Erlotinib-resistant Cell Lines Research Articles

Abstract 5958: Exploring AKR1C3 as a therapeutic target to overcome erlotinib resistance in lung adenocarcinoma

Abstract Introduction: Erlotinib has demonstrated promising efficacy in lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor mutation. However, the development of resistance to erlotinib poses a significant challenge in achieving long-lasting treatment responses. Aldo-keto reductase family 1 member C3 (AKR1C3) is associated with tumor progression, drug resistance, and unfavorable prognosis. This study aims to assess the potential of AKR1C3 as a therapeutic target for overcoming erlotinib resistance in LUAD. Methods: AKR1C3 expression was assessed in paired tumor tissues from 100 LUAD patients using reverse transcription quantitative polymerase chain reaction. Both in vitro and in vivo experiments were categorized into four treatment groups: a saline control (CTR), erlotinib (ER), an AKR1C3 inhibitor 3-(4-trifluoromethyl)phenylamino)benzoic acid (BA), and the combination of erlotinib with the AKR1C3 inhibitor (COM). An erlotinib-resistant cell line was established from the LUAD cell line HCC4006. The MTS assay was used to evaluate cell proliferation and viability. The combined effect of erlotinib and the AKR1C3 inhibitor was assessed using Synergyfinder and CompuSyn calculation software. Both cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of erlotinib resistance to LUAD were established in NSG mice. The mice received daily oral gavage administration of the drugs for 31 days, while tumor volume was monitored biweekly. Results: AKR1C3 expression was significantly upregulated by more than three-fold in LUAD tumor samples compared to normal counterparts (p < 0.0001). After 24 hours of treatment in erlotinib-resistant cells, the ER and BA groups exhibited inhibition rates of 7% and 9%, respectively, while the COM group demonstrated a substantially higher inhibition rate of 45%. Synergyfinder and CompuSyn analysis revealed a synergy score of 12 and a combination index of 0.5 for the COM group, indicating a robust synergistic effect between the two drugs. In the CDX model, the COM group exhibited a tumor volume reduction of 25% in erlotinib-resistant cells and 64% in parental cells compared to the CTR group. In the PDX model, the COM group displayed a significant reduction of 50.4% in tumor volume compared to the CTR group. There was no significant difference in the weight of body and internal organs among the four treatment groups in both xenograft models. Conclusions: AKR1C3 overexpression was observed in the tumor tissues of LUAD patients. The combination of erlotinib and AKR1C3 inhibitor showed promising efficacy in overcoming erlotinib resistance in both in vitro and in vivo experiments. These findings highlight AKR1C3 as a potential therapeutic target for managing erlotinib resistance in LUAD patients. Targeting AKR1C3 represents a novel approach to overcome resistance and enhance treatment outcomes in LUAD patients undergoing erlotinib therapy. Citation Format: William C. Cho, Kwan P. Li, Chi F. Wong, King Y. Fung, James C. Chow, Ka M. Cheung, Jeffrey C. Chan, Eunice Y. Lau. Exploring AKR1C3 as a therapeutic target to overcome erlotinib resistance in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5958.

Abstract 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3290.

Regulation of epidermal growth factor receptor tyrosine kinase inhibitor resistance via Ambra1-mediated autophagy in non-small cell lung cancer.

