Resistant Non-small Cell Lung Cancer Research Articles

LncRNAs LCETRL3 and LCETRL4 at chromosome 4q12 diminish EGFR-TKIs efficiency in NSCLC through stabilizing TDP43 and EIF2S1

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are effective targeted therapy drugs for advanced non-small cell lung cancer (NSCLC) patients carrying sensitized EGFR mutations. The rapid development of EGFR-TKIs resistance represents a major clinical challenge for managing NSCLC. The chromosome 4q12 is the first genome-wide association study (GWAS)-reported locus associated with progression-free survival (PFS) of NSCLC patients treated with EGFR-TKIs. However, the biological significance of the noncoding transcripts at 4q12 in NSCLC remains elusive. In the present study, we identified two 4q12 long noncoding RNAs (lncRNAs) LCETRL3 and LCETRL4 which could significantly dimmish EGFR-TKIs efficiency. In line with their oncogenic role, evidently higher LCETRL3 and LCETRL4 levels were observed in NSCLC tissues as compared with normal specimens. Importantly, lncRNA LCETRL3 can interact with oncoprotein TDP43 and inhibit ubiquitination and degradation of TDP43. Similarly, lncRNA LCETRL4 can bind and stabilize oncoprotein EIF2S1 through reducing ubiquitin-proteasome degradation of EIF2S1. In particular, elevated levels of LCETRL3 or LCETRL4 in NSCLC cells resulted in stabilization of TDP43 or EIF2S1, increased levels of NOTCH1 or phosphorylated PDK1, activated AKT signaling and, thus, EGFR-TKIs resistance. Taken together, our data revealed a novel model that integrates two lncRNAs transcribed from the 4q12 locus into the regulation of EGFR-TKIs resistance in NSCLC. These findings shed new light on the importance of functionally annotating lncRNAs in the GWAS loci and provided insights to declare novel druggable targets, i.e., lncRNAs, which may unlock the therapeutic potential of EGFR-TKIs resistant NSCLC in the clinic.

Targeted Self-Emulsifying Drug Delivery Systems to Restore Docetaxel Sensitivity in Resistant Tumors.

The use of chemotherapeutic agents such as docetaxel (DTX) in anticancer therapy is often correlated to side effects and the occurrence of drug resistance, which substantially impair the efficacy of the drug. Here, we demonstrate that self-emulsifying drug delivery systems (SEDDS) coated with enoxaparin (Enox) are a promising strategy to deliver DTX in resistant tumors. DTX partition studies between the SEDDS pre-concentrate and the release medium (water) suggest that the drug is well retained within the SEDDS upon dilution in the release medium. All SEDDS formulations show droplets with a mean diameter between 110 and 145 nm following dilution in saline and negligible hemolytic activity; the droplet size remains unchanged upon sterilization. Enox-coated SEDDS containing DTX exhibit an enhanced inhibition of cell growth compared to the control on cells of different solid tumors characterized by high levels of FGFR, which is due to an increased DTX internalization mediated by Enox. Moreover, only Enox-coated SEDDS are able to restore the sensitivity to DTX in resistant cells expressing MRP1 and BCRP by inhibiting the activity of these two main efflux transporters for DTX. The efficacy and safety of these formulations is also confirmed in vivo in resistant non-small cell lung cancer xenografts.

Design, Synthesis, and Biological Evaluation of Novel EGFR PROTACs Targeting Del19/T790M/C797S Mutation.

The tertiary epidermal growth factor receptor (EGFR) C797S mutation predominates in the acquired mutational resistance in cancer patients to third-generation EGFR inhibitors. Small-molecule inhibitors targeting the EGFR C797S mutation have been developed with good efficiency. However, these compounds may still induce new EGFR mutations to evade the inhibition pathway. One EGFR protein degrader based on an allosteric inhibitor has shown some benefits of degrading the EGFR L858R/T790M/C797S triple mutant. However, the degrader of the other important triple EGFR mutation Del19/T790M/C797S has not been reported. Here we present the design and synthesis of a series of EGFR proteolysis-targeting chimeras (PROTACs) that can rapidly and potently induce EGFR degradation in Ba/F3 cells expressing the EGFRDel19/T790M/C797S mutant. One representative compound 6h time- and dose-dependently induced EGFR degradation with a DC50 of 8 nM. It also showed good antiproliferation activity (IC50 = 0.02 μM) against Ba/F3-EGFRDel19/T790M/C797S cells. 6h may serve as a lead compound to develop therapeutic agents for the treatment of resistant non-small cell lung cancer patients with EGFR C797S mutants.

