Sensitive Non-small Cell Lung Cancer Cell Lines Research Articles

The Role of Serine Protease 8 in Mediating Gefitinib Resistance in Non-small Cell Lung Cancer.

This investigation aims to explore the expression levels of serine protease 8 (PRSS8) in gefitinib-resistant Non-Small Cell Lung Cancer (NSCLC) cell lines (PC9/GR) and elucidate its mechanism of action. We measured PRSS8 expression in gefitinib-resistant (PC9/GR) and sensitive (PC9) NSCLC cell lines using Western blot analysis. PRSS8-specific small interfering RNA (PRSS8-siRNA), a recombinant plasmid, and a corresponding blank control were transfected into PC9/GR cells. Subsequently, Western blot analyses were conducted to assess the expression levels of PRSS8, phosphorylated AKT (p-AKT), AKT, phosphorylated mTOR (p-mTOR), mTOR, and various apoptosis-related proteins within each group. Additionally, a cell proliferation assay utilizing Cell Counting Kit-8 (CCK8) was performed on each group treated with gefitinib. PRSS8 expression was markedly higher in PC9/GR cells compared to PC9 cells (p < 0.05). The group treated with PRSS8-siRNA exhibited significantly reduced protein expression levels of PRSS8, p-AKT, p-mTOR, β-catenin, and BCL-2 compared to the control siRNA (Con-siRNA) group, whereas expressions of Caspase9 and Bax were significantly increased. In the untransfected PC9/GR cells, protein expressions of PRSS8, p-AKT, pmTOR, and BCL-2 were significantly elevated when compared with the plasmid-transfected group, which also showed a significant reduction in Bax expression. The proliferative activity of the PRSS8-siRNA group postgefitinib treatment was significantly diminished at 24, 48, and 72 hours in comparison to the Con-siRNA group. The findings indicate that PRSS8 contributes to the acquisition of resistance to gefitinib in NSCLC, potentially through regulation of the AKT/mTOR signaling pathway.

Chemosensitizing effect of pentoxifylline in sensitive and multidrug-resistant non-small cell lung cancer cells.

Aim: Multidrug resistance (MDR) is frequent in non-small cell lung cancer (NSCLC) patients, which can be due to its fibrotic stroma. This work explores the combination of pentoxifylline, an anti-fibrotic and chitinase 3-like-1 (CHI3L1) inhibitor drug, with conventional chemotherapy to improve NSCLC treatment. Methods: The effect of pentoxifylline in the expression levels of P-glycoprotein (P-gp), CHI3L1 and its main downstream proteins, as well as on cell death, cell cycle profile, and P-gp activity was studied in two pairs of sensitive and MDR counterpart NSCLC cell lines (NCI-H460/NCI-H460/R and A549/A549-CDR2). Association studies between CHI3L1 gene expression and NSCLC patients' survival were performed using The Cancer Genome Atlas (TCGA) analysis. The sensitizing effect of pentoxifylline to different drug regimens was evaluated in both sensitive and MDR NSCLC cell lines. The cytotoxicity of the drug combinations was assessed in MCF10A non-tumorigenic cells. Results: Pentoxifylline slightly decreased the expression levels of CHI3L1, β-catenin and signal transducer and activator of transcription 3 (STAT3), and caused a significant increase in the G1 phase of the cell cycle in both pairs of NSCLC cell lines. A significant increase in the % of cell death was observed in the sensitive NCI-H460 cell line. TCGA analysis revealed that high levels of CHI3L1 are associated with low overall survival (OS) in NSCLC patients treated with vinorelbine. Moreover, pentoxifylline sensitized both pairs of sensitive and MDR NSCLC cell lines to the different drug regimens, without causing significant toxicity to non-tumorigenic cells. Conclusion: This study suggests the possibility of combining pentoxifylline with chemotherapy to increase NSCLC therapeutic response, even in cases of MDR.

Abstract 4675: Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [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 4675.

Meta-analysis of microarray data to determine gene indicators involved in cisplatin resistance in non-small cell lung cancer.