To explore the molecular mechanisms related to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) resistance, along with potential therapeutic targets and strategies. The autophagy and Beclin 1 regulator 1 (Ambra1) short hairpin ribonucleic acid (shRNA) lentivirus vector and Ambra1 overexpression plasmid, constructed with a plasmid cloning deoxyribonucleic acid (pcDNA) 3.1 vector, were used to down-regulate and up-regulate Ambra1 expression in the human lung adenocarcinoma erlotinib-resistant cell line (PC9/ER), respectively, as well as to screen stable transgenic cell lines. The IC50 of Erlotinib in these cell lines were measured to determine their resistance status. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure messenger ribonucleic acid (mRNA) expression of resistance-related genes like multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung drug-resistant-related protein (LRP). Western blot was performed to analyze the protein expressions of the autophagy-related genes Beclin 1, LC3II/I, and p62. Each stable transgenic line formed a tumor under the skin in nude mice; the mice with subcutaneous tumorigenesis of PC9/ER cells and shAmbra1-PC9/ER cells were subsequently treated with rapamycin (RAPA) and chloroquine (CQ), respectively. The mRNA expressions of MDR1, MRP1, and LRP in each tumor tissue sample were detected by qRT-PCR. The protein expressions of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), forkhead box O3 (FoxO3a), and phosphorylated forkhead box O3 (p-FoxO3a) in the AMPK/FoxO3a signaling pathway were analyzed via Western blot. The qRT-PCR result revealed that the level of Ambra1 in EGFR-TKI-resistant cells had increased. This was further exacerbated by the overexpression of Ambra1 and was reduced after its inhibition. Additionally, Ambra1 upregulated the mRNA expression of drug-resistant genes and the expression of autophagy-related proteins. Subcutaneous tumorigenesis of RAPA-treated shAmbra1-PC9/ER cells resulted in increased expression of drug resistance-related genes and a concomitant decrease in p-AMPK and increase in p-FoxO3a. The results revealed that Beclin-1/β-actin, p62/β-actin, and LC3II/I in the model group were all significantly increased compared to the control group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly higher in the pcDNA-Ambra1 group, with P<0.05. Compared to the model group, Beclin-1/β-actin, p62/β-actin, and LC3II/I were all significantly decreased in the shAmbra1 group, with P<0.05. Thus, these data suggest that Ambra1 promotes cellular autophagy. In addition, subcutaneous tumorigenesis of CQ-treated shAmbra1-PC9/ER cells resulted in reduced expression of drug resistance-related genes, and a concomitant increase in p-AMPK and decrease in p-FoxO3a. The results of this study revealed that Ambra1-mediated autophagy regulated EGFR-TKI resistance in NSCLC, most probably through the AMPK/FoxO3a signaling pathway.

Oral Squamous Cell Carcinoma Cells with Acquired Resistance to Erlotinib Are Sensitive to Anti-Cancer Effect of Quercetin via Pyruvate Kinase M2 (PKM2).

Oral squamous cell carcinoma (OSCC) frequently carries high epidermal growth factor receptor (EGFR) expression. Erlotinib, a small molecule tyrosine kinase inhibitor (TKI), is an effective inhibitor of EGFR activity; however, resistance to this drug can occur, limiting therapeutic outcomes. Therefore, in the current study, we aimed to unveil key intracellular molecules and adjuvant reagents to overcome erlotinib resistance. First, two HSC-3-derived erlotinib-resistant cell lines, ERL-R5 and ERL-R10, were established; both exhibited relatively higher growth rates, glucose utilization, epithelial-mesenchymal transition (EMT), and invasiveness compared with parental cells. Cancer aggressiveness-related proteins, such as N-cadherin, Vimentin, Twist, MMP-2, MMP-9, and MMP-13, and the glycolytic enzymes PKM2 and GLUT1 were upregulated in ERL-R cells. Notably, ERL-R cells were sensitive to quercetin, a naturally-existing flavonol phytochemical with anti-cancer properties against various cancer cells. At a concentration of 5 μM, quercetin effectively arrested cell growth, reduced glucose utilization, and inhibited cellular invasiveness. An ERL-R5-derived xenograft mouse model confirmed the growth-inhibitory efficacy of quercetin. Additionally, knock-down of PKM2 by siRNA mimicked the effect of quercetin and re-sensitized ERL-R cells to erlotinib. Furthermore, adding quercetin blocked the development of erlotinib-mediated resistance by enhancing apoptosis. In conclusion, our data support the application of quercetin in anti-erlotinib-resistant OSCC and indicate that PKM2 is a determinant factor in erlotinib resistance and quercetin sensitivity.

A novel combination treatment of antiADAM17 antibody and erlotinib to overcome acquired drug resistance in non-small cell lung cancer through the FOXO3a/FOXM1 axis.

After the identification of specific epidermal growth factor receptor (EGFR)-activating mutations as one of the most common oncogenic driver mutations in non-small cell lung cancer (NSCLC), several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) with different clinical efficacies have been approved by various health authorities in the last two decades in targeting NSCLC harboring specific EGFR-activating mutations. However, most patients whose tumor initially responded to the first-generation EGFR-TKI developed acquired resistance. In this study, we developed a novel combination strategy, "antiADAM17 antibody A9(B8) + EGFR-TKIs", to enhance the efficacy of EGFR-TKIs. The addition of A9(B8) was shown to restore the effectiveness of erlotinib and overcome acquired resistance. We found that when A9(B8) antibody was treated with erlotinib or gefitinib, the combination treatment synergistically increased apoptosis in an NSCLC cell line and inhibited tumor growth in vivo. Interestingly, the addition of A9(B8) could only reduce the survival of the erlotinib-resistant NSCLC cell line and inhibit the growth of erlotinib-resistant tumors in vivo but not gefitinib-resistant cells. Furthermore, we revealed that A9(B8) overcame erlotinib resistance through the FOXO3a/FOXM1 axis and arrested the cell cycle at the G1/S phase, resulting in the apoptosis of cancer cells. Hence, this study establishes a novel, promising strategy for overcoming acquired resistance to erlotinib through the FOXO3a/FOXM1 axis by arresting the cell cycle at the G1/S phase.

