LK2 Cells Research Articles

Opposing Functions of Maspin Are Regulated by Its Subcellular Localization in Lung Squamous Cell Carcinoma Cells.

Mammary serine protease inhibitor (maspin) is a tumor suppressor protein downregulated during carcinogenesis and cancer progression; cytoplasmic-only maspin expression is an independent, unfavorable prognostic indicator in patients with lung squamous cell carcinoma (LUSC). We hypothesized that the cytoplasmic-only localization of maspin has tumor-promoting functions in LUSC. The subcellular localization of maspin and the invasive capability of LUSC cell lines were investigated using RNA sequencing (RNA-seq), Western blotting, and siRNA transfection. Maspin mRNA and protein expression were suppressed in LK-2 and RERF-LC-AI cells. Cell invasion significantly increased in response to siRNA-mediated maspin knockdown in KNS-62 cells expressing both nuclear and cytoplasmic maspin. In LK-2 cells, both nuclear and cytoplasmic maspin were re-expressed, and cell invasion and migration were significantly decreased. In contrast, re-expressed maspin in RERF-LC-AI cells was detected only in the cytoplasm (cytMaspin), and cell invasion and migration were significantly promoted. RNA-seq and downstream analyses revealed that increased cytMaspin expression downregulated the genes associated with cell adhesion and activated PYK2 and SRC, which play important roles in cancer progression. Our study demonstrates a novel biological function of cytMaspin in enhancing the invasive capabilities of LUSC cells. Understanding cytoplasm-to-nuclear maspin translocation dysregulation may develop novel therapeutic approaches to improve the prognosis of patients with LUSC.

Curcumin Inhibits the Progression of Non-small Cell Lung Cancer by Regulating DMRT3/SLC7A11 Axis.

Non-small cell lung cancer (NSCLC) is a fatal malignancy all over the world. Emerging studies have shownthat curcumin might repress NSCLC progression by regulating ferroptosis, but the underlying mechanism remains unclear. 16HBE, LK-2, and H1650 cell viability was detected using Cell Counting Kit-8 assay. LK-2 and H1650 cell proliferation, apoptosis, and angiopoiesis were measured using 5-ethynyl-2'-deoxyuridine, flow cytometry, and tube formation assay. Superoxide dismutase, Malondialdehyde, Glutathione, and lactate dehydrogenase levels in LK-2 and H1650 cells were examined using special assay kits. Fe+ level was assessed using an iron assay kit. Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) and solutecarrierfamily7member11(SLC7A11) protein levels were detected using western in NSCLC tissues, adjacent matched normal tissues, 16HBE cells, LK-2 cells, H1650 cells, and xenograft tumor tissues. Glutathione peroxidase 4, Acyl-CoA Synthetase Long Chain Family Member 4, and transferrinreceptor1 protein levels in LK-2 and H1650 cells were examined by western blot assay. DMRT3 and SLC7A11 levels were determined using real-time quantitative polymerase chain reaction. After JASPAR prediction, binding between DMRT3 and SLC7A11 promoter was verified using Chromatin immunoprecipitation and dual-luciferase reporter assays in LK-2 and H1650 cells. Role of curcumin on NSCLC tumor growth was assessed using the xenograft tumor model in vivo. Curcumin blocked NSCLC cell proliferation and angiopoiesis, and induced apoptosis and ferroptosis. DMRT3 or SLC7A11 upregulation partly abolished the suppressive role of curcumin on NSCLC development. In mechanism, DMRT3 was a transcription factor of SLC7A11 and increased the transcription of SLC7A11 via binding to its promoter region. Curcumin inhibited NSCLC growth in vivo by modulating DMRT3. Curcumin might constrain NSCLC cell malignant phenotypes partly through the DMRT3/SLC7A11 axis, providing a promising therapeutic strategy for NSCLC.

