Non-small Cell Lung Cancer PC-9 Research Articles

Bee venom prompts the inhibition of gefitinib on proliferation, migration, and invasion of non-small cell lung cancer cells via EGFR-mediated autophagy

It has been confirmed that bee venom (BV) can inhibit tumor metastasis of lung cancer cells induced by epidermal growth factor, suggesting the inhibitory role of BV on the regulation of epidermal growth factor receptor (EGFR), and may synergistically promote the anti-lung cancer effect of EGFR tyrosine kinase inhibitor gefitinib. This paper aims to ascertain the therapeutic potentials of BV combined with gefitinib against non-small cell lung cancer (NSCLC) in vitro. As results, the content of the main component melittin in air-dried BV was determined by HPLC. Subsequently, it was found that BV significantly inhibited the proliferation of NSCLC PC-9 and NCI-H1299 cells, but not generated apparent toxicity to human normal lung epithelial BEAS-2B cells. Meanwhile, the combination of BV and gefitinib also significantly inhibited the proliferation of these two cells, and suppressed the migration and invasion of PC-9 cells. By bioinformatics analysis and molecular docking, it was predicted that the main component melittin in BV could act on the cell membrane and transmembrane protein EGFR. Ultimately, Western blot assays showed BV alone or combined with gefitinib significantly decreased the protein expression of phosphorylated EGFR (p-EGFR) and the protein expression ratio of p-EGFR to EGFR, and increased the protein expression ratio of LC3-II to LC3-I in PC-9 cells or epidermal growth factor-activated PC-9 cells. The results demonstrated that BV could prompt the inhibition of gefitinib on proliferation, migration, and invasion of NSCLC cells via EGFR-mediated autophagy, showing the synergistic anti-NSCLC potential when combined with gefitinib.

Solasonine promotes apoptosis of non-small cell lung cancer cells by regulating the Bcl-2/Bax/caspase-3 pathway

To investigate the effect of solasonine, an active component of Solanum nigrum, on proliferation and apoptosis of non-small cell lung cancer PC9 cells. PC9 cells were treated with 2, 5, 10, 15, 20, or 25 μmol/L solasonine, and the changes in cell proliferation were examined using CCK-8 assay. Tetramethyl rhodamine ethyl ester (TMRE) was used to detect the changes in mitochondrial membrane potential, and caspase-3/7 detection kit and GreenNucTM caspase-3/Annexin V-mCherry kit for live cell were used to analyze the changes in caspase-3 of the cells. Annexin V-FITC/PI double staining was employed to analyze the apoptosis rate of the cells. The effect of PTEN inhibitors on solasonine-induced cell apoptosis was examined by detecting apoptosis-related protein expressions using Western blotting. Solasonine treatment for 24, 48, and 72 h significantly lowered the viability of PC9 cells. The cells treated with solasonine for 24 h showed significantly decreased mitochondrial membrane potential and increased cell apoptosis with enhanced caspase-3/7 and caspase-3 activities and expression of cleaved caspase-3. Solasonine treatment significantly decreased phosphorylation levels of PI3K and Akt, increased the protein expressions of PTEN and Bax, and lowered the expression of Bcl-2 protein in the cells. Solasonine inhibits proliferation and induces apoptosis of PC9 cells by regulating the Bcl-2/Bax/caspase-3 pathway and its upstream proteins.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

A Wnt/β-catenin signaling inhibitor, IMU1003, suppresses the emergence of osimertinib-resistant colonies from gefitinib-resistant non-small cell lung cancer cells

Drug resistance has become a challenge in effective longterm molecular targeted therapy. Longterm non-small cell lung cancer (NSCLC) treatments with the first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) shorten the effective duration of the third-generation EGFR-TKI, osimertinib, via genetic or epigenetic mechanisms in addition to the gatekeeper mutation T790M. This study reproduced this persistence in vitro using gefitinib-resistant NSCLC PC-9 cells (GR cells) and revealed that pharmacological nuclear localization inhibition of β-catenin suppressed the osimertinib resistance. Osimertinib effectively reduced GR cell survival but left significantly more resistant colonies than parental PC-9 cells. The nuclear fraction of β-catenin was enriched in GR cells during acquisition of osimertinib resistance. A chemical nuclear localization inhibitor of β-catenin, IMU1003, dramatically decreased the emergence of osimertinib-resistant colonies. Forced nuclear localization of β-catenin reduced IMU1003 efficacy. Thus, suppression of the nuclear β-catenin function may overcome the transgenerational EGFR-TKI-resistance.