Integrin β3 Promotes Resistance to EGFR-TKI in Non-Small-Cell Lung Cancer by Upregulating AXL through the YAP Pathway.

Integrin β3 plays a key role in the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), but the development of integrin β3 inhibitors has been stalled due to the failure of phase III clinical trials for cancer treatment. Therefore, it is imperative to find a potentially effective solution to the problem of acquired resistance to EGFR-TKI for patients with integrin-β3 positive non-small-cell lung cancer (NSCLC) by exploring novel downstream targets and action mechanisms of integrin β3. In the present study, we observed that the expression of integrin β3 and AXL was significantly upregulated in erlotinib-resistant NSCLC cell lines, which was further confirmed clinically in tumor specimens from patients with NSCLC who developed acquired resistance to erlotinib. Through ectopic expression or knockdown, we found that AXL expression was positively regulated by integrin β3. In addition, integrin β3 promoted erlotinib resistance in NSCLC cells by upregulating AXL expression. Furthermore, the YAP pathway, rather than pathways associated with ERK or AKT, was involved in the regulation of AXL by integrin β3. To investigate the clinical significance of this finding, the current well-known AXL inhibitor R428 was tested, demonstrating that R428 significantly inhibited resistance to erlotinib, colony formation, epithelial–mesenchymal transformation and cell migration induced by integrin β3. In conclusion, integrin β3 could promote resistance to EGFR-TKI in NSCLC by upregulating the expression of AXL through the YAP pathway. Patients with advanced NSCLC, who are positive for integrin β3, might benefit from a combination of AXL inhibitors and EGFR-TKI therapy.

Abstract 3248: Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets

Abstract Glioblastoma (GBM) is a devastating primary brain tumor with &amp;lt;5% 5-year survival. CDKN2A deletion (~60%) and EGFR amplification (~55%) mutations frequently co-occur in these tumors. EGFR is an attractive therapeutic target due to its mutational frequency and availability of brain-penetrant tyrosine kinase inhibitors (TKI). Several EGFR TKI have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used a panel of ten genetically engineered mouse astrocyte lines harboring Cdkn2a deletion and EGFRvIII, a common (~30%) activating mutation. Resistant cells were generated via long-term exposure to gefitinib or erlotinib, either in vitro or in vivo. Both transcriptomic (RNAseq) and proteomic (multiplexed inhibitor beads with mass spectrometry, MIB-MS) experiments showed that cell lines clustered primarily by resistance phenotype and secondarily by method of resistance development when analyzed using principal component analysis and unsupervised hierarchical clustering. Kinases involved in proliferation and differentiation signaling pathways (ex: Pdgfrb, Pdk2, Tnik, Mapk3, Fgfr2) were upregulated in both RNAseq and MIB-MS datasets and thus represent putative druggable targets for dual kinase inhibition. Analysis of commonly upregulated kinases and their commercially available inhibitors revealed dovitinib and dasatinib, two brain-penetrant drugs approved for other cancer indications, as candidates for dual inhibition with an EGFR TKI. Resistant cell lines were all more sensitive to dovitinib than their drug-naïve parents; however, sensitivity to dasatinib varied. BLISS analysis of dual treatment with EGFR TKI neratinib and dasatinib or dovitinib revealed synergistic drug interactions in most lines. Additionally, drug-naïve cells displayed a robust, acute proteomic response to EGFR TKI afatinib over 48h, while the response of resistant lines was significantly blunted. This model system can also be used to examine acute vs. long-term kinome response to EGFR TKI. Acute response was examined by treating drug-naïve cells with afatinib over 48h, and long-term kinome rewiring was observed by comparing untreated cells to gefitinib- and erlotinib-resistant cell lines. Combing both RNAseq datasets for kinases upregulated in both drug-naïve cells over a 48h EGFR TKI treatment course and in resistant cell lines compared to their sensitive parents reveals 21 and 13 common kinases, respectively, at p&amp;lt;0.001. Eight of these kinases (Cdk19, Ddr1, Kalrn, Khk, Mapk3, Pink1, Tnik, Ulk2) appear in both the in vitro and in vivo datasets, indicating a conserved kinome response regardless of method of resistance generation. Overall, integrated kinome profiling in GBM models with defined mutational profiles provides a powerful framework to identify novel therapeutic targets that could significantly alter current treatment paradigms. Citation Format: Abigail K. Shelton, Erin Smithberger, Madison Butler, Allie Stamper, Ryan E. Bash, Steve P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3248.