LncRNA SNHG12 in extracellular vesicles derived from carcinoma-associated fibroblasts promotes cisplatin resistance in non-small cell lung cancer cells

ABSTRACT Non-small-cell lung cancer (NSCLC) is defined as the most universally diagnosed class of lung cancer. Cisplatin (DDP) is an effective drug for NSCLC, but tumors are prone to drug resistance. The current study set out to evaluate the regulatory effect of long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) in extracellular vesicles (EVs) derived from carcinoma-associated fibroblasts (CAFs) on DDP resistance in NSCLC cells. Firstly, NSCLC cells were treated with EVs, followed by detection of cell activity, IC50 values, cell proliferation and apoptosis, and Cy3-SNHG12. We observed that CAFs-EVs promoted IC50 values and cell proliferation and inhibited apoptosis. In addition, we learned that lncRNA SNHG12 carried by CAFs-EVs into NSCLC facilitated DDP resistance of NSCLC cells. Furthermore, ELAV like RNA binding protein 1 (HuR/ELAVL1) binding to lncRNA SNHG12 and X-linked inhibitor of apoptosis (XIAP) was verified and RNA stability of XIAP was also verified CAFs-EVs promoted RNA stability and transcription of XIAP, while silencing HuR could partially-reverse this promoting effect. Further joint experimentation showed that silencing XIAP partially inhibited DDP resistance in NSCLC cells. Additionally, the tumor growth and the positive rate of Ki67 and HuR were detected, which showed that CAFs-oe-EVs promoted the tumor and the positive rate of Ki67, as well as the levels of lncRNA SNHG12, HuR, and XIAP in vivo. Collectively, our findings indicated that lncRNA SNHG12 carried by CAFs-EVs into NSCLC cells promoted RNA stability and XIAP transcription by binding to HuR, thus augmenting DDP resistance in NSCLC cells.

Patritumab Deruxtecan: Paving the Way for EGFR-TKI–Resistant NSCLC

HER3 is ubiquitously expressed in EGFR-mutant non-small cell lung cancer (NSCLC) irrespective of resistant mechanisms to EGFR tyrosine kinase inhibitors, thus garnering attention as a valuable therapeutic target. In this issue of Cancer Discovery, Jänne and colleagues highlight early clinical data supporting patritumab deruxtecan as a potentially appreciable agent for previously treated EGFR-mutant NSCLC.See related article by Jänne et al., p. 74.

Abstract P042: Phase I/II first-in-human study of TT-10 (A2AB inhibitor) as a single agent in subjects with advanced selected solid tumors

Abstract Immunotherapy has emerged as a potent tool for cancer treatment by activating the immune system to recognize and attack cancer cells. Immune checkpoint blockage has shown notable clinical success in several malignancies such as advanced non-small cell lung cancer, melanoma, advanced kidney cancer and several other indications, as demonstrated with the approval of CTLA-4 and PD-1/PD-L1 checkpoint inhibitors. However, the efficacy of checkpoint inhibitors to date is limited to only a subset of patients, even within the same cancer type. Thus, the continued focus in the cancer immunotherapy field is to identify new immunosuppressive pathways that allow cancer cells to evade immune surveillance. One emerging pathway of interest is the purinergic pathway, in which adenosine is recognized as a key metabolic checkpoint, and high levels of extracellular adenosine have been evidenced to mediate profound tumor resistance. In this first-in-human Phase I/II study, we explore selective inhibition of the adenosine A2A receptor (A2AR) with TT-10, an oral immune-oncology agent, as a treatment option for subjects with select solid tumors. TT-10 is being evaluated as a single agent, in an open-label, non-randomized, multicenter, dose-escalation (phase 1) and dose-expansion (phase 2) study in subjects with renal cell cancer (RCC), castrate resistant prostate cancer (CRPC) and non-small cell lung cancer (NSCLC) (NCT# NCT04969315). Phase 1 (Cohort A) will explore ascending doses of TT-10, taken orally (10, 20, 50, 100, 200 mg, BID), utilizing a 3+3 design in 28-day cycles to identify the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (R2PD). Enrollment will be open to all three indications. Phase 2 will begin once RP2D is determined and will enroll up to 60 subjects, with approximately 15 subjects per cohort (B.) RCC, (C.) CRPC, (D.) NSCLC (E.) Exploratory Tumor Biopsy). The primary objectives of this study are to evaluate the safety, tolerability, and MTD or the RP2D of TT-10 monotherapy. Secondary objectives are to obtain a preliminary estimate of efficacy, evaluate PK and anti-tumor activity of TT-10 monotherapy, with exploratory objectives inclusive of identification of candidate biomarkers and biomarkers that might predict response, as well as, exploring if adenosine signature and/or receptor expression in tumor tissue at baseline might predict response. Subjects must have locally advanced, recurrent or metastatic neoplastic disease that is not curable by currently available, failure to respond to standard therapy or for whom no appropriate therapies are available, and have a life expectancy of ≥ 3 months to be eligible for the trail. Select subjects that would like to participate in Cohort E.) Exploratory Tumor Biopsy must have an accessible tumor for pre and post dose biopsies. Enrollment of phase 1 is expected to start October 2021. Citation Format: Sushant Kumar, Kasim Mookhtiar, Desa Rae Pastore, Brian Schwartz, Vijay Reddy. Phase I/II first-in-human study of TT-10 (A2AB inhibitor) as a single agent in subjects with advanced selected solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P042.