Lung cancer is a major cause of cancer-related mortality worldwide, with a 5-year survival rate of approximately 22%. Cisplatin is one of the standard first-line chemotherapeutic agents for non-small cell lung cancer (NSCLC), but its efficacy is often limited by the development of resistance. Despite extensive research on the molecular mechanisms of chemoresistance, the underlying causes remain elusive and complex. We analyzed three microarray datasets to find the gene signature and key pathways related to cisplatin resistance in NSCLC. We compared the gene expression of sensitive and resistant NSCLC cell lines treated with cisplatin. We found 274 DEGs, including 111 upregulated and 163 downregulated genes, in the resistant group. Gene set enrichment analysis showed the potential roles of several DEGs, such as TUBB2B, MAPK7, TUBAL3, MAP2K5, SMUG1, NTHL1, PARP3, NTRK1, G6PD, PDK1, HEY1, YTHDF2, CD274, and MAGEA1, in cisplatin resistance. Functional analysis revealed the involvement of pathways, such as gap junction, base excision repair, central carbon metabolism, and Notch signaling in the resistant cell lines. We identified several molecular factors that contribute to cisplatin resistance in NSCLC cell lines, involving genes and pathways that regulate gap junction communication, DNA damage repair, ROS balance, EMT induction, and stemness maintenance. These genes and pathways could be targets for future studies to overcome cisplatin resistance in NSCLC.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 428.

Abstract 1088: Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity

Abstract It is anticipated that there will be 235,760 cases and 131,880 deaths due to lung cancer in the US in 2021. Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are currently being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. Sphingosine kinase 2 (SK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P), promoting cancer cell proliferation. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SK2. However, the role of SK2 in TKI resistance is not established. To determine if SK2 causes TKI resistance, we have studied its role in NSCLC and TKI resistance. We hypothesize that SK2 may be involved in inducing TKI- resistance in NSCLC cells and is positively correlated with tumor progression. We studied SK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. Our study investigated SK2 expression in drug resistant cells versus parental cells using qPCR and Western Blotting. The effects of SK2 inhibitor, Opaganib, on NSCLC cell proliferation was studied through an MTT assay. We have also studied its expression in early- and late-stage tumors using Immunohistochemistry (IHC). In drug resistant cells, qPCR studies showed that SK2 was upregulated in H2170 by 1.30-fold (ER) and 1.5-fold (OR); in H358 by 1.36-fold (ER) and 1.17-fold (OR); in PC9 by 3.3-fold (ER) and 1.8-fold (OR) and in H3255 by 4.3-fold (OR) compared to parental cells. A western blotting experiment demonstrated that SK2 was upregulated significantly by 1.7-fold and 1.5-fold in H358OR and H2170OR cells, respectively, compared to parental cells. These results suggest that SK2 may play a role in TKI resistance, as displayed by SK2 upregulation in TKI-resistant cells. MTT assay data showed 71% inhibition of cell growth by Osimertinib and SK2 inhibitor in H2170OR. The inhibition due to the combination of the drug and inhibitor was more than additive by 9% compared to the drug and inhibitor individually. This indicates SK2s potential to overcome TKI- Resistance. To analyze SK2 expression in cancer progression, IHC experiments were performed on 63 early and 48 late stage lung cancer patient sections. We observed upregulation of SK2 in late- stage lung cancer (p&amp;lt;0.05), indicating its role in cancer progression. In conclusion, SK2 may be a key biomarker in combating TKI-resistance and play a role in cancer progression in NSCLC. Thus, SK2 may be a promising candidate for molecular targeted therapy against NSCLC. Citation Format: Namrata Umeshchandra Dube, Katie Lam, Neelu Puri. Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity [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 1088.