Progesterone receptor membrane component 2 expression leads to erlotinib resistance in lung adenocarcinoma cells.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) provide a favorable treatment outcome in patients with EGFR mutation-positive non-small cell lung cancer. However, most of such patients become resistant to EGFR-TKIs within a year. Thus, clarifying the mechanism of acquired resistance to EGFR-TKIs has been a research focus. Here, we demonstrated that the expression of progesterone receptor membrane component 2 (PGRMC2) was upregulated in an erlotinib-resistant cell line, PC9/ER, compared with the parental PC9 lung cancer cells. Our previous study showed that PGRMC1 is responsible for acquired resistance to erlotinib; however, PGRMC2 has not been discussed yet. Thus, the aim of this study was to determine the role of PGRMC2 in acquired resistance to erlotinib. Transfection with PGRMC2 siRNA significantly enhanced the sensitivity to erlotinib in PC9/ER cells. Furthermore, knockdown of PGRMC2 reduced the expression of p21, which is known as cell-cycle inhibitor and antiproliferative effector. These results suggest that PGRMC2 partially contributes to erlotinib resistance in PC9/ER cells, and that investigation into the effect of PGRMC2 on apoptosis and the cell cycle are warranted.

Estrogen Receptor β Is Involved in Acquired Resistance to EGFR-tyrosine Kinase Inhibitors in Lung Cancer.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has posed serious clinical problems in the treatment of lung adenocarcinoma (LADC) patients harboring relevant EGFR mutations. In this study, we explored the role of estrogen receptor β (ERβ) in the development of acquired resistance to EGFR-TKIs in human LADC. First, the role of ERβ in erlotinib resistance of LADC cell lines (PC9/ER) was examined. Then, the immunolocalization of ERβ in 28 LADC patient samples treated with EGFR-TKIs was investigated. Cytoplasmic ERβ was upregulated in erlotinib resistant cell lines. EGFR-TKIs sensitivity increased with ERβ inhibition in PC9/ER cells. ERK1/2 and AKT activities were both markedly increased by specific ERβ agonists even under erlotinib treatment of PC9/ER cells. Cytoplasmic ERβ immunoreactivity was significantly associated with clinical response to EGFR-TKIs. Cytoplasmic ERβ in LADC cells was involved in the development of resistance to EGFR-TKIs.

Efficacy of newly discovered DNA aptamers targeting AXL in a lung cancer cell with acquired resistance to Erlotinib.

BackgroundAccumulating evidences indicate that AXL overexpression or activation is associated with cancer progression and acquired resistance to targeted anti-cancer drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Despite recent development of several drugs that target multiple receptor tyrosine kinases (RTKs), drugs that selectively target AXL signaling are extremely rare. Short nucleic acid aptamers are non-immunogenic molecules with high binding affinity and specificity to their target molecules that could potentially be used as a novel cancer treatment.MethodsModified-DNA aptamers were selected on the basis of its ability to bind recombinant human AXL. AXL aptamers were selected for their inhibition of AXL and then selected aptamers were tested for their use to overcome acquired resistant to EGFR-TKI on a lung cancer cell with acquired resistance to erlotinib.ResultsThese new AXL aptamers inhibited cell viability to an extent of 30–40% in HCC827/ER cells with acquired resistance to erlotinib. The possible mechanism of overcoming the acquired resistance may be by inhibiting the activation of Akt and Erk. Although, aptamers effectively decreased cell viability of erlotinib‐resistant cell line, the combination of aptamers and erlotinib did not synergistically decrease the survival of the resistant cell line.ConclusionsWe developed newly modified DNA aptamers that selectively bind to AXL receptors, and assessed their efficacy in a human lung cancer cell with acquired resistance to EGFR-TKI.