The MEK/ERK/miR-21 Signaling Is Critical in Osimertinib Resistance in EGFR-Mutant Non-Small Cell Lung Cancer Cells.

Simple SummaryOur study provided data that the inhibition of MEK/ERK signaling could overcome Osimertinib resistance both in vitro and in vivo. Mechanistically, MEK inhibitor Trametinib suppressed the tumorigenic properties of NSCLC cells by reducing the generation of CAFs. The trametinib-mediated anti-cancer function was also associated with the significantly suppressed level of miR-21, of which primary targets included PDCD4, as shown in this study and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes.Background: The third-generation epidermal growth factor receptor (EGFR) inhibitor, Osimertinib, is used to treat non-small cell lung cancer (NSCLC) patients with tyrosine kinase inhibitor (TKI) resistance caused by acquired EGFR T790M mutation. However, patients eventually develop resistance against Osimertinib with mechanisms not yet fully clarified. Activated alternative survival pathways within the tumor cells and cancer-associated fibroblasts (CAFs) have been proposed to contribute to Osimertinib resistance. MET and MEK inhibitors may overcome EGFR-independent resistance. Another acquired resistance mechanism of EGFR-TKI is the up-regulation of the RAS/RAF/MEK/ERK signaling pathway, which is the key to cell survival and proliferation; this may occur downstream of various other signaling pathways. In this report, we reveal the possible regulatory mechanism and inhibitory effect of the MEK inhibitor trametinib applied to MEK/ERK/miR-21 axis and PDCD4 in Osimertinib resistance. We found a possible regulatory role of PDCD4 in ERK signaling. PDCD4 is a new type of tumor suppressor that has multiple functions of inhibiting cell growth, tumor invasion, metastasis, and inducing apoptosis. Previous bioinformatics analysis has confirmed that PDCD4 contains the binding site of miR-21 and acts as a tumor suppressor in the regulation of various processes associated with the development of cancer, including cell proliferation, invasion, metastasis, and neoplastic transformation. Based on the above analysis, we hypothesized that the tumor suppressor PDCD4 is one of the effective inhibitory targets of miR-21-5p. Methods: The expression between EGFR and ERK2 in lung adenocarcinoma was evaluated from the TCGA database. Osimertinib-sensitive and resistant NSCLC cells obtained from patients were used to co-culture with human lung fibroblasts (HLFs) to generate CAF cells (termed CAF_R1 and CAF_S1), and the functional roles of these CAF cells plus the regulatory mechanisms were further explored. Then, MEK inhibitor Trametinib with or without Osimertinib was applied in xenograft model derived from patients to validate the effects on growth inhibition of Osimertinib-resistant NSCLC tumors. Result: ERK2 expression correlated with EGFR expression and higher ERK2 level was associated with worse prognosis of patients and Osimertinib resistance. CAFs derived from Osimertinib-resistant cells secreted more IL-6, IL-8, and hepatocyte growth factor (HGF), expressed stronger CAF markers including α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP) plus platelet-derived growth factor receptor (PDGFR), and enhanced stemness and Osimertinib resistance in NSCLC cells. Meanwhile, increased MEK/ERK/miR-21 expressions were found in both CAFs and NSCLC cells. MEK inhibitor Trametinib significantly abrogated the abovementioned effects by modulating β-catenin, STAT3, and ERK. The xenograft model showed combining Osimertinib and Trametinib resulted in the most prominent growth inhibition of Osimertinib-resistant NSCLC tumors. Conclusions: Our results suggested that MEK/ERK/miR-21 signaling is critical in Osimertinib resistance and CAF transformation of NSCLC cells, and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes.

The Role of EREG/EGFR Pathway in Tumor Progression.