MicroRNA expression profiling and biomarker validation in treatment-naïve and drug resistant non-small cell lung cancer

BackgroundIn the absence of targetable mutations or immune checkpoints, cisplatin-doublet chemotherapy remains the standard of care in non-small cell lung cancer (NSCLC). Drug resistance has however become a significant clinical challenge. Exploring a role for small non-coding microRNAs (miRNA) as biomarker candidates in cisplatin resistant (CisR) lung cancer is lacking and warrants further investigation.MethodsmiRNA expression profiling was assessed in a panel of cisplatin sensitive and resistant NSCLC cell lines and validated by qPCR. Modulation of altered miRNAs was studied using antagomiRs and pre-miRs while functional assays were used to assess cisplatin response. The translational relevance of these miRNAs as potential biomarkers was assessed in serum and matched normal and tumour lung tissues from chemo-naïve NSCLC patients, in addition to xenograft formalin-fixed paraffin-embedded (FFPE) tumours derived from cisplatin sensitive and resistant cell lines.ResultsDifferential expression of a 5-miR signature (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286) demonstrated their ability to distinguish between normal and tumour lung tissue and between NSCLC histologies. In squamous cell carcinoma (SqCC), tissue miRNA expression was associated with poor survival. miR-4286 showed promise as a blood-based diagnostic biomarker that could distinguish between adenocarcinoma and SqCC histologies. In a xenograft model of cisplatin resistance, using 7-9 week old female NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl), a 5-miRNA panel showed altered expression between sensitive and resistant tumours.ConclusionsThis study identified a panel of miRNAs which may have diagnostic and prognostic potential as novel biomarkers in lung cancer and furthermore, may have a predictive role in monitoring the emergence of resistance to cisplatin.

Dissecting the m6A methylation affection on afatinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Afatinib is the first-line anti-cancer agent for treatment of NSCLC. However, unexpected resistance has been a major obstacle for its clinical efficacy. In this study, we dissected afatinib resistance from the perspective of N6-Methyladenosine (m6A) modification. First, we depicted the m6A modification profiles for the afatinib resistant and sensitive NSCLC cell lines (H1299 and A549). We found that the sum enrichment scores of the resistant cell line (H1299) was much higher than that of the sensitive cell line (A549). Next, we identified the functionally m6A-modified genes, which were the intersection of the differentially m6A methylated genes and the differentially expressed genes between H1299 and A549, as well as negative correlation between m6A modification levels and gene expression levels. In addition, functional enrichment analysis of the functionally m6A-modified genes indicated that m6A methylation might modify cell cycle to affect afatinib response. Furthermore, the functionally m6A-modified genes were over-represented in the putative drug resistance-associated genes and the FDA-approved drug targets, and had significantly higher average degree and clustering coefficient than other genes in protein-protein interaction (PPI) network. We also identified five network modules, which were all related to drug resistance functions. Finally, survival analysis demonstrated that m6A modification could affect prognosis of NSCLC patients. In conclusion, we conducted a first attempt to dissect m6A methylation affection on afatinib resistance in NSCLC, and brought inspiration for the study of epigenetic roles in drug resistance.

An effective drug sensitizing agent increases gefitinib treatment by down regulating PI3K/Akt/mTOR pathway and up regulating autophagy in non-small cell lung cancer

Gefitinib is one of commonly used first-line treatment options for patients with positive EGFR mutation in non-small cell lung cancer (NSCLC). However, most patients with gefitinib treatment relapse over time due to the loss of drug sensitivity. Compound Kushen injection (CKI) has been used to treat lung cancer, including EGFR-mutated NSCLC. In this report, we examined the anti-cancer and drug sensitivity increased activities of CKI in gefitinib less sensitive NSCLC cell lines H1650 and H1975. Bioinformatics analysis was applied to uncover gene regulation and molecular mechanisms of CKI. Our results indicated that when associating with gefitinib in a dose-dependent fashion, CKI demonstrated the ability to inhibit the proliferation and to increase the sensitivity to gefitinib treatment in gefitinib less sensitive cell lines. This could be the results of down regulation of the PI3K/Akt/mTOR pathway and up regulation of autophagy, which were identified as the potential primary targets of CKI to increase gefitinib treatment effect.