Abstract 6043: Discovery and characterization of a p300-selective degrader with potent anti-tumor activity in CBP mutant cancers

Abstract Although precision medicine strategies for the treatment of human cancers are clinically successful, they have also revealed that only a small fraction of cancers carry actionable alterations in oncogenic drivers. To expand the druggable genome while maintaining an emphasis on genetically targeted therapies, recent efforts have focused on exploiting synthetic lethal relationships. Paralogous protein pairs are particularly compelling targets in this context due to the clear mechanistic basis for the synthetic lethality. As such, functional genomics screens as well as work from Ogiwara et al. have shown compelling rationale for targeting the histone acetylase transferase p300 in the context of CBP-deficient cancer types. We discovered potent heterobifunctional degraders with demonstrated selectivity for p300 over CBP. Our compound exhibits degradation of p300 within 2 hours (DC50 < 10nM, Dmax > 90%) with minimal impact on CBP through 48 h (DC50 >1uM, Dmax = 30%). Utilizing CRISPR-generated isogenic cell lines depleted for either CBP or p300, we observed significant growth inhibition in CBP knockout (KO) cells (gIC50 = 17nM) with minimal impact on growth of p300 KO (gIC50 = >10uM) or parental wild-type cells (gIC50 > 10uM). This translated to downstream pharmacology, where we observed a rapid and potent inhibition of global H3K27 acetylation in the CBP deficient context but a significantly attenuated response in p300 deleted or parental cells. We subsequently employed computational methods to identify and characterize CBP alterations, defining a putative loss-of-function (LoF) phenotype and confirming this sensitivity pharmacologically in cancer cell types harboring these endogenous mutations. Results mirrored what we observed with CBP depletion in these endogenous CBP LoF cell lines, with potent growth inhibition seen in multiple cell lines including LK-2 (gIC50 = 8nM), H1703 (gIC50 = 9nM), and TE-8 cells (gIC50 = 23nM). To confirm the in vitro results in an in vivo setting, we evaluated the response in mice harboring H1703 xenografts. Once daily oral administration of our compound led to almost complete degradation of p300 within H1703 xenograft tumors, resulting in a pronounced inhibition of tumor growth. Lastly, we profiled p300 degradation in human bone marrow derived myeloid progenitor colony-forming assays where we observed a significant reduction in toxicity (IC50 = 3.9uM) compared to a dual CBP/p300 inhibitor (IC50 = 121nM) or degrader (IC50 = 16nM), supporting the hypothesis that a p300-specific mechanism will offer an improved therapeutic index while maintaining anti-tumor efficacy in a biomarker population Taken together, our results suggest that a p300-selective degrader has the potential to serve as an effective therapeutic modality in CBP-mutated cancers. Citation Format: Mike R. Russell, Cassandra L. Lowenstein, Xuqing Zhang, Jeremy Roach, Jianing Song, Rakesh Nagilla, Nathan Kendsersky, Shreyas Joshi, Peter Orth, Matt Tudor, Qiaolin Deng, Clemente Aguilar-Bonavides, Elham Behshad, Sudeep Banjade, Zhihua Sui, Corey Strickland, Larry Jolivette, Helai P. Mohammad. Discovery and characterization of a p300-selective degrader with potent anti-tumor activity in CBP mutant cancers [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 6043.

Acrolein suppresses anticancer drug-induced toxicity mediated by activating claudin-1 and Nrf2 axis in a spheroid model of human lung squamous cell carcinoma cells

Tobacco smoke contains various carcinogenic ingredients such as nicotine, acrolein, and benzopyrene; however, their effects on cancer treatment are not fully understood. Claudin-1 (CLDN1), a component of tight junctions, is involved in the increased resistance to anticancer drugs. In this study, we found that acrolein increases the mRNA and protein levels of CLDN1 in RERF-LC-AI cells derived from human lung squamous cell carcinoma (SCC). Acrolein increased the p-extracellular signal-regulated kinase (ERK) 1/2 levels without affecting the p-Akt level. The acrolein-induced elevation of CLDN1 expression was attenuated by U0126, a mitogen-activated protein kinase kinas (MEK) inhibitor. These results indicate that the activation of MEK/ERK pathway is involved in the acrolein-induced elevation of CLDN1 expression. In a spheroid model, acrolein suppressed the accumulation and toxicity of doxorubicin (DXR), which were rescued by CLDN1 silencing. The acrolein-induced effects were also observed in lung SCC-derived EBC-1 and LK-2 cells. Acrolein also increased the expression level of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates antioxidant and detoxifying genes, which were inhibited by CLDN1 silencing. In spheroid cells, the levels of reactive oxygen species were enhanced by acrolein, which was inhibited by CLDN1 silencing. Taken together, acrolein may reduce the anticancer drug-induced toxicity in human lung SCC cells mediated by high CLDN1 expression followed by the upregulation of Nrf2 signaling pathway.