Aberrant activation of the epidermal growth factor receptor (EGFR/ERBB1) by erythroblastic leukemia viral oncogene homolog (ERBB) ligands contributes to various tumor malignancies, including lung cancer and colorectal cancer (CRC). Epiregulin (EREG) is one of the EGFR ligands and is low expressed in most normal tissues. Elevated EREG in various cancers mainly activates EGFR signaling pathways and promotes cancer progression. Notably, a higher EREG expression level in CRC with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) is related to better efficacy of therapeutic treatment. By contrast, the resistance of anti-EGFR therapy in CRC was driven by low EREG expression, aberrant genetic mutation and signal pathway alterations. Additionally, EREG overexpression in non-small cell lung cancer (NSCLC) is anticipated to be a therapeutic target for EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, recent findings indicate that EREG derived from macrophages promotes NSCLC cell resistance to EGFR-TKI treatment. The emerging events of EREG-mediated tumor promotion signals are generated by autocrine and paracrine loops that arise from tumor epithelial cells, fibroblasts, and macrophages in the tumor microenvironment (TME). The TME is a crucial element for the development of various cancer types and drug resistance. The regulation of EREG/EGFR pathways depends on distinct oncogenic driver mutations and cell contexts that allows specific pharmacological targeting alone or combinational treatment for tailored therapy. Novel strategies targeting EREG/EGFR, tumor-associated macrophages, and alternative activation oncoproteins are under development or undergoing clinical trials. In this review, we summarize the clinical outcomes of EREG expression and the interaction of this ligand in the TME. The EREG/EGFR pathway may be a potential target and may be combined with other driver mutation targets to combat specific cancers.

Identification of lncRNA-miRNA-mRNA Networks Linked to Non-small Lung Cancer Resistance to Inhibitors of Epidermal Growth Factor Receptor.

Background: Tyrosine kinase inhibitors that act against epidermal growth factor receptor (EGFR) show strong efficacy against non-small cell lung cancer (NSCLC) involving mutated EGFRs. However, most such patients eventually develop resistance to EGFR-TKIs. Numerous researches have reported that messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) may be involved in EGFR-TKI resistance, but the comprehensive expression profile and competitive endogenous RNA (ceRNA) regulatory network between mRNAs and ncRNAs in EGFR-TKI resistance of NSCLC are incompletely known. We aimed to define a ceRNA regulatory network linking mRNAs and non-coding RNAs that may mediate this resistance. Methods: Using datasets GSE83666, GSE75309 and GSE103352 from the Gene Expression Omnibus, we identified long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs differentially expressed between NSCLC cells that were sensitive or resistant to EGFR-TKIs. The potential biological functions of the corresponding differentially expressed genes were analyzed based KEGG pathways. We combined interactions among lncRNAs, miRNAs and mRNAs in the RNAInter database with KEGG pathways to generate transcriptional regulatory ceRNA networks associated with NSCLC resistance to EGFR-TKIs. Kaplan-Meier analysis was used to assess the ability of core ceRNA regulatory sub-networks to predict the progression-free interval and overall survival of NSCLC. The expression of two core ceRNA regulatory sub-networks in NSCLC was validated by quantitative real-time PCR. Results: We identified 8,989 lncRNAs, 1,083 miRNAs and 3,191 mRNAs that were differentially expressed between patients who were sensitive or resistant to the inhibitors. These DEGs were linked to 968 biological processes and 31 KEGG pathways. Pearson analysis of correlations among the DEGs of lncRNAs, miRNAs and mRNAs identified 12 core ceRNA regulatory sub-networks associated with resistance to EGFR-TKIs. The two lncRNAs ABTB1 and NPTN with the hsa-miR-150–5p and mRNA SERPINE1 were significantly associated with resistance to EGFR-TKIs and survival in NSCLC. These lncRNAs and the miRNA were found to be down-regulated, and the mRNA up-regulated, in a resistant NSCLC cell line relative to the corresponding sensitive cells. Conclusion: In this study, we provide new insights into the pathogenesis of NSCLC and the emergence of resistance to EGFR-TKIs, based on a lncRNA-miRNA-mRNA network.

Virtual screening of Osimertinib and Dacomitinib Analogues with potential activity on EGFR (T790M and l858R Mutations) for non-small cell lung cancer treatment


 
 
