR-mutant tumors compared with EGFR-wild type tumors without major toxicity or weight loss. These results suggested that safety of TAE226 within the treatment dose for EGFR-mutant cells in vivo. Conclusions: We indicated that TAE226 showed anti-tumor effect on EGFR-mutant cells and overcame resistance to gefitinib. Our results suggest that TAE226 will be expected as a novel strategy for NSCLC with EGFR mutation regardless of the presence of secondary T790M mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1616.

The antitumor effect of orally active heat shock protein 90 Inhibitor, 17-DMAG, on the growth of gefitinib-resistant non-small cell lung cancer

e22064 Background: It has been reported that NSCLC cell lines with activating mutations of EGFRgene were significantly sensitive to Heat Shock Protein 90 (Hsp 90 ) ihibitors regardless of the secondary T790M mutation. Methods: We examined the antitumor effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), the orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. Results: In MTS assay, the IC50 values for 3 cell lines with EGFR mutation (PC-9, RPC-9, and NCI-H1975) ranged from 0.15 to 0.28 μM. RPC-9 is a gefitinib-resistant lung cancer cell line with the secondary T790M mutation established from PC-9. By contrast, those of 7 EGFR-wild type cell lines ranged from 1.63 to 28.5 μM. Western blot analysis revealed that mutant EGFRs were more readily depleted than wild-type EGFRs after the treatment of 17-DMAG. Furthermore, expression of phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 was depleted after exposure to low concentrations of 17-DMAG in EGFR-mutant cells. Celeaved PARP expression confirmed apoptosis in response to 17-DMAG in EGFR-mutant cells. In animal models, 17-DMAG significantly reduced the growth of EGFR-mutant cell lines PC-9 and RPC-9 compared with controls and induced degradation of mutant EGFR. In contrast, 17-DMAG did not inhibit the growth of EGFR wild type cell lines compared with controls. Conclusions: These results suggested that 17-DMAG could become a novel therapeutic agent for patients with lung tumors expressing mutant EGFR and having developed clinical resistance to gefitinib. No significant financial relationships to disclose.

Possible correlation between SOCS1 methylation and EGFR exon 19 mutation but not exon 21 mutation in Chinese NSCLC patients

18113 Background: Suppressor of cytokine signaling-1 (SOCS1) and -3 (SOCS3) interact with the epidermal growth factor receptor (EGFR) to bring about the ubiquination and degradation of the receptor. Silencing of SOCS1 and SOCS3 by methylation was postulated to regulate the IL-6R/JAK-mediated STAT3 activation. Its relationship to EGFR and EGFR mutations in lung cancer remains unclear. Here, we examined the relationship between EGFR mutations and methylation of SOCS1 and SOCS3 in Chinese NSCLC patients. Methods: EGFR mutations were assessed in DNAs from microdissected tumor cells. PCR products were purified and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystem) and the ABI 3100 Genetic Analyzer. For methylation analyses of SOCS1 and SOCS3, DNAs were extracted from paraffin sections of tumor tissues, bisulfite treated and analyzed by methylation- specific PCR (MSP). Results: Data is available from 9 lung cancer cell lines and 20 tumor samples. Five EGFR-wild type cell lines had methylation for SOCS1. We found EGFR exon 19 mutation in 5 (25%) tumors, exon 21 mutation (L858R) in 2 (10%) tumors, and 1 (5%) tumor had double mutations. Four of 5 (80%) tumors with exon 19 mutation and none (0%) of the tumors with exon 21 mutation had SOCS1 methylation, comparing to only 2 of 12 (17%) EGFR-wild type tumors had SOCS1 methylation. SOCS3 methylation was not detected in any of the primary tumors or cell lines. Conclusions: Our preliminary findings suggest that SOCS1 methylation might be positively correlated with EGFR exon 19 mutation but exclusive from exon 21 mutation. A following study with larger sample size is currently under way. SOCS1 methylation may explain the better tumor response associated with EGFR exon 19 mutation. No significant financial relationships to disclose.

Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol‐3 kinase pathways in non‐small cell lung cancer cells

In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k-ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib-sensitivity in egfr wild type cells. Activating k-ras mutations were specifically detected in gefitinib-resistant cells, suggesting that the occurrence of k-ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib-sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib-resistance of NSCLC cell lines. Gefitinib-resistant NSCLC cell lines, showing EGFR-independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co-treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy.

337 Velcade, an NF-kb inhibitor, enhances tamoxifen sensitivity in Akt-induced resistant breast cancer xenograft tumors

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC50 values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 μM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 μM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.