Surface chemistry driven selective anticancer potential of functional silver nanoparticles toward lung cancer cells

Decades of nanomedicine research has demonstrated the importance of physicochemical properties to tightly regulate cytotoxicity of nanoparticles. Wherein, the tailor-made surface corona of nanoparticles may dictate nano-bio interfacial interactions toward therapeutic benefits without any significant nanotoxicity. Therefore, in the current research, selective anticancer effects originating from the specific surface chemistry of silver nanoparticles (AgNPs) is demonstrated employing lung cells. The AgNPs are synthesized by bioactive curcumin (Cur) to form a stable corona around AgNPs, and further surface functionalized with an antibiotic isonicotinic acid hydrazide (INH, isoniazid). These NPs were subjected to various structural and spectroscopy analysis for deeper understanding of the surface chemistry, followed by the anticancer potential assessment on LK-2 lung cancer cells. The higher production of reactive oxygen species (ROS) was responsible for therapeutic effects on LK-2 cells. The TUNEL and apoptotic dye studies revealed and confirmed the role of apoptosis in the ROS-mediated selective death of lung cancer cells over human foetus lung diploid fibroblasts (WI-38). Furthermore, the involvement of mitochondria in mediating the effects of AgNP's toxicity is demonstrated. The study provides a deeper understanding regarding the mechanism of anti-cancer actions of the formulated functional AgNPs, and it has potential to open new avenues for tailor-made surfaces chemistry driven nanomedicine development.

Abstract 3783: GSTO2, a novel tumor suppressor gene of lung squamous cell carcinoma, regulates mitochondria function via the p38/β-catenin signaling pathway

Abstract Glutathione S-transferase omega 2 (GSTO2) is one of regulators of GSH/GSSG balance, and its polymorphism showed strong associations with lung functions as well as the risk of chronic obstructive pulmonary disease. Recently, we found that GSTO2 was exclusively expressed in airway basal cells, Clara cells and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was lost in lung squamous cell carcinoma (LSCC). In the present study, we restored GSTO2 expression in LSCC cell lines (LK-2 and H520) to clarify the significance of GSTO2 loss in LSCC. In both LSCC cell lines used, GSTO2 overexpression significantly inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2-transfected LK-2 cells formed smaller tumors in nude mice than mock-transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2-transfected LK-2 cells than in those injected with mock-transfected cells. Metabolomic analyses using the XF96 extracellular flux analyzer revealed that GSTO2 overexpression suppressed mitochondrial oxidative phosphorylation but did not affect glycolysis. Upon JC-1 dye staining, GSTO2-transfected cells showed decreased mitochondrial membrane potential compared with mock-transfected cells. Since β-catenin has been reported as a novel regulator of the OXPHOS in hepatocytes, we next examined the effect of GSTO2 expression on β-catenin expression in LSCC and found that GSTO2 overexpression suppressed the expression of β-catenin. Because p38 phosphorylation was accelerated in both GSTO2-transfected cells, we examined the involvement of the p38 signaling pathway in the GSTO2-mediated downregulation of β-catenin as well as mitochondrial membrane potential in LSCC.When GSTO2-transfected cells were treated with SB203580, a specific inhibitor of p38 MAPK, β-catenin expression and mitochondrial membrane potentialwere restored. Finally, we examine whether DNA methylation of the GSTO2 could explain the loss of GSTO2 expression in LSCC. When human LSCC cell lines were treated with 5-aza-2′-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced. Bisulfite sequencing showed that the promoter region of the GSTO2 was frequently methylated in LSCC tissues than that of normal tissue. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the cell growth and progression of LSCC, probably by modulating oxidative phosphorylation in mitochondria via the p38/β-catenin signaling pathway. Citation Format: Ryusuke Sumiya, Masayoshi Terayama, Teruki Hagiwara, Kazuaki Nakata, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Kawamura I. Yuki. GSTO2, a novel tumor suppressor gene of lung squamous cell carcinoma, regulates mitochondria function via the p38/β-catenin signaling pathway [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 3783.