Abstract 832: An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC

Abstract Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p&amp;lt;0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models. Citation Format: Louise A. Koopman, Maarten L. Janmaat, Kirstine Jacobsen, Mikkel Green Terp, Elke Gresnigt-van den Heuvel, Ulf Forssman, Andreas Lingnau, Paul W. Parren, Henrik Ditzel, Esther C. Breij. An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 832.

Targeting the cancer stem cell marker, aldehyde dehydrogenase 1, to circumvent cisplatin resistance in NSCLC.

Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors in vitro. Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of cisplatin.

Abstract 3142: Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the world. Non-small cell lung cancer (NSCLC) accounts for ~85% of the cases. NSCLC patients frequently harbor causal gene mutations. Epidermal Growth Factor Receptor (EGFR), an NSCLC causal gene, is mutated in 10-35% of NSCLC patients. Patients with EGFR activating mutation are treated with a tyrosine-kinase inhibitor, Erlotinib, specifically targeting EGFR. However, most patients develop Erlotinib resistance within a year. Although multiple mechanisms are involved in the development of Erlotinib resistance, the role of microRNAs in mediating Erlotinib resistance is largely unexplored. MicroRNAs (miRNAs) are small non-coding RNAs that regulate normal cellular physiology. In cancers, miRNAs are severely dysregulated, contributing to multiple cancer processes, including drug response. Despite the involvement of miRNAs in cancer, their direct role as drivers of drug resistance remain understudied, therefore, there is a critical need to understand the role of miRNAs in inducing Erlotinib resistance. To this end, we propose an unbiased two-prong approach to identify the miRNAs that drive the development of Erlotinib resistance. Therefore, miRNAs are either being (i) overexpressed, or (ii) silenced in Erlotinib sensitive cells with the hypothesis that perturbed miRNA levels will drive Erlotinib resistance. Erlotinib sensitive NSCLC cell lines, EKVX and H322M were identified from the NCI-60 Developmental Therapeutics Project and their Erlotinib dose response curves were established. To perform the miRNA overexpression screen, EKVX and H322M cells stably expressing renilla and firefly luciferase genes were generated, which will be used to monitor cell number and transfection efficiency, respectively. The luciferase-expressing cells will be transfected with a library of 2,019 individually arrayed human miRNAs and cell growth in the presence of Erlotinib will be monitored. The second prong of the study will identify miRNAs, that when lost, confer Erlotinib resistance. 400-fold coverage of small guide RNA (sgRNA) library of the CRISPR-Cas9 system was transduced in Cas9 overexpressing EKVX cells, to knockout ~21,000 human encoded genes (~1800 miRNA genes). Cells are being cultured in the presence of 75% or 90% growth inhibitory concentrations (GI75 or GI90) of Erlotinib so that only cells with sgRNAs against genes critical for Erlotinib response, survive and grow due to acquired resistance. DNA from these cells will be harvested, the sgRNAs sequenced, and compared to the sgRNAs present in Erlotinib-untreated cells. Successful completion of this project will identify miRNAs that drive Erlotinib resistance and may contribute towards development of miRNA therapeutics to enhance Erlotinib sensitivity of NSCLC tumors. Citation Format: Arpita S. Pal, Alejandra Agredo, Andrea L. Kasinski. Aberrantly expressed microRNAs drive the development of acquired Erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2017-3142

Epithelial–Mesenchymal Transition Predicts Polo-Like Kinase 1 Inhibitor–Mediated Apoptosis in Non–Small Cell Lung Cancer