 Introduction: Tyrosine kinase inhibitors (TKIs) drugs act on epidermal growth factor (EGFR) receptors to treat Non-small cell lung cancer (NSCLC). However, mutations on EGFR receptors T790M and L858R allow just a global response rate (GRR) of 80% with Osimertinib, while Erlotinib and Gefitinib only 10%.
 Objective: To identify promising molecules analogues to tyrosine kinase inhibitor (TKIs) drugs with the potential capacity to bind the native and mutated EGFR receptor (T790M and L858R) to avoid mutational resistance in NSCLC.
 Methods: Virtual screening by molecular docking between analogues of Osimertinib (DB09330) and Dacomitinib (DB11963) drugs retrieved of DRUGBANK database and receptors of native EGFR and mutated on L585R and T790M obtained of Protein Data Bank was performed, using PyRx software. Finally, toxicological prediction was made using GUSAR.
 Results: Analogues studied, DB03878, DB04739, DB07280 and DB06876 achieved significant affinity (-9,1 y -8,3 Kcal/mol) on mutated T790M EGFR compared with osimertinib (-7,6 Kcal/mol). Similarly, DB08091, DB08730, DB07220 and DB06920 achieved significant affinity (between 9.4 and -8.9 Kcal/mol) on L858R EGFR mutated compared with dacomitinib (-7.0 Kcal/mol). Overall, there were predominance of Van der Waals forces and links π-alkyl. Also, two analogues were safe with category IV according to predictions, DB08730 and DB03878.
 Conclusions: Eight TKI analogues showed superior binding energy over EGFR compared to reference drugs. According to toxicological predictions only 2 analogues were selected as promising TKI-type safe candidates for the treatment of resistant NSCLC.
 
 

Cisplatin resistance of NSCLC cells involves upregulation of visfatin through activation of its transcription and stabilization of mRNA

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment.

Qing-Kai-Ling Injection Acts Better Than Shen-Fu Injection in Enhancing the Antitumor Effect of Gefitinib in Resistant Non-Small Cell Lung Cancer Models.

Patients with EGFR gene mutation often obtain de novo resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or develop secondary resistance to EGFR-TKIs after taking EGFR-TKI therapy. Traditional Chinese medicine (TCM) with different treatment principles, in combination with EGFR-TKIs, plays an important role in the treatment of cancers including resistant non-small cell lung cancer (NSCLC). However, inappropriate use of TCM herbs may induce resistance to gefitinib. Therefore, it is of a great value to evaluate which TCM treatment principle should be combined with EGFR-TKIs, and which one should be avoided, and find out the potential mechanisms. The lentiviral transfection assay was used for overexpression of PIK3CA mutation gene in PC-9 cells to construct PC-9-PIK3CA-mutation (PC-9-PIK3CA-M) cells. Cell proliferation, apoptosis, and the expression of EGFR/PI3K/AKT and EGFR/RAS/RAF/ERK in PC-9-PIK3CA-M and H1975 cells treated by the typical cooling-heat drug, Qing-kai-ling (QKL) and Tan-re-qing (TRQ), or the typical warming-yang drug, Shen-fu (SF) and gefitinib treatment, were detected by MTT, Annexin V/PI double labeling, and Western blot assays, respectively. Tumor xenograft and immunohistochemistry experiments were carried out to confirm the in vitro findings. PC-9-PIK3CA-M cells were less sensitive to gefitinib, when compared with PC-9 cells. QKL injection and TRQ injection, not SF injection, combined with gefitinib induced significantly increased cell growth inhibition and apoptosis in PC-9-PIK3CA-M and H1975 cells. SF injection antagonized the effect of gefitinib in promoting cancer cell apoptosis. QKL injection and TRQ injection increased the sensitivity of gefitinib by inhibiting the phosphorylation of AKT or ERK in H1975 and PC-9-PIK3CA-M cells. Similar findings were observed in vivo in H1975 xenograft mouse model. QKL and TRQ, with cooling-heat TCM treatment principle, should be combined with gefitinib in the treatment of NSCLC. Furthermore, warming-yang drug SF should be avoided to be used together with EGFR-TKIs.

P68.02 Losartan Enhances Lung Squamous Cell Carcinoma’s Sensitivity to Cisplatin Treatment By Promoting Mesenchymal to Epithelial Transformation