Brusatol Inhibits Proliferation, Migration, and Invasion of Nonsmall Cell Lung Cancer PC-9 Cells

Objective: The purpose of this study was to investigate the inhibitory effect of brusatol, a nigakilactone extracted from Brucea javanica, on lung cancer for development of therapeutic drugs. We explored the effects of brusatol on the proliferation, migration, and invasion of lung cancer PC-9 cells in vitro and analyzed the mechanisms involved. Materials and Methods: MTT assay was used to determine the effect of brusatol on the proliferative capacity of PC-9 and H1975 cells in vitro. The half-maximal inhibitory concentrations (IC50) were calculated and used as a reference for subsequent experiments. Variations in the number and size of tumor cell clusters were monitored by the colony formation assay as evidence for the effect of brusatol on cell proliferation. The effect of brusatol on the migration and invasion of PC-9 cells was evaluated using wound healing and transwell assays, respectively. Apoptosis in lung cancer cells was detected using the Annexin V-FITC/propidium iodide assay. The correlated anticancer mechanism was detected using Western blotting. Results: The IC50 values of brusatol acting on PC-9 and H1975 cells were 1.58 ± 0.30 mM and 31.34 ± 2.72 mM, respectively, according to the MTT experiment. In addition, brusatol suppressed PC-9 cell proliferation, migration, and invasion, as well as induced apoptosis, which may be related to the downregulation of epidermal growth factor receptor (EGFR), β-catenin, Akt, and STAT3 expression. Conclusions: Brusatol showed potent anticancer activity against lung cancer PC-9 cells, inhibiting the proliferative capacity and metastatic potential of PC-9 cells. Its anticancer effect may be related to the downregulation of EGFR, β-catenin, Akt, and STAT3.

Diverse Mechanisms of Resistance against Osimertinib, a Third-Generation EGFR-TKI, in Lung Adenocarcinoma Cells with an EGFR-Activating Mutation.

Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is used as a first-line treatment for patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, the mechanisms underlying its anticancer activity, particularly the subsequent development of acquired resistance, are unclear. Herein, we investigated the mechanisms underlying the development of osimertinib resistance by treating NSCLC PC-9 cells (harboring an EGFR-activating mutation) with osimertinib, thereby developing five resistant cell lines, i.e., AZDR3, AZDR6, AZDR9, AZDR11, and AZDR14. The amplification of wild-type EGFR in AZDR3 cells and wild-type EGFR and KRAS in AZDR6 cells was also studied. AZDR3 cells showed dependence on EGFR signaling, in addition to afatinib sensitivity. AZDR9 cells harboring KRASG13D showed sensitivity to MEK inhibitors. Furthermore, combination treatment with EGFR and IGF1R inhibitors resulted in attenuated cell proliferation and enhanced apoptosis. In AZDR11 cells, increased Bim expression could not induce apoptosis, but Bid cleavage was found to be essential for the same. A SHP2/T507K mutation was also identified in AZDR14 cells, and, when associated with GAB1, SHP2 could activate ERK1/2, whereas a SHP2 inhibitor, TNO155, disrupted this association, thereby inhibiting GAB1 activation. Thus, diverse osimertinib resistance mechanisms were identified, providing insights for developing novel therapeutic strategies for NSCLC.