To identify new therapeutic targets for non-small cell lung cancer (NSCLC), we systematically searched two cancer cell line databases for sensitivity data on a broad range of drugs. We identified polo-like kinase 1 (PLK1) as the most promising target for further investigation based on a subset of sensitive NSCLC cell lines and inhibitors that were in advanced clinical development. To identify potential biomarkers of response of NSCLC to PLK1 inhibition and mechanisms of PLK1 inhibitor-induced apoptosis, integrated analysis of gene and protein expression, gene mutations, and drug sensitivity was performed using three PLK1 inhibitors (volasertib, BI2536, and GSK461364) with a large panel of NSCLC cell lines. The NSCLC cell lines had different sensitivities to PLK1 inhibition, with a minority demonstrating sensitivity to all three inhibitors. PLK1 inhibition led to G2-M arrest, but only treatment-sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. NSCLC lines with high epithelial-mesenchymal transition (EMT) gene signature scores (mesenchymal cell lines) were more sensitive to PLK1 inhibition than epithelial lines (P< 0.02). Likewise, proteomic profiling demonstrated that E-cadherin expression was higher in the resistant cell lines than in the sensitive ones (P< 0.01). Induction of an epithelial phenotype by expression of the miRNA miR-200 increased cellular resistance to PLK1 inhibition. Also, KRAS mutation and alterations in the tight-junction, ErbB, and Rho signaling pathways correlated with drug response of NSCLC. In this first reported large-scale integrated analysis of PLK1 inhibitor sensitivity, we demonstrated that EMT leads to PLK1 inhibition sensitivity of NSCLC cells. Our findings have important clinical implications for mesenchymal NSCLC, a significant subtype of the disease that is associated with resistance to currently approved targeted therapies.

Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines

Abstract Rationale: ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms. Methods: The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA. Results: By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity. Conclusion: We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels. Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2015-4

Abstract 4355: A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1

Abstract Purpose: We endeavored 1) to understand the contribution of TGFβ signaling on expression of a 13-gene microRNA (miRNA) signature and 2) to assess the activity of the signature miRNA genes on mRNA targets that impact the mechanism of activity of erlotinib, the mechanisms by which resistance to erlotinib arises, and the influence of epithelial-to-mesenchymal transition (EMT) on sensitivity to erlotinib. Background: Previous findings from this lab identified a 13-gene miRNA signature that can predict response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in non-small cell lung cancer (NSCLC) cell lines. Bioinformatic analysis of the miRNA signature showed a convergence of the 13 miRNA genes on the TGFβ signaling pathway. We hypothesize that TGFβ signaling controls expression of the miRNAs comprising a signature of response to erlotinib in NSCLC. Methods: Two signature miRNA genes (miR-140-3p and miR-141) were chosen for study because they demonstrate opposing expression levels in erlotinib-resistant and -sensitive NSCLC cell lines. Promoters of both miRNA genes harbor potential Smad Binding Elements (SBEs), binding sites of the TGFβ-responsive Smad transcription factors. TGFβ activity was assessed in erlotinib-resistant (A549) and -sensitive (PC9) NSCLC cell lines using western blot analysis of proteins of interest in the TGFβ signaling cascade and quantitative real-time PCR for expression of miR-140 and -141. The activity of the potential SBEs controlling each of the two miRNA genes was measured by sub-cloning relevant promoter regions into luciferase-reporter constructs. The resulting miR-140 and -141 promoter constructs were transfected into A549 and PC9 cells, and luciferase activity was measured in the presence of TGFβ, the TGFβ inhibitor, SB-431542, and pharmacological inhibitors of EGFR and MAPK. Results: TGFβ acts through the Smad-responsive, canonical signaling pathway in A549 cells. In PC9 cells, TGFβ utilizes the Ras/MAPK pathway for similar signaling endpoints. Analysis of endogenous miRNA expression supports the hypothesis that TGFβ, in the presence and absence of SB-431542 treatment, alters the expression of both microRNAs in both cell lines. Analysis of the influence of the possible SBE sites in each miRNA gene promoter suggests a cell-line specific response. Conclusion: TGFβ can control the expression of signature microRNA genes using different downstream effectors in erlotinib-sensitive and in erlotinib-resistant cell lines. We hypothesize that the erlotinib sensitivity signature miRNA genes can control sensitivity to erlotinib and EMT. Citation Format: Madeline J. Krentz, Rebecca Astley, Andrew Stacy, Esther P. Black. A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2014-4355

Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR.