Angiotensin II type 1 receptor (AT1R) overexpression has been associated with poor prognosis in patients with some solid organ cancers. The angiotensin II/AT1R axis is closely related to tumor proliferation, angiogenesis, invasion and metastasis. Platinum resistant non-small cell lung cancer shows significantly higher type I angiotensin II receptor (AGTR1) mRNA expression than their corresponding parent cells. Addition of angiotensin II receptor blocker (ARB) to platinum-based first-line chemotherapy may contribute to prolonged survival in patients with locally advanced squamous cell lung cancer. The TCGA database was analyzed for expression of AGTR1 in tumor tissue from resected lung squamous cell carcinoma (N=497) and correlated with overall survival. Multiple human lung squamous cancer cell lines (SK-MES-1, H520, H1703) with primary mesenchymal phenotype [Fig. 1D] were evaluated invitro for AGTR1 expression [Fig. 1C] and treated with losartan or combination losartan and cisplatin to assess cell survival and EMT expression by western blot. Patients with stage I, II, and III lung squamous cell carcinoma (LUSC) demonstrated improved overall survival with low expression of AGTR1(P =0.0056) [Fig 1A]. When stratified by stage, stage II exhibited low AGTR1 expression and was associated with a better prognosis (P =0.0032). Expression of AGTR1 positively correlated with EMT expression of Vimentin (r=0.57, P <0.0001), ZEB1 (r=0.43, P <0.0001) and ZEB2 (r=0.59, P <0.0001) [Fig 1B]. SK-MES-1, H520 and H1703 express abundant AGTR1. Vimentin expression of SK-MES-1 decreased with losartan treatment [Fig. E] in a dose dependent fashion (0, 0.1, 0.2, 0.5μM). E-cad expression of H520 increased and Vimentin expression decreased with increasing losartan treatment [Fig. F]. Compared to cisplatin treatment alone, combination of Losartan and cisplatin significantly improved the cytotoxic effect in SK-MES-1(P=0.0006), H520 (P =0.0053) and H1703(P =0.0234) [Fig. G]. AGTR1 expression correlates with survival outcomes of lung squamous cell carcinoma in humans and is associated with a more mesenchymal phenotype in vivo and in vitro. Losartan treatment appears to promote a mesenchymal to epithelial transformation and enhances the anti-tumor effect of chemotherapy (cisplatin) compared to chemotherapy alone in lung squamous cancer cell.

P71.04 MiR-1243 Increases Radiosensitivity in EGFR-TKI Resistant Non-Small Cell Lung Cancer Cells

Acquired resistance is an important issue which limits the application of EGFR-TKI in advanced non-small cell lung cancer (NSCLC). Our data showed nearly half of EGFR-mutant patients develop local relapse or limited metastases at disease progression. Previous studies have confirmed the value of radiotherapy in advanced lung cancer patients. EGFR-TKI resistant cells with T790M exhibited enhanced sensitivity to radiation. Studies have shown that microRNAs are involved in DNA damage repair mechanisms and thus affect the sensitivity of radiotherapy. Therefore, it is important to explore the detailed mechanism on miRNAs regulating radiosensitivity in EGFR-TKI resistant NSCLC. We performed miRNA microarray to screen miRNAs differentially expressed in the paired gefitinib-sensitivity PC-9 cells and gefitinib-resistant PC-9-GR cells (T790M mutation). Furthermore, we use bioinformatics to screen out target proteins and verify the expression differences of these target proteins. The proliferation of lung cancer cells was determined by CCK-8 assay. Proteins and the phosphorylation of proteins were evaluated by western blot assay. Colony formation assay showed PC-9-GR cells were more sensitive to radiation than PC-9 cells. MiRNA microarray showed 156 differentially expressed miRNAs between PC-9 and PC-9-GR cells. miR-1243 was in the top10 differentially expressed miRNAs and it was up-regulated in PC-9-GR cells. Stable knock-down miR-1243 PC-9-GR cells were further constructed using, termed as PC-9-GR-miR1243KD. CCK-8 assay showed similar proliferation kinetics between PC-9-GR and PC-9-GR-miR1243KD. It was found that PC-9-GR-miR1243KD cells showed a significant reduction in clone formation compared with PC-9-GR and PC-9-GR-NC cells after radiation, indicating higher radiosensitivity. Bioinformatics showed miR-1243 may function as a tumor oncogene to regulate apoptosis of gefitinib resistant cell with T790M mutation. SPOCK1 was one of its targeting gene. Western blots showed SPOCK1 was high expression in PC-9-GR-miR1243KD cells. This study found novel molecular mechanism might correlate with increase of radiosensitivity in EGFR-TKI resistant NSCLC cells. It could be a new therapeutic strategy for patients in advanced NSCLC to aid expansion of the effectiveness of TKI treatment through radiotherapy.

RESTRICTING EXTRACELLULAR CA2+ ON GEFITINIB RESISTANT NON-SMALL CELL LUNG CANCER CELLS REVERSES ALTERED EPIDERMAL GROWTH FACTOR MEDIATED CA2+ RESPONSE, WHICH CONSEQUENTLY ENHANCES GEFITINIB SENSITIVITY