Application of decoy oligodeoxynucleotides strategy for inhibition of cell growth and reduction of metastatic properties in nonresistant and erlotinib-resistant SW480 cell line.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer.

DNA Polymerase Alpha Subunit B Is a Binding Protein for Erlotinib Resistance in Non-Small Cell Lung Cancer.

Simple SummaryNon-small-cell lung carcinoma (NSCLC) covers for almost 85% of all lung cancers and a major contributor to the overall cancer death rate. Erlotinib is used to treat NSCLC via inhibition of epithelial growth factor receptor (EGFR) kinase activity. Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. We performed DARTS LC-MS/MS with SWATH of DIA analysis and identified a novel binding protein of Erlotinib that may underlie NSCLC resistance. Our study indicated that Erlotinib binds POLA2 in addition to EGFR. This was confirmed by DARTS and CETSA results. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

1745P SARS-CoV-2 infects metabolically-primed epithelial cells in lung cancer models

The novel coronavirus SARS-CoV-2 is the cause of the respiratory illness COVID-19—a global pandemic affecting over 4 million individuals worldwide. Viruses efficiently replicate by hijacking host cell machinery to obtain macromolecules and energy by similar mechanisms as cancer cells. Since viral infection is known to alter cellular nutrient requirements, this study explores the metabolites and metabolic pathways associated with SARS-CoV-2 infection. Bulk and single-cell seequencing data from cell lines and tumor samples were retrieved from publically available datasets. Transcriptional data were retrieved from publically available datasets of gefitinib- and erlotinib-resistant EGFR-mutant cell lines and Calu3 and A549 cells mock treated or infected with SARS-CoV-2. Single-cell RNAseq datasets of EGFR-mutant PC-9 mock and osimertinib treated were downloaded from GEO. 225 metabolites were profiled in CCLE cell lines using LC-MS. To identify metabolic features of cells able to be infected by SARS-CoV-2 via the ACE2 receptor, metabolites associated with ACE2 expression were investigated. ACE2 expression positively correlates with glutamine in upper aerodigestive tract cell lines. Consistent with this, ACE2 expression was examined against a list of 253 metabolism-associated genes and GLUL, which encodes an enzyme (glutamine synthetase) responsible for conversion of glutamate to glutamine, was significantly positively correlated in NSCLC, HNSCC, and SCLC cell lines and confirmed in human tumor datasets. Additionally, GLS, which encodes the enzyme (glutaminase) that catalyzes the opposing reaction, is negatively correlated with ACE2 expression. Further, we analyzed RNA sequencing data from NSCLC cell lines infected with SARS-CoV-2 for 24 hours and revealed that upon infection there is a down regulation of GLUL signifying a metabolic-shift away from glutamine as the cells undergo EMT. We show that SARS-CoV-2 targeting of ACE-2 expressing, metabolically-primed epithelial cells is advantageous to exploit the abundance of glutamine to synthesize nucleotides for rapid replication and viral spread.

Exosomal Transfer of lncRNA H19 Promotes Erlotinib Resistance in Non-Small Cell Lung Cancer via miR-615-3p/ATG7 Axis.

BackgroundDrug resistance restrains the effect of drug therapy in non-small cell lung cancer (NSCLC). However, the mechanism of the acquisition of drug resistance remains largely unknown. This study aims to investigate the effect of exosomal lncRNA H19 on erlotinib resistance in NSCLC and the underlying mechanism.MethodsHCC827 and A549 cells were continuously grafted into erlotinib-containing culture medium to establish erlotinib-resistant cell lines. The expression of H19 and miR-615-3p was detected by qRT-PCR. The protein levels of MMP2, MMP9, CD9, CD63 and ATG7 were measured by Western blot. Cell viability and proliferation were determined by Cell Counting Kit-8 (CCK-8) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, respectively. Migration and invasion were assessed by transwell assay. Xenograft tumor models were used to investigate the effect of H19 on erlotinib resistance in vivo. Online software and dual-luciferase reporter assay were used to predicate the downstream targets and confirm the targeted relationships.ResultsH19 was upregulated in erlotinib-resistant cells, and knockdown of H19 inhibited cell proliferation, migration and invasion in erlotinib-resistant cells. Extracellular H19 can be packaged into exosomes. Exosomes containing H19 induced erlotinib resistance of sensitive cells, while knockdown of H19 abolished this effect. miR-615-3p was a target of H19 and can bind to ATG7. Exosomal H19 affected erlotinib resistance of erlotinib-resistant NSCLC cells via targeting miR-615-3p to regulate ATG7 expression. In addition, the serum exosomal H19 was upregulated in patients with erlotinib resistance. Furthermore, downregulated H19 decreased the resistance of tumor cells to erlotinib in vivo.ConclusionOur study demonstrated that exosomal H19 facilitated erlotinib resistance in NSCLC via miR-615-3p/ATG7 axis, which might provide a potential target for the diagnosis and treatment of NSCLC.