Stratification of lung squamous cell carcinoma based on ferroptosis regulators: Potential for new therapeutic strategies involving ferroptosis induction

ObjectivesLung squamous cell carcinoma (LSCC) exhibits poor response to treatment compared with other lung cancer subtypes, resulting in worse prognosis. Therefore, new therapeutic strategies are required for advanced LSCC. Ferroptosis is a recently discovered nonapoptotic cell death caused by intracellular lipid peroxidation that can bring about effective cell death in cancer cells resistant to apoptosis. Hence, ferroptosis is a potential therapeutic strategy for refractory cancer. Materials and MethodsIn this study, we performed clinicopathological and molecular analyses on tumor specimens from 270 patients with squamous cell lung cancer, focusing on the expression of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1), which are known to be key regulators of ferroptosis, and the accumulation of 4-hydroxynoneral (4-HNE), a lipid peroxidation marker. ResultsImmunohistochemistry revealed that patients with low 4-HNE accumulation and low levels of GPX4 or FSP1 had significantly worse prognoses than other patients (P = 0.001). This stratification was an independent prognostic predictor (P = 0.003). A dramatic cell death synergistic effect was observed on LSCC-derived LK-2 and EBC1 cells treated with GPX4 and FSP1 inhibitors. This effect was completely inhibited by treatment with the ferroptosis inhibitor. Notably, this was not the case in LK-2 cells treated with the apoptosis inhibitor, and in these cells, ferroptosis was induced. ConclusionFerroptosis regulators GPX4 and FSP1 are associated with lung squamous cell cancer cancer’s prognosis. We present the clinicopathological and molecular basis of novel therapeutic strategies for refractory LSCC.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

ATF4 promotes lung cancer cell proliferation and invasion partially through regulating Wnt/β-catenin signaling.

Activating transcription factor 4 (ATF4) is a member of the cAMP response element binding (CREB) protein family and has been reported to participate in cancer progression; however, its molecular mechanism is not fully understood. In this study, we investigated the function of ATF4 in non-small cell lung cancer and its molecular regulation. We detected cytoplasmic and nuclear ATF4 expression in lung cancer A549, H1299, and LK2 cells, and the total expression of ATF4 was higher than that in HBE cells (p < 0.05). Higher nuclear ATF4 expression was detected in all these cells compared to cytoplasmic ATF4 expression (p < 0.05). Overexpression of ATF4 in A549 cells significantly promoted cancer cell growth and invasion (p < 0.05). Expression of Wnt signaling molecules, including β-catenin, MMP7, and cyclin D1, and the activity of canonical Wnt signaling were also significantly promoted by ATF4 (p < 0.05). ICG001, a canonical Wnt signaling inhibitor that selectively inhibits β-catenin/ cyclic adenosine monophosphate response element binding protein (CBP) interaction, significantly inhibited cancer cell invasion and Wnt signaling. The function of ATF4 was also significantly inhibited by ICG001 (p < 0.05). However, compared to treatment with ICG001, the invasion ability of cancer cells treated with both ICG001 and ATF4 cDNA significantly increased (p < 0.05), which indicates that the function of ATF4 was not dependent only on Wnt/β-catenin signaling. The function of ATF4 in the regulation of β-catenin expression was not significantly affected by ICG001 (p > 0.05). The function of ATF4 to promote the activity of Wnt/β-catenin signaling in cancer cells was abolished by treatment with ICG001 (p > 0.05). These results indicate that ATF4 may contribute to lung cancer progression at least partly by regulating Wnt/β-catenin signaling.

PROPORTION OF ANTICANCER ACTIVITY OF PROTEIN AND PROTEIN HYDROLYSATE FROM EPIPHYTIC BACTERIA

This research aimed to explore the anticancer potential in the protein and hydrolysate protein produced by epiphytic bacteria associated with marine algae on the in vitro model of lung cancer LK-2. Protein was isolated from the bacterium Enterobacter unhas strain SG-A1 in which has an association with brown algae, Sargassum sp. A fractionation method has used in separating the protein. It was used as a dialysis process added by Tris HCl to conduct pre-purified of the fractions, and the pepsin enzyme was used in producing protein hydrolysate (pH 2.0, 37 C) at 3:100 ratio of enzyme-substrate. Pre-screening of toxicity can be conducted using BSLT or Brine Shrimp Lethality Test. The cytotoxic effects of protein and protein hydrolysate on LK-2 cell lines during 16 h were determined by the yellow tetrazolium salt 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The BSLT test revealed that the hydrolysate fractions were very toxic. It showed a higher magnitude of decreasing (significance p < 0.05) in cell viability in LK-2 cells. These findings suggested that the hydrolysate of protein produced by epiphytic bacteria brown algae associated, Sargassum sp. has a high probability of being used as cancer therapy agents.