Abstract A027: Exploring the effect of hypoxia and spatial interactions on the dynamics between gefitinib resistant and naïve NSCLC cell lines

Abstract Hypoxia and the development of spatially heterogeneous hypoxic and/or necrotic regions in non-small cell lung cancer (NSCLC) and other solid tumors is associated with chemotherapy and radiation resistance. One mechanism by which this could occur is through modification of the cell-cell interactions (or games) between the resistant and naïve cells within the tumor. Cell-cell interactions can modify the fitness of cells and thereby change the evolutionary dynamics of a tumor. Hypoxia may, in this way, change the game dynamics between cells and directly support maintenance of a population of resistant cells. To understand the mechanism by which hypoxic heterogeneity modulates evolutionary dynamics and therapy response, we combine computational modeling and in vitro experiments under a spatial hypoxia gradient. We utilize in-house game assay protocols to probe the evolutionary games between cell types. The experimental system is adapted from Carmona-Fontaine's MEMIC plate. This system allows us to create stable oxygen and nutrient gradients for 12 evolutionary replicates of NSCLC PC-9 cells. Fluorescence labeled gefitinib-resistant PC-9 cells and naïve parental PC-9 cells are co-cultured in a closed chamber with limited availability to oxygen and nutrient supply at one end. A green hypoxia probe is used to verify the establishment of a hypoxia gradient and a red Annexin V probe monitors apoptosis. Once microenvironmental gradients are established due to cellular consumption of oxygen and nutrients, images of cells are taken every 1.5 hours for 5 days. We carry out multiple experiments in which we vary the initial proportions of parental and resistant cells. Computationally, green and red fluorescence from the images captured by the microscopy system is quantified by an in-house image processing pipeline. We apply a random forest machine learning classifier, illumination correction, and object identification to each image to gather fluorescence counts for each well. These counts are used to compute the background rate of cell death and absolute vs. relative oxygen levels. The subsequent cell counts over time were used to calculate the growth rate (fitness) of the cell types in different environments and therefore the strength of environmental and frequency dependent interactions between the cell types. This framework allows for tightly coupled computational and in vitro experiments aimed at understanding and predicting the clinical implications of hypoxia resistance across a range of tumor types and microenvironments. We have measured the effect of hypoxia upon game interactions between gefinib-resistant and susceptible NSCLC cells. The evolutionary game changes with changing levels of hypoxia–and for the first time we are able to write down a continuous equation representing the game as a function of oxygen. Citation Format: Mina Dinh, Bezhou Feng, Jeff Maltas, Emily Dolson, Masahiro Hitomi, Steph Owen, Jacob Scott. Exploring the effect of hypoxia and spatial interactions on the dynamics between gefitinib resistant and naïve NSCLC cell lines [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A027.

Cell-penetrating peptides containing the progesterone receptor polyproline domain inhibits EGF signaling and cell proliferation in lung cancer cells.

Non-small cell lung cancer (NSCLC) accounts for the majority (80–85%) of all lung cancers. All current available treatments have limited efficacy. The epidermal growth factor receptor (EGFR) plays a critical role in the development and progression of NSCLC, with high EGFR expression associated with increased cell proliferation and poor prognosis. Thus, interfering with EGFR signaling has been shown to effectively reduce cell proliferation and help in the treatment of NSCLC. We previously demonstrated that the progesterone receptor (PR) contains a polyproline domain (PPD) that directly interacts with Src homology 3 (SH3) domain-containing molecules and expression of PR-PPD peptides inhibits NSCLC cell proliferation. In this study, we investigated whether the introduction of PR-PPD by cell-penetrating peptides (CPPs) could inhibit EGF-induced cell proliferation in NSCLC cells. PR-PPD was attached to a cancer-specific CPP, Buforin2 (BR2), to help deliver the PR-PPD into NSCLC cells. Interestingly, addition of BR2-2xPPD peptides containing two PR-PPD repeats was more effective in inhibiting NSCLC proliferation and significantly reduced EGF-induced phosphorylation of Erk1/2. BR2-2xPPD treatment induced cell cycle arrest by inhibiting the expression of cyclin D1 and CDK2 genes in EGFR-wild type A549 cells. Furthermore, the combination treatment of EGFR-tyrosine kinase inhibitors (TKIs), including Gefitinib or Erlotinib, with BR2-2xPPD peptides further suppressed the growth of NSCLC PC9 cells harboring EGFR mutations as compared to EGFR-TKIs treatment alone. Importantly, BR2-2xPPD peptides mediated growth inhibition in acquired Gefitinib- and Erlotinib- resistant lung adenocarcinoma cells. Our data suggests that PR-PPD is the minimal protein domain sufficient to inhibit NSCLC cell growth and has the potential to be developed as a novel NSCLC therapeutic agent.