BackgroundNon-small cell lung cancer (NSCLC) expressed high levels of epidermal growth factor receptor (EGFR). Gefitinib (Iressa) has demonstrated clinical efficacy in NSCLC patients harboring EGFR mutations or refractory to chemotherapy. However, most of NSCLC patients are with wild type EGFR, and showed limited response to gefitinib. Therefore, to develop new effective therapeutic interventions for NSCLC is still required. Our previous study showed Marsdenia tenacissima extract (MTE) restored gefitinib efficacy in the resistant NSCLC cells, but whether MTE acts in the gefitinib-sensitive NSCLC cells is the same as it in the resistant one is unknown.MethodsDose response curves for gefitinib and MTE were generated for two sensitive NSCLC cell lines with mutant or wild type EGFR status. Three different sequential combinations of MTE and gefitinib on cell growth were evaluated using IC50 and Combination Index approaches. The flow cytometric method was used to detect cell apoptosis and cell cycle profile. The impact of MTE combined with gefitinib on cell molecular network response was studied by Western blotting.ResultsUnlike in the resistant NSCLC cells, our results revealed that low cytotoxic dose of MTE (8 mg/ml) combined gefitinib with three different schedules synergistically or additively enhanced the growth inhibition of gefitinib. Among which, MTE → MTE + gefitinib treatment was the most effective one. MTE markedly prompted cell cycle arrest and apoptosis caused by gefitinib both in EGFR mutant (HCC827) and wild type of NSCLC cells (H292). The Western blotting results showed that MTE → MTE + gefitinib treatment further enhanced the suppression of gefitinib on cell growth and apoptosis pathway such as ERK1/2 and PI3K/Akt/mTOR. This combination also blocked the activation of EGFR and c-Met which have cross-talk with each other. Unlike in gefitinib-resistant NSCLC cells, MTE alone also demonstrated certain unexpected modulation on EGFR related cell signal pathways in the sensitive cells.ConclusionOur results suggest that MTE is a promising herbal medicine to improve gefitinib efficacy in NSCLC regardless of EGFR status. However, why MTE acted differently between gefitinib-sensitive and -resistant NSCLC cells needs a further research.

MiR-130a Overcomes Gefitinib Resistance by Targeting Met in Non-Small Cell Lung Cancer Cell Lines

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most common cause of lung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is used for its treatment; however, drug resistance is a major obstacle. Expression of Met has been associated with both primary and acquired resistance to gefitinib, but the mechanisms regulating its expression are not fully understood. Recently, miRNAs such as miR-130a have been shown to play a role in gefitinib resistance, but importance in NSCLC and relationships with Met have not been fully explored. Here we show that miR-130a is over-expressed in gefitinib- sensitive NSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Moreover, miR-130a expression was negatively correlated with that of Met. Further analysis revealed that over-expression of miR-130a increased cell apoptosis and inhibited proliferation of NSCLC cells treated with gefitinib, whereas lowering the expression of miR-130a decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in both gefitinib-sensitive and -resistant NSCLC cell lines, suggesting that miR-130a overcomes gefitinib resistance. We also demonstrated that miR-130a binds to the 3'-UTR of Met and significantly suppresses its expression. Finally, our results showed that over-expressing Met could "rescue" the functions of miR-130a regarding cell apoptosis and proliferation after cells are treated with gefitinib. These findings indicate that the miR-130a/Met axis plays an important role in gefitinib resistance in NSCLC. Thus, the miR-130a/Met axis may be an effective therapeutic target in gefitinib-resistant lung cancer patients.