Curcumin and isonicotinic acid hydrazide functionalized gold nanoparticles for selective anticancer action

Besides physicochemical properties, careful design of biomolecular surface corona can dictate therapeutic potential of nanoparticles. Therefore, we demonstrate a new approach to improve the anticancer potential of gold nanoparticles (AuNPs) by developing a strong corona of curcumin and isonicotinic acid hydrazide (INH) around them. Here, curcumin reduced AuNPsCur are surface-functionalized with biologically active INH (AuNPsCur@INH). Our investigations on human lung squamous carcinoma (LK-2) and human lung fibroblast (TIG-120) cells indicate selective toxicity of functionalized nanoparticles. Conjugation of curcumin and INH on the surface of AuNPs induced anticancer action through reactive oxygen species (ROS) generation. The further extension of this study revealed apoptosis and morphological changes in LK-2 and TIG-120 cells, respectively. Moreover, the anticancer potential of these nanoparticles is compared with standard cisplatin. The outcomes of the work determine that the facile functional surfaces on nanoparticles can be important in controlling the biological activities of nanoparticles.

Abstract 4716: Glutathione S transferase omega 2 (GSTO2) is a novel tumor suppressor gene of human lung squamous cell carcinoma

Abstract Lung cancer is one of the most common malignant diseases and the leading cause of cancer death globally. The clinical, pathologic, and genomic characteristics of lung cancer are very diverse. Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancers and can be histologically classified into lung adenocarcinoma and squamous cell carcinoma (SCC). Lung adenocarcinoma is generally located in alveolar or endobronchial, whereas LSCC frequently arises in bronchi and bronchiole. Molecular-targeted drugs have been developed for lung adenocarcinoma; however, there is currently no definite clinical implication for lung SCC (LSCC). Therefore, to increase the cure and survival rates of lung cancers, developing type-specific diagnostic/prognostic markers and treatment methods might improve this situation. One of the major risk factors of LSCC is smoking, which causes aberrant DNA methylation, abnormal gene expression, and eventually malignant transformation. We previously identified glutathione S-transferase omega 2 (GSTO2) as one of DNA methylation target genes in esophageal SCC (Otsubo T, Oncotarget, 8, 84434-84448, 2017). We reported that GSTO2 was exclusively expressed in nethermost basal cells which are believed to function as stem cells in normal esophageal mucosa. Furthermore, GSTO2 overexpression in esophageal SCC cells significantly inhibited cell growth in vitro and in vivo. (Terayama M, Carcinogenesis, in press). In present study, we investigated the expression and function of GSTO2 in normal lung and LSCC. In normal bronchi and bronchiole containing a variety of epithelial cell populations such as ciliated cells, non-ciliated, columnar Clara cells, and basal cells, GSTO2 was expressed in cytokeratin 5/6-positive airway basal cells. In contrast, all 94 LSCC specimens examined in this study showed no GSTO2 expression. To clarify the significance of GSTO2 silencing LSCC, we restored GSTO2 expression in LK-2 LSCC cells. Overexpression of GSTO2 significantly inhibited cell growth compared with the mock-transfected LK-2 cells. In colony formation assay, GSTO2-transfected cells failed to form colonies, whereas mock-transfected cells formed colonies efficiently. When human LSCC cell lines (LK-2 and EBC-1) were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, the GSTO2 transcription was induced, indicating that aberrant hypermethylation of the GSTO2 in LSCC was likely the cause of the downregulated expression. Considering its expression in basal cells which are proposed to be the precursor cells of LSCC, the silencing of GSTO2 by DNA hypermethylation may contribute malignant transformation of LSCC. Citation Format: Ryusuke Sumiya, Masayoshi Terayama, Teruki Hagiwara, Keigo Sekihara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Yuki I. Kawamura. Glutathione S transferase omega 2 (GSTO2) is a novel tumor suppressor gene of human lung squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4716.

Phase II study of TAK228 in patients with advanced non-small cell lung cancer (NSCLC) harboring NFE2L2 and KEAP1 mutations.