Up-Regulation of p53/miR-628-3p Pathway, a Novel Mechanism of Shikonin on Inhibiting Proliferation and Inducing Apoptosis of A549 and PC-9 Non-Small Cell Lung Cancer Cell Lines.

Shikonin (SHK) is a pleiotropic agent with remarkable cell growth inhibition activity against various cancer types, especially non–small cell lung cancer (NSCLC), but its molecular mechanism is still unclear. Our previous study found that miR-628-3p could inhibit the growth of A549 cells and induce its apoptosis. Bioinformatics analysis predicted that miR-628-3p promoter sequence contained p53 binding sites. Considering the regulatory effect of SHK on p53, we speculate that SHK may inhibit the growth and induce apoptosis of NSCLC cells by up-regulating miR-628-3p. CCK-8 and EdU assay confirmed the inhibitory effect of SHK on A549 and PC-9 cells. Meanwhile, quantitative reverse transcription–polymerase chain reaction and Western blot showed that SHK could promote the expression of p53 and miR-628-3p in a dose-dependent manner. Overexpression of p53 or miR-628-3p can inhibit the growth and promote apoptosis of A549 and PC-9 cells, while silencing p53 or miR-628-3p has the opposite effect. Dual luciferase reporting assay and ChIP (chromatin immunoprecipitation) assay further verified the direct interaction between p53 and the promoter of miR-628-3p. Gene knockdown for p53 or miR-628-3p confirmed that SHK inhibits the growth and induces apoptosis of A549 and PC-9 cells at least partly by up-regulating p53/miR-628-3p signaling pathway. Therefore, these novel findings provide an alternative approach to target p53/miR-628-3p axis and could be used for the development of new treatment strategies for NSCLC.

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

Long non‑coding RNA MEG3 inhibits cell migration and invasion of non‑small cell lung cancer cells by regulating the miR‑21‑5p/PTEN axis.

Long non-coding RNAs (lncRNAs) are involved in the occurrence and progression of numerous types of cancer. The aim of the present study was to evaluate the effect of the lncRNA maternally expressed gene 3 (MEG3) on the migration and invasion of non-small cell lung cancer (NSCLC) H1299 and PC9 cells. Reverse transcription-quantitative (RT-q)PCR analysis showed that MEG3 was downregulated in NSCLC PC9 and H1299 cells. Additionally, bioinformatics analysis indicated that MEG3 sponges microRNA (miR)-21-5p; miR-21-5p was predicted to target the phosphatase and tensin homolog (PTEN) 3′-untranslated region sequence. MEG3 overexpression led to miR-21-5p suppression and PTEN upregulation in PC9 and H1299 cells, as detected by RT-qPCR. Subsequently, western blot analysis confirmed that MEG3 overexpression enhanced PTEN expression levels and inhibited the PI3K/AKT signaling pathway in NSCLC cells. These effects were attenuated by miR-21-5p. Dual luciferase assay supported the sponging effect of MEG3 on miR-21-5p and validated the direct interaction between miR-21-5p and PTEN. Furthermore, Transwell assay demonstrated that MEG3 overexpression had an inhibitory effect on cell migration and invasion. MEG3 overexpression also mediated epithelial-to-mesenchymal transition by significantly enhancing E-cadherin and decreasing N-cadherin, Vimentin and matrix metalloprotein 9 expression levels in NSCLC cells, as indicated by western blot analysis. These changes were partially reversed by an miR-21-5p mimic. These results indicated that MEG3 acted as a tumor suppressor that inhibited NSCLC cell migration and invasion via sponging miR-21-5p, which, in turn, enhanced the expression levels of PTEN, in part via the PI3K/AKT signaling pathway. The results of the present study have suggested the potential of MEG3 as a novel therapeutic target for NSCLC treatment.