TOPIC: Lung Cancer TYPE: Original Investigations PURPOSE: Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. METHODS: We analyzed the intracellular calcium ([Ca2+]i) signals in PC-9 cells and PC-9/GR (Gefitinib Resistant) cells under following conditions. We observed hEGF (200ng/ml)-mediated [Ca2+]i oscillations response. Calcium influx, endoplasmic reticulum (ER) calcium contents, hEGF (200 ng/ml)-mediated [Ca2+]i oscillations, and effects of inhibitors were studied in absence of extracellular calcium ([Ca2+]e). We compared gefitinib-induced cytotoxicity and cell viability according to the presence or absence of [Ca2+]e. RESULTS: NSCLC PC-9 cells and gefitinib resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. CONCLUSIONS: These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity. CLINICAL IMPLICATIONS: resistant mechanism in EGFR TKI DISCLOSURES: no disclosure on file for Misung Kim; no disclosure on file for Sohui Kim; no disclosure on file for Minsuk Kim; No relevant relationships by Seihoon Yang, source=Web Response

MiR-519d-3p enhances the sensitivity of non-small-cell lung cancer to tyrosine kinase inhibitors.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Tyrosine kinase inhibitors (TKIs) are currently the most effective chemotherapy for NSCLC. However, most cancer patients develop TKI resistance at tumor relapse stage. We firstly measured the expression change of miR-519d-3p in TKI resistance NSCLC cells. We then ectopically expressed miR-519-3p in TKI resistant cells to study its functional impact on cell proliferation, migration, invasion and cell sensitivity to gefitinib. The downstream target of miR-519-3p was identified by bioinformatics and validated in luciferase reporter assay and western blotting analysis. We also studied the reversing effect of the candidate target in NSCLC cells expressing miR-519d-3p. Lastly, we compared the miR-519d-3p level in NSCLC patients with good or poor response to gefitinib. miR-519d-3p level was downregulated in TKI resistant NSCLC cells. The restoration of miR-519d-3p in these NSCLC cells inhibited cell proliferation, invasion and migration; enhanced cell sensitivity to gefitinib. EPAS1 was identified and validated as downstream target of miR-519d-3p. Co-expressing EPAS1 antagonized the inhibitory effect of miR-519d-3p on NSCLC cells. MiR-519d-3p was downregulated in NSCLC patients with poor response to gefitinib. Targeting miR-519d-3p/EPAS1 axis may provide alternative treatment for TKI-resistant NSCLC.

KCa channel blockers increase effectiveness of the EGF receptor TK inhibitor erlotinib in non-small cell lung cancer cells (A549)

Non-small cell lung cancer (NSCLC) has a poor prognosis with a 5 year survival rate of only ~ 10%. Important driver mutations underlying NSCLC affect the epidermal growth factor receptor (EGFR) causing the constitutive activation of its tyrosine kinase domain. There are efficient EGFR tyrosine kinase inhibitors (TKIs), but patients develop inevitably a resistance against these drugs. On the other hand, KCa3.1 channels contribute to NSCLC progression so that elevated KCa3.1 expression is a strong predictor of poor NSCLC patient prognosis. The present study tests whether blocking KCa3.1 channels increases the sensitivity of NSCLC cells towards the EGFR TKI erlotinib and overcomes drug resistance. mRNA expression of KCa3.1 channels in erlotinib-sensitive and -resistant NSCLC cells was analysed in datasets from Gene expression omnibus (GEO) and ArrayExpress. We assessed proliferation and migration of NSCLC cells. These (live cell-imaging) experiments were complemented by patch clamp experiments and Western blot analyses. We identified three out of four datasets comparing erlotinib-sensitive and -resistant NSCLC cells which revealed an altered expression of KCa3.1 mRNA in erlotinib-resistant NSCLC cells. Therefore, we evaluated the combined effect of erlotinib and the KCa3.1 channel inhibition with sencapoc. Erlotinib elicits a dose-dependent inhibition of migration and proliferation of NSCLC cells. The simultaneous application of the KCa3.1 channel blocker senicapoc increases the sensitivity towards a low dose of erlotinib (300 nmol/L) which by itself has no effect on migration and proliferation. Partial erlotinib resistance can be overcome by KCa3.1 channel blockade. The sensitivity towards erlotinib as well as the potentiating effect of KCa3.1 blockade is further increased by mimicking hypoxia. Our results suggest that KCa3.1 channel blockade may constitute a therapeutic concept for treating NSCLC and overcome EGFR TKI resistance. We propose that this is due to complementary mechanisms of action of both blockers.