9607 Background: Despite past efforts, no targeted therapies exist for squamous cell lung cancer (LUSC) pts. We identified a heretofore untargeted oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~15% of LUSCs. NFE2L2 encodes NRF2, a transcription factor involved in the oxidative stress response and targeted for degradation by KEAP1. NFE2L2 mutations (mut) occur only in an exon 2 hotspot (Neh2 domain), which is the binding site for KEAP1. Mutations in this region disrupt KEAP1 binding, leading to NRF2 nuclear translocation and increased mTOR signaling via RagD. We report translational studies and results from a phase 2 trial of the oral TORC1/2 inhibitor TAK228 in biomarker-selected pts. Methods: Cell line and xenograft experiments were performed using LK-2 LUSC (NFE2L2 E79K mut), A549 ADCL (KRAS G12S + KEAP1 loss), and SK-MES-1 LUSC cells (NFE2L2/KEAP1 WT) treated with TAK-228, everolimus, rapamycin, or deforolimus. Pts with stage IV LUSC harboring NFE2L2 or KEAP1 mut and ADCL harboring KRAS + KEAP1 co-mut were treated on an NCI CTEP phase 2 study of TAK228 3mg po qd (NCT02417701). Primary endpoint: ORR. Secondary endpoint: PFS. The study used a Simon 2-stage design for each cohort with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK228 exhibited differential anti-tumor activity over TORC1 rapalogs in LK-2 and A549 cells. TAK228 alone was cytotoxic at sub-[μM] (IC50 68nM) in LK-2 cells; all other rapalogs had IC50s &gt; 10μM. This was associated with marked decrease in TORC1/2 &amp; MAPK signaling (decreased pS6, pAKT, pERK). Anti-tumor response was seen in LK-2 and A549 xenografts treated with TAK228. No anti-tumor/growth inhibitory responses were seen with any other rapalog. N = 21 evaluable pts have been treated (10 NFE2L2, 6 KEAP1, 5 KRAS+NFE2L2/KEAP1). Median age = 70; median prior lines tx = 2, smokers = 100%, median pack yrs = 39. Most common AEs included hyperglycemia (72%), fatigue (32%), diarrhea (32%), decreased appetite (32%). In NFE2L2 mut LUSC pts, ORR = 20% (2/10 confirmed PR), DCR = 100% with median PFS = 8.9mos (95%CI 7-NR). In KEAP1 mut LUSC pts, ORR = 17% (1/6 confirmed PR), DCR = 67% with PFS range = 1.8-8.6 mos. In KRAS + NFE2L2/KEAP1 mut ADCL pts, ORR = 0% and DCR = 0%. Conclusions: TAK228 is tolerable with differential activity in NFE2L2 (primary endpoint met) and KEAP1 mutant LUSC. A randomized phase 2 trial of TAK228 + docetaxel vs. SoC chemotherapy in advanced LUSC pts with NFE2L2/KEAP1 mut is in development (LungMAP S1900D) as is an NCI CTEP phase 1/1b trial of TAK228 + CB-839 in advanced NSCLC patients with NFE2L2/KEAP1 mut (NCI #10327). Clinical trial information: NCT02417701 .

Targeting NFE2L2/KEAP1 mutations in advanced NSCLC with the TORC1/2 inhibitor TAK-228.

9085 Background: Despite efforts over the past decade, no targeted therapy options exist for SQCLC pts. We have identified a heretofore untargeted oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~20% of patients with SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through RagD (Shibata Cancer Res 2010). We report translational studies and results from an ongoing phase 2 trial of the oral TORC1/2 inhibitor TAK-228. Methods: Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells (NFE2L2 E79K mut) treated with TAK-228, everolimus, rapamycin, or deforolimus. Pts with stage IV SQCLC harboring NFE2L2 or KEAP1 mutations and NSCLC harboring KRAS + NFE2L2 or KEAP1 co-mutations are treated on an NCI CTEP phase 2 study of TAK228 3mg po qd (continuous 28 day cycles; NCT02417701). Primary endpoint: ORR. Secondary endpoints: PFS. The study utilizes a Simon 2-stage design for each cohort with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK-228 exhibited increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK-228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs exhibited IC50s &gt; 10μM. This was associated with marked decrease in pS6, pAKT, and pERK. Tumor response (-55%) was seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog. N = 15 evaluable pts have been treated on study (7 NFE2L2, 3 KEAP1, 5 KRAS + NFE2L2 or KEAP1). 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; there were no unexpected AEs. Within SQCLC NFE2L2 pts ORR = 29% (2/7 PR) and DCR = 100%. Prolonged duration of response is present, with PFS = 10.5mo, 11.4mo (18.2mo tx beyond PD), 6mo (8mo tx beyond PD), 5.5mo+, 4.5mo, and 1.5mo+. Within SQCLC KEAP1 pts DCR = 66% (2SD, 1PD) with PFS = 7.4mo+, 3mo+, 1mo. Data for the KRAS + NFE2L2/KEAP1 arm will be presented. Conclusions: TAK228 is tolerable with evidence of pre-clinical activity, radiographic responses, and prolonged DOR in biomarker-selected NSCLC pts. Accrual is ongoing. Clinical trial information: NCT02417701.