Progesterone receptor membrane component 1 leads to erlotinib resistance, initiating crosstalk of Wnt/\u03b2-catenin and NF-\u03baB pathways, in lung adenocarcinoma cells

In non-small-cell lung cancer, mutation of epidermal growth factor receptor (EGFR) stimulates cell proliferation and survival. EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib are used as first-line therapy with drastic and immediate effectiveness. However, the disease eventually progresses in most cases within a few years due to the development of drug resistance. Here, we explored the role of progesterone membrane component 1 (PGRMC1) in acquired resistance to erlotinib and addressed the molecular mechanism of EGFR-TKI resistance induced by PGRMC1. The erlotinib-sensitive cell line PC9 (derived from non-small-cell lung cancer) and the erlotinib-resistant cell line PC9/ER were used. In proteomic and immunoblotting analyses, the PGRMC1 level was higher in PC9/ER cells than in PC9 cells. WST-8 assay revealed that inhibition of PGRMC1 by siRNA or AG-205, which alters the spectroscopic properties of the PGRMC1-heme complex, in PC9/ER cells increased the sensitivity to erlotinib, and overexpression of PGRMC1 in PC9 cells reduced their susceptibility to erlotinib. In the presence of erlotinib, immunoprecipitation assay showed that AG-205 suppressed the interaction between EGFR and PGRMC1 in PC9/ER cells. AG-205 decreased the expression of β-catenin, accompanied by up-regulation of IκBα (also known as NFKBIA). Furthermore, AG-205 reduced the expression of β-TrCP (also known as BTRC), suggesting that PGRMC1 enhanced the crosstalk between NF-κB (also known as NFKB) signaling and Wnt/β-catenin signaling in an erlotinib-dependent manner. Finally, treatment with the Wnt/β-catenin inhibitor XAV939 enhanced the sensitivity of PC9/ER cells to erlotinib. These results suggest that PGRMC1 conferred resistance to erlotinib through binding with EGFR in PC9/ER cells, initiating crosstalk between the Wnt/β-catenin and NF-κB pathways.

Molecular mechanism of acquired drug resistance in the EGFR-TKI resistant cell line HCC827-TR.

BackgroundThe first‐line standard treatment of non‐small cell lung cancer (NSCLC) with EGFR mutation is EGFR‐tyrosine kinase inhibitors (TKIs). However, most patients will develop acquired resistance after 9–13 months. This study investigated novel molecular mechanisms of acquired resistance to EGFR‐TKIs to identify a potential new treatment for EGFR‐TKI resistant NSCLC patients.MethodsWe established an EGFR‐TKI resistant cell line (HCC827‐TR) by culturing the HCC827‐P cell line through continuous erlotinib culture. We used Sanger sequencing, RT‐PCR, and western blot to rule out known resistance mechanisms in HCC827‐TR cells, including EGFR‐T790M and MET, PTEN, or EGFR expression changes. Next‐generation sequencing was performed and identified differentially expressed genes between two cell lines and examined the genes with GO and KEGG pathway database analyses. We also examined the molecular alterations in COSMIC and GDSC databases and performed hazard predictions using SIFT, PolyPhen‐2, Mutation Taster, and CADD.ResultsOur results identified FGF2 as a differentially expressed gene with a G101T point mutation in HCC827‐TR cells that showed high mutation frequency and hazard score. HCC827‐TR cells showed elevated FGF2 compared to parental cells. It is noteworthy that treatment with the FGFR inhibitor AZD4547 could restore the sensitivity of HCC872‐TR cells to erlotinib.ConclusionsAn erlotinib‐resistant cell line HCC827‐TR was successfully constructed and we identified the EGFR‐TKI resistance mechanism involving the FGF2 gene mutation. Targeted inhibition of the FGF2/FGFR signaling pathway may effectively restore the sensitivity of the resistant cells to erlotinib. These results suggest a novel treatment strategy for EGFR‐TKI resistant NSCLC patients.Key points Significant findings of the study: Identifies a novel molecular mechanism for EGFR‐TKI acquired resistance. What this study adds: A potential novel strategy for the treatment of EGFR‐TKI resistant NSCLC patients.