JARID2 promotes stemness and cisplatin resistance in non-small cell lung cancer via upregulation of Notch1

Increased stemness is causally linked to development of drug resistance in cancers. JARID2 is a member of the Jumonji family of proteins and regulates differentiation of embryonic stem cells. However, the role of JARID2 in lung cancer stemness and drug resistance is still unclear. In this study, we investigated the expression of JARID2 in parental and cisplatin (CDDP) resistant non-small cell lung cancer (NSCLC) cells. The function of JARID2 in modulating CDDP sensitivity of NSCLC cells was determined. It was found that JARID2 is upregulated in CDDP resistant NSCLC cells, which depends on SOX2 expression. JARID2 overexpression promotes CDDP resistance in NSCLC cells, whereas JARID2 depletion restores CDDP sensitivity in CDDP resistant NSCLC cells. Moreover, JARID2 overexpression enhances cancer stem cell-like properties in NSCLC cells, which is coupled with increased expression of cancer stem cell markers. Mechanistically, JARID2-induced stemness and CDDP resistance is mediated by upregulation of Notch1. In clinical settings, high expression of JARID2 is significantly associated with advanced TNM stage, shorter overall survival, and poor chemotherapeutic response. These findings point toward an important role of JARID2 in CDDP resistance and stemness of NSCLC and provide a promising target for overcoming CDDP resistance.

Exosomal miR-136-5p Derived from Anlotinib-Resistant NSCLC Cells Confers Anlotinib Resistance in Non-Small Cell Lung Cancer Through Targeting PPP2R2A.

BackgroundAnlotinib resistance is a challenge for advanced non-small cell lung cancer (NSCLC). Understanding the underlying mechanisms against anlotinib resistance is of great importance to improve prognosis and treatment of patients with advanced NSCLC.MethodsRT-qPCR assay was used to assess the level of miR-136-5p in anlotinib-resistant NSCLC cells and exosomes derived from anlotinib-resistant NSCLC cells. In addition, miR-136-5p level in tumor tissues from patients who exhibited a poor response to anlotinib therapy and patients who were therapy naïve or patients who exhibited a positive response to anlotinib therapy was detected by RT-qPCR assay.ResultsIn this study, we found that high levels of plasma exosomal miR-136-5p is correlated with clinically poor anlotinib response. In addition, anlotinib-resistant NSCLC cells promoted parental NSCLC cell proliferation via transferring functional miR-136-5p from anlotinib-resistant NSCLC cells to parental NSCLC cells via exosomes. Moreover, exosomal miR-136-5p could endow NSCLC cells with anlotinib resistance by targeting PPP2R2A, leading to the activation of Akt pathway. Furthermore, miR-136-5p antagomir packaging into anlotinib-resistant NSCLC cell-derived exosomes functionally restored NSCLC cell anlotinib sensitivity in vitro. Animal studies showed that A549/anlotinib cell-derived exosomal miR-136-5p agomir promoted A549 cell anlotinib resistance in vivo.ConclusionCollectively, these findings indicated that anlotinib-resistant NSCLC cell-derived exosomal miR-136-5p confers anlotinib resistance in NSCLC cells by targeting PPP2R2A, indicating miR-136-5p may act as a potential biomarker for anlotinib response in NSCLC.

Arsenic sulfide reverses cisplatin resistance in non-small cell lung cancer in vitro and in vivo through targeting PD-L1.

BackgroundRecent studies have found that programmed death ligand 1 (PD‐L1) might be involved in chemotherapy resistance in non‐small cell lung cancer (NSCLC). Arsenic sulfide (As4S4) has been recognized to have antitumor activities and enhance the cytotoxic effect of chemotherapy drugs. In this study, we aimed to verify the relationship between PD‐L1 and cisplatin (DDP) resistance and identify whether As4S4 could reverse DDP resistance through targeting PD‐L1 in NSCLC.MethodsThe effect of As4S4 and DDP on cell proliferation and apoptosis was investigated in NSCLC cell lines. The expression of p53 and PD‐L1 proteins was measured by western blotting analysis. The levels of miR‐34a‐5p, miR‐34a‐3p and PD‐L1 in cells were measured by real‐time qPCR analysis. Mouse xenograft models were established by inoculation with A549/DDP (DDP‐resistant) cells.ResultsDepletion of PD‐L1 inhibited DDP resistance in A549/DDP and H1299/DDP cells. As4S4 was capable of sensitizing A549/DDP cells to DDP by enhancing apoptosis. As4S4 upregulated p53 expression and downregulated PD‐L1 expression in A549/DDP cells. As4S4 increased miR‐34a‐5p level in A549/DDP cells. Inhibition of p53 by PFT‐α partially restored the levels of PD‐L1 and miR‐34a‐5p. Pretreatment with PFT‐α suppressed the apoptosis rate induced by cotreatment of As4S4 and DDP in A549/DDP cells. Cotreatment of DDP and As4S4 notably reduced the tumor size when compared with DDP treatment alone in vivo.ConclusionsUpregulation of PD‐L1 was correlated with DDP resistance in NSCLC cells. Mechanistic analyses indicated that As4S4 might sensitize NSCLC cells to DDP through targeting p53/miR‐34a‐5p/PD‐L1 axis.