Abstract IA22: Tracking metabolic compartmentation and epigenetic metabolism in human lung cancer

Abstract Although metabolic reprogramming has been recognized as a hallmark of human cancers, our understanding of metabolic heterogeneities/compartmentation and regulation of epigenetics in human tumors is largely unknown. This is crucial to elucidating metabolic checkpoints and strategizing therapeutic target(s) for human cancer. The slow progress is at least in part due to the lack of relevant models and tools for studying these processes. We have established patient-derived organotypic tissue cultures (OTC) that recapitulate the in vivo metabolic reprogramming of human cancer while retaining native patient tumor microenvironment and 3D architecture 1,2. The matched pairs of cancerous (CA) and non-cancerous (NC) lung OTC from the same patient enable reprogrammed cancer tissue metabolism and on-/off-target drug responses for individual patients to be defined. We have deployed this system along with multiplexed Stable Isotope-Resolved Metabolomics (mSIRM) to probe the efficiency of different substates that fuel de novo synthesis of purine nucleotides in matched patients’ CA versus NC lung OTC 3. We found for seven non-small cell lung cancer (NSCLC) patients that 13C6-glucose was the preferred substrate over D3-serine, D2-glycine, or 13C2-glycine in supporting purine synthesis in CA tissues but not or less so in matched NC tissues. Kinetic modeling of the tracer data along with gene/protein expression data suggests that cytoplasmic activation and dynamic compartmentation of the glucose-to-serine pathway as well as reversal of mitochondrial serine to glycine fluxes can account for this preference for glucose. In all seven patients’ CA tissues, c-MYC was overexpressed and its suppression in NSCLC PC9 cells blocked de novo serine synthesis from glucose, enhanced serine-glycine exchanges, and reduced the efficiency of glucose versus serine incorporation into ATP/GTP. However, serine rather than glucose was the preferred substrate for purine synthesis in NSCLC cell lines but c-MYC could alter this preference for serine towards glucose in fueling purine synthesis. This tissue and cell line difference should be considered in designing serine starvation-based cancer therapy. To track metabolism that modulates epigenetic regulation, we employed a fast, sensitive, and robust Fourier transform-mass spectrometric method to analyze S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels in A549 cells under anti-cancer methylseleninic acid (MSA) and selenite treatments. We found that SAM/SAH levels were reduced by both treatments and MSA but not selenite increased the SAM to SAH ratio. These changes were accompanied by reduced proliferation, central metabolism, and H3K27Me3 levels in MSA and less so in selenite-treated A549 cells. Thus, the tool for tracking SAM/SAH metabolism along with the SIRM approach opens the opportunity for interrogating epigenetic metabolism and its regulation of growth-promoting central metabolic events. Funding: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL). Refs: 1 Sellers, K. et al. Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation. The Journal of Clinical Investigation 125, 687-698, doi:10.1172/JCI72873 (2015). 2 Fan, T. W., Lane, A. N. &amp; Higashi, R. M. Stable Isotope Resolved Metabolomics Studies in ex vivo Tissue Slices. Bio-protocol 6, e1730 (2016). 3 Fan, T. W. M. et al. De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues. The Journal of biological chemistry 294, 13464-13477, doi:10.1074/jbc.RA119.008743 (2019). Citation Format: Teresa W-M Fan, Andrew N. Lane, Jason A. Brandon, Joonseon Yang, Chi V. Dang, Richard M. Higashi. Tracking metabolic compartmentation and epigenetic metabolism in human lung cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA22.

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.

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. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive 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. 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.

Reduced expression of CYLD promotes cell survival and inflammation in gefitinib-treated NSCLC PC-9 cells: Targeting CYLD may be beneficial for acquired resistance to gefitinib therapy.