Protein tyrosine kinase 2: a novel therapeutic target to overcome acquired EGFR-TKI resistance in non-small cell lung cancer

BackgroundProtein tyrosine kinase 2 (PTK2) expression has been reported in various types of human epithelial cancers including lung cancer; however, the role of PTK2 in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) has not been elucidated. We previously reported that pemetrexed-resistant NSCLC cell line PC-9/PEM also acquired EGFR-TKI resistance with constitutive Akt activation, but we could not find a therapeutic target.MethodsCell viability in EGFR-mutant NSCLC cell lines was measured by the WST-8 assay. Phosphorylation antibody array assay for receptor tyrosine kinases was performed in PC-9 and PC-9/PEM cell lines. We evaluated the efficacy of EGFR and PTK2 co-inhibition in EGFR-TKI-resistant NSCLC in vitro. Oral defactinib and osimertinib were administered in mice bearing subcutaneous xenografts to evaluate the efficacy of the treatment combination in vivo. Both the PTK2 phosphorylation and the treatment combination efficacy were evaluated in erlotinib-resistant EGFR-mutant NSCLC cell lines.ResultsPTK2 was hyperphosphorylated in PC-9/PEM. Defactinib (PTK2 inhibitor) and PD173074 (FGFR inhibitor) inhibited PTK2 phosphorylation. Combination of PTK2 inhibitor and EGFR-TKI inhibited Akt and induced apoptosis in PC-9/PEM. The combination treatment showed improved in vivo therapeutic efficacy compared to the single-agent treatments. Furthermore, erlotinib-resistant NSCLC cell lines showed PTK2 hyperphosphorylation. PTK2 inhibition in the PTK2 hyperphosphorylated erlotinib-resistant cell lines also recovered EGFR-TKI sensitivity.ConclusionPTK2 hyperphosphorylation occurs in various EGFR-TKI-resistant NSCLCs. Combination of PTK2 inhibitor and EGFR-TKI (defactinib and osimertinib) recovered EGFR-TKI sensitivity in the EGFR-TKI-resistant NSCLC. Our study result suggests that this combination therapy may be a viable option to overcome EGFR-TKI resistance in NSCLC.

P2.14-13 Elevated Expression of Eukaryotic Translation Initiation Factor 3 Subunit C Contributes to EGFR-TKI Resistance in Lung Adenocarcinoma

The treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, in patients with EGFR-mutant lung adenocarcinoma causes tumor regression and improved prognosis. However, drug resistance and tumor relapse eventually occur, and the molecular mechanisms remain to be fully revealed. In this study, we elucidated the novel mechanism of EGFR-TKI resistance and clarified the clinical significance using erlotinib-resistant lung adenocarcinoma cell lines and patient specimens. The erlotinib-resistant cell line, PC9/ER was obtained from PC9 cell line by exposing to increasing concentrations of erlotinib for 6 months. The sensitivity of erlotinib was evaluated by the MTT assay. In order to identify erlotinib-resistance related factors, we performed a proteomics analysis, followed by western blotting. To evaluate the roles of the candidate factor, we performed gene knockdown analysis using siRNAs. The autophagy activity was assessed by the amount of LC3B-II protein or visualization of autophagosomes using LC3B-RFP expression vector. Immunohistochemical staining was performed on tumor biopsies obtained from lung adenocarcinoma patients with activating mutation in EGFR, and we examined the correlation between the staining intensity and clinical EGFR-TKI resistance. Initially, we performed the sequencing analysis of EGFR exons 18 to 21, which cover the tyrosine kinase domain, and found that there was no difference between PC9 and PC9/ER cells. The proteomics analysis revealed ribosomal and translation-related proteins were abundantly identified in PC9/ER cells, compared with PC9 cells. Among them, we found that eukaryotic translation initiation factor 3 subunit C (eIF3c) was overexpressed in PC9/ER cells. The knockdown of eIF3c expression with siRNA improved the sensitivity of EGFR-TKI in PC9/ER cells. Additionally, we observed that LC3B-II increased in PC9/ER cells, but decreased by the knockdown of eIF3c. Consistently, the overexpression of eIF3c upregulated the number of autophagosomes. Moreover, the inhibition of autophagy with chloroquine attenuated erlotinib-resistance in PC9/ER cells. Finally, the frequency of eIF3c-positive samples was higher in biopsy specimens from the patients’ refractory to EGFR-TKI therapy than in those from the patients prior to the treatment with EGFR-TKI. Furthermore, patients with eIF3c-positive tumors had shorter progression-free survival in EGFR-TKI treatment than eIF3c-negative patients. The increased eIF3c expression conferred the resistance to EGFR-TKI in lung adenocarcinoma cells via enhancement of autophagy. Moreover, the patients with increased eIF3c expression in biopsy samples showed poor prognosis. The inhibition of eIF3c could be a new therapeutic strategy for overcoming EGFR-TKI resistance in lung adenocarcinoma patients.