Erlotinib-induced autophagy in epidermal growth factor receptor mutated non-small cell lung cancer

PurposeErlotinib is a commonly used tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). Autophagy is a catabolic process in response to stress and deprivation of nutrients. This study aims to investigate whether autophagy confers acquired resistance to erlotinib treatment in NSCLC. MethodsFour NSCLC cell lines (HCC827, HCC4006, H358 and H1975) with different epidermal growth factor receptor (EGFR) mutation status (exon 19 deletion, exon 19 deletion, wild-type and L858R/T790M respectively) were selected. MTT assay, crystal violet staining and Annexin-V assay were performed to determine cell viability and apoptosis. Autophagic proteins were detected by Western blot. Acidic vesicular organelle (AVO) formation was determined by acridine orange staining. Autophagy inhibitor (chloroquine) and RNA interference were used to demonstrate the biological effect of erlotinib-induced autophagy. ResultsIn line with EGFR mutation status, it was shown that both HCC827 and HCC4006 cells were sensitive to erlotinib, while H358 and H1975 cell lines were resistant. Erlotinib treatment at clinically relevant concentrations induced autophagy (increased LC3II expression, Atg-5/Atg12 conjugation, formation of AVO and p62 degradation) in sensitive NSCLC cell lines, via p53 nuclear translocation, AMPK activation and mTOR suppression. Addition of chloroquine, as an autophagy inhibitor, enhanced erlotinib sensitivity in sensitive cells. Similarly, silencing of Atg5 or Beclin-1 significantly increased sensitivity to erlotinib in both sensitive cell lines. In contrast, there was no induction of autophagy in resistant H358 and H1975 cell lines upon erlotinib exposure. ConclusionsErlotinib can induce both apoptosis and autophagy in sensitive NSCLC cell lines with activating EGFR mutation (exon 19 del). Inhibition of autophagy can further enhance sensitivity to erlotinib in EGFR-mutated NSCLC, suggesting that autophagy may serve as a protective mechanism.

Mixed micelles of PEG2000-DSPE and vitamin-E TPGS for concurrent delivery of paclitaxel and parthenolide: Enhanced chemosenstization and antitumor efficacy against non-small cell lung cancer (NSCLC) cell lines

Concurrent combination of chemotherapeutic drugs is a promising alternative to single-agent therapies in cancer. In the present study, paclitaxel and parthenolide were loaded into mixed micelles and tested against taxol sensitive (A549) and resistant (A549-T24) NSCLC cell lines. Combination chemotherapy was further evaluated by isobologram analyses and combination index calculations. Drugs were loaded into micelles by the film casting method using PEG2000-DSPE and vitamin E-TPGS. Micelle characterization studies included the determination of particle size, encapsulation efficiency, in vitro release kinetics, as well as 1H NMR analysis. The in vitro release of both drugs was slower from the mixed micelles, which maintained an encapsulation efficiency >95% and chemical stability over a storage period of 45days. The IC50 of paclitaxel and parthenolide determined by MTT assay were 108.6nM and 21μM, respectively, while the combination had an IC50 of 64.15nM in A549 cells. In the taxol resistant cell lines, the IC50 values of paclitaxel and parthenolide were 233nM and 32μM, respectively, while the combination had an IC50 of 128nM. The efficacy of paclitaxel and parthenolide against both cell lines significantly increased when the drugs were coencapsulted in mixed micelles. Mixed micelles caused 79% cell death, which was significantly higher than the 46% cell death caused by the drugs in solution against taxol sensitive cell lines. In taxol resistant cell lines, the cell death caused by mixed micelles was 70% as compared to 45% cell death caused by un-encapsulated drugs. Co-encapsulation of parthenolide with paclitaxel in mixed micelles increased the anticancer activity of paclitaxel against resistant and sensitive lung cancer cell lines.

SULF2 methylation is prognostic for lung cancer survival and increases sensitivity to topoisomerase-I inhibitors via induction of ISG15

The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients and now also demonstrate a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (HR = 0.63, p = 0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through siRNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin. NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared to SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H) expressing NSCLC cell lines were 134-fold more sensitive to camptothecin than SULF2U and low ISG15 (ISG15L) expressing cell lines. Topotecan, a soluble analogue of camptothecin and FDA approved anti-cancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (p < 0.002). Similarly, high ISG15 expression that is comparable to the topotecan sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared to normal lung indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized topotecan therapy.