The application of tyrosine kinase inhibitors (TKIs) to the epidermal growth factor receptor (EGFR) has been proven to be highly effective for non-small-cell lung cancer (NSCLC). However, patients often evolve into acquired resistance. The secondary mutations in EGFR account for nearly half of the acquired resistance. While the remaining 50% of patients exhibit tolerance to EGFR-TKIs with unclear mechanism(s). Cylindromatosis (CYLD), a deubiquitinase, functions as a tumor suppressor to regulate cell apoptosis, proliferation, and immune response, and so on. The role of CYLD in NSCLC EGFR-TKI resistance remains elusive. Here, we found CYLD was upregulated in PC-9 cells, whereas downregulated in PC-9 acquired gefitinib-resistant (PC-9/GR) cells in response to the treatment of gefitinib, which is consistent with the results in the Gene Expression Omnibus database. Overexpression of CYLD promoted a more apoptotic death ratio in PC-9/GR cells than that in PC-9 cells. In addition, silencing the expression of CYLD resulted in an increase of the expression level of interleukin-6, transforming growth factor-β and tumor necrosis factor-α, which may contribute to acquired resistance of PC-9 cells to gefitinib. Taken together, our data in vitro demonstrate that PC-9/GR cells downregulated CYLD expression, enhanced subsequent CYLD-dependent antiapoptotic capacity and inflammatory response, which may provide a possible target for acquired gefitinib-resistant treatment in NSCLC.

CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression.

The underlying molecular mechanisms of tumorigenesis and progression of non-small cell lung cancer (NSCLC) are not yet fully elucidated. In the present study, in vitro functional dissections suggest that siRNA-mediated silencing of CCNE2 profoundly attenuated the proliferative and colony-formative abilities of NSCLC PC9 and HCC827 cells, while forced overexpression of CCNE2 significantly strengthened the proliferative and colony-formative capabilities of these cells. Intriguingly, by ChIP and luciferase reporter gene assays, we observed that CARM1 is recruited to the promoter regions of CCNE2 gene and acts as a transcriptional activator. Mechanically, the asymmetric di-methylation of H3R17me2a and H3R26me2a, as the catalytic substrates of CARM1, were highly enriched at the core promoter regions of CCNE2 gene, thereby activating the expression of CCNE2. In vitro and in vivo rescue experiments demonstrated that restoration of CCNE2 expression significantly abolished the CARM1 shRNA-mediated inhibition of cell proliferation, indicating that the oncogenic function of CARM1, at least partially, depended on the activation of CCNE2. Inhibition of CARM1 enzymatic activity could significantly repress CCNE2 expression in NSCLC cells. In addition, the expression of CARM1 was significantly elevated and positively correlated with CCNE2 levels in 20 cases of NSCLC patients. Both CARM1 and CCNE2 are highly associated with shorter 10-year overall survival of at a large cohort of 461 cases of NSCLC patients from the Kaplan-Meier plotter database. To summarize, these findings provide compelling evidence that CARM1 could promote NSCLC progression via activation of CCNE2, paving the way for future therapeutic strategies in NSCLC.

Hypoxia/pH dual-responsive nitroimidazole-modified chitosan/rose bengal derivative nanoparticles for enhanced photodynamic anticancer therapy

Most of the traditional photosensitizers (PSs) used in photodynamic therapy (PDT) have poor tumor targeting and phototoxic side effects. Drug delivery systems can be designed to be triggered to release payload PSs in response to intracellular hypoxia and acidic environment of tumors with controllable photoactivity to achieve enhanced therapeutic effects with minimal side-effects. In this work, we synthesized nitroimidazole-modified chitosan (Cs-NA) and prepared Cs-NA/rose bengal ω-carboxyheptyl ester (a rose bengal derivative, RBD) complex nanoparticles (Cs-NA/RBD) for pH and hypoxia double-responsive RBD delivery. Cs-NA/RBD possessed appropriate particle size of about 86.9 nm and zeta potential of +33.4 mV, showed good stability in cell culture medium, and demonstrated increased accumulative release at acidic and hypoxia condition by about 60% compared to that at neutral pH. The cellular uptake efficiency of Cs-NA/RBD in non-small cell lung cancer PC9 cells increased more than 10 times compared with Cs-NA/RB. The anti-proliferative effects and the generation of reactive oxygen species (ROS) in PC9 cells indicated the notable photodynamic therapeutic effects of Cs-NA/RBD. These results suggested that Cs-NA/RBD could be a promising nanoplatform for enhanced controllable photodynamic anticancer therapy.

Docosahexaenoic Acid Serving As Sensitizing Agents And Gefitinib Resistance Revertants In EGFR Targeting Treatment.