P1.03-46 LncRNA H19 Downregulation Promoted Resistance to EGFR-TKIs Through Regulating AKT and SRC Activating in NSCLC Cells

Most NSCLC patients with EGFR activating mutations initially respond to EGFR-TKI treatment. However, acquired resistance to EGFR-TKIs inevitably occurs after treatment for several months. There are evidences that suggest lncRNAs could play important roles in EGFR-TKI resistance, but limited data is available now. In previous study, we found that LncRNA H19 was downregulated in EGFR-TKI-resistant cells. We established two erlotinib-resistant cell lines HCC827-ER and PC9-ER. Real-time quantitative PCR (qPCR) was used to detect H19 levels. We detected gene mutations in the ER cell lines through whole-exome sequencing. We used H19 and H19 shRNA lentiviral expression vector to overexpress or knockdown its expression. MTT cytotoxic and clone formation experiments were performed to assess cell growth inhibition. The apoptosis analysis was evaluated by PARP and caspase-3 degradation. Protein levels in AKT and SRC were examined by western blotting. H19 expression was lower in HCC827-ER and PC9-ER cells than in their parental sensitive (PS) cells. We did not detect mutations in EGFR T790M, PI3KCA, or KRAS by whole-exome sequencing of ER cell lines. MTT and clone formation assays showed H19 knockdown caused resistance to erlotinib in PS cells, whereas H19 overexpression not only enhanced erlotinib sensitivity in PS cells but also recapitulated erlotinib sensitivity in HCC827-ER and PC9-ER cells. Treatment with erlotinib increased PARP and caspase-3 cleavage in H19-ovexpressing ER and PS cells. We also found erlotinib treatment decreased levels of phosphorylated AKT and SRC in PS cells, and further lowered their levels in H19-overexpressing PS cells. On the contrast, erlotinib treatment had no effect on levels of phosphorylated AKT and SRC in H19-knockdown PS cells. Finally, we measured the expression of H19 using qPCR in 20 fresh frozen EGFR-mutant NSCLC specimens. Lower expression of H19 was observed in EGFR-TKI-resistant tissue samples. H19 was downregulated in erlotinib-resistant NSCLC cells. H19 inhibited AKT and SRC activating to restore sensitivity to erlotinib, which could provide new insight into resistance mechanisms to EGFR-TKIs.

SRC and MEK Co-inhibition Synergistically Enhances the Anti-tumor Effect in Both Non-small-cell Lung Cancer (NSCLC) and Erlotinib-Resistant NSCLC

Non-small-cell lung cancer (NSCLC) is the predominant form of lung cancer, and it is regulated by a complex signal transduction network. Single-agent targeted therapy often results in acquired resistance, which leads to treatment failure. In this study, we demonstrated that a combination of the kinase inhibitors trametinib and bosutinib can synergistically suppress the growth of NSCLC by inhibiting both the mitogen-activated protein kinase (MAPK) and proto-oncogene tyrosine-protein kinase (SRC) pathways. The combination was profiled against a panel of 22 NSCLC cell lines, including one erlotinib-resistant cell line, and this combination was found to show synergistic effects against 16 cell lines. NSCLC cell lines (HCC827, HCC827-erlotinib-resistant, and H1650) were treated with trametinib, bosutinib, or a combination of these drugs. The drug combination inhibited colony formation and induced cell apoptosis. A mechanism study showed that the phosphorylation of multiple kinases in the epidermal growth factor receptor (EGFR) signaling pathway in NSCLC was down-regulated. In addition, the combination significantly attenuated tumor growth of HCC827 xenografts with low toxicity. Our findings provide a theoretical basis for further study of the combination of MAPK and SRC pathway inhibitors in NSCLC, especially in the treatment of erlotinib-resistant NSCLC.