ObjectiveDue to the resistance of cancer cells, chemotherapy has been severely restricted. Docosahexaenoic acid (DHA) has been broadly identified as the chemo-sensitizing agent and revertant of multidrug resistance owing to its pleiotropic characteristics; however, it has not been well interpreted. The purpose of this research was to identify the anticancer role of DHA and its combination with the chemotherapeutic agent Gefitinib in non-small cell lung cancer (NSCLC).MethodsHuman chemo-sensitive NSCLC PC-9 cells and the Gefitinib-resistant counterpart PC-9/GR cells were adopted to assess the effects of the integrated DHA and Gefitinib treatments in vitro and vivo, for which the combination index (CI), apoptosis rate and the epithelial growth factor receptor (EGFR) pathway were analyzed.ResultsComparing with the control cells, the DHA-treated PC-9/GR cells triggered the increase of drug absorption and sensitivity, suggesting that the sensitivity of chemotherapeutic drug could be induced by DHA. Moreover, the elevation of phosphorylation levels of EGFR and the downstream extracellular signal-regulated kinase (ERK) in the cellular lysates were induced by the DHA+Gefitinib treatment. Additionally, the long-term Gefitinib stimulated PC-9 model revealed that DHA could revert the Gefitinib resistance.ConclusionThis is the first research that indicated the novel biochemical effect of DHA, which can help in overcoming the resistance of EGFR-TKI in NSCLC cells and broaden the horizon of the DHA supplementation during the NSCLC therapy.

EP1.03-11 Mechanisms of Gefitinib Plus Pemetrexed on Human Non-Small Cell Lung Cancer

Resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) is often acquired in non-small cell lung cancer (NSCLC) patients during treatment. We previously demonstrated that combined treatment with EGFR-TKI gefitinib plus chemotherapy improved progression-free survival (PFS) in NSCLC patients carrying sensitive EGFR mutations. Pharmacological interaction between gefitinib and pemetrexed was evaluated in NSCLC cell line PC-9 using MTT assay. The influence of combined treatment with gefitinib plus pemetrexed on gene expression profiles and signaling pathways has been investigated using microarray and Ingenuity Pathway Analysis (IPA). Synergistic inhibitory effect between gefitinib and pemetrexed was observed in NSCLC cell line PC-9. Figure 1A suggested representative proliferation inhibitory effects of gefitinib, pemetrexed and combined treatment for 48 hours. Figure 1B showed CI values of concurrent gefitinib-pemetrexed treatment in PC-9 NSCLC cell line. CI values at ED50, ED75 and ED90 were shown. Furthermore, widespread gene expression changes and critical signaling pathways were induced significantly by combined treatment in PC-9 cells. Figure 2A was heatmap of gene expression prolifes in human NSCLC PC-9 cell line treated with gefitinib (blue), pemetrexed (purple) or gefitinib-pemetrexed combination (orange) with the criteria P<0.05 and ▏fold change ▏>1.5. Genes and samples were listed in rows and columns, respectively. A colour standard for data normalization was shown at the bottom with green representing downregulated genes while red representing upregulated genes. In Figure 2B, pathway enrichment of differential expressed genes was analysed using Ingenuity Pathway Analysis (IPA). Signaling pathways shown here were based on a P<0.0001. Figure 2C showed heatmap of critical pathways affected by combined treatment as compared to gefitinib single treatment. Heatmap colour represented the Z-score of signalling pathways. Z-score>0 meant the signalling pathway was stimulated by related treatment while Z-score<0 meant the signalling pathway was inhibited by related treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gene expression profiles revealed potential signaling pathways contributing to the synergism.

De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues

Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [13C6]glucose, D2-glycine, [13C2]glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro Surprisingly, [13C6]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated [13C6]glucose, D3-serine, and [13C2]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.

Abstract 1281: T790M gatekeeper mutation emerges via de novo at the early stages of erlotinib treament in PC9 non-small cell lung cancer cells

Abstract The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors used in a subset of non-small cell lung cancer (NSCLC) patients harboring activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detect the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, confirming that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more dominantly resistant to erlotinib than parental PC9 cells by acquiring T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance. Citation Format: Sujin Kim, Angela KJ Park, Jeonghee Cho. T790M gatekeeper mutation emerges via de novo at the early stages of erlotinib treament in PC9 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1281.