AC138128.1 an Intronic lncRNA originating from ERCC1 Implies a Potential Application in Lung Cancer Treatment.

Lung cancer is one of the most devastating tumors with a high incidence and mortality worldwide. Polymorphisms and expression of ERCC1 commonly predicted the occurrence and prognosis of lung cancer. However, few studies have focused on long non-coding RNAs related to ERCC1 though some studies reminded the importance of its post-transcriptional regulation. In the present study, an intronic lncRNA AC138128.1 originated from ERCC1 was firstly identified in microarray chip and database, and its possibility as a novel biomarker to predict lung cancer treatment was further discussed. Firstly, the qRT-PCR data showed that AC138128.1 expression was much lower in lung cancer comparing with its para-cancer tissues, which further analyzed by ROC curve. Similarly, the difference was also verified in 16HBE, A549 and LK2 cells. Then AC138128.1 expression was found to have an increasing trend in a dose or time-dependent manner after cisplatin treatment. Finally, the subcellular distribution of AC138128.1 reminded that AC138128.1 was mainly expressed in the nucleus. Interestingly a positive relationship between AC138128.1 and ERCC1 expression was only found in cancer tissues, which reminded AC138128.1 may be involved in the regulation of ERCC1. Therefore, as a preliminary exploration of the lncRNA originated from ERCC1, the present study suggested AC138128.1 is of potential value in predicting platinum analogue benefit in lung cancer.

Hes3 Enhances the Malignant Phenotype of Lung Cancer through Upregulating Cyclin D1, Cyclin D3 and MMP7 Expression.

Hes3 is a basic helix-loop-helix factor gene, which was found to be involved in neural cell differentiation. Expression and clinicopathological significance of Hes3 in non-small cell lung cancer was not clear. In this study, we used immunohistochemistry to examine Hes3 expression in normal human lung and non-small cell lung cancer tissues. Hes3 expression was detected in cytoplasm and nucleus. Hes3 expression in bronchial epithelial cells and epithelial cells of submucosal glands was relatively weak and the positive rate was of 30.3% (10/33). Hes3 expression in non-small cell lung cancer tissues (51.8% (58/112)) was significantly higher than that in normal lung tissues (p < 0.05). Hes3 expression in cancer tissues was significantly associated with poor differentiation, advanced TNM stages, lymph node metastasis, and a shorter patient survival time (p < 0.05). In vitro study showed that overexpression of Hes3 in A549 cells significantly promoted cancer cell proliferation and invasion, while inhibition of Hes3 expression significantly downregulated cancer cell proliferation and invasion (p < 0.05). Western blotting showed that overexpression of Hes3 significantly upregulated expression of Cyclin D1, Cyclin D3, and MMP7 in A549 cells, while inhibition of Hes3 expression in LK2 cells significantly downregulated the expression of these molecules (p < 0.05). These results indicated that Hes3 may contribute to the malignant phenotype of non-small cell lung cancer, possibly through regulation of Cyclin D1, Cyclin D3, and MMP7, and may be a promising cancer marker.

X-ray irradiation induced Disabled-2 gene promoter de-methylation enhances radiosensitivity of non-small-cell lung carcinoma cells

BackgroundDisabled-2 (Dab2) is known as a tumor suppressor as well as a Wnt pathway inhibitor. We previously reported that Dab2 was down-regulated due to gene promoter hypermethylation in lung cancer. Here, we aim to study if X-ray irradiation can induce de-methylation of the Dab2 gene and subsequently up-regulate its expression, and also to attempt to suppress the malignant biological behavior of and enhance the radiosensitivity in lung cancer cells with hypermethylation of the Dab2 gene.MethodsImmunostaining was performed to investigate the relationship between Dab2 expression and lung cancer clinicopathological characteristics. Bisulfite sequencing PCR (BSP) was used to evaluate the methylation status of lung cancer cells with or without X-ray treatment. Real-time PCR and western Blot were performed to investigate the expression of Dab2, Wnt pathway factors, DNMTs and methyl CpG binding protein 2 (MeCP2). Colony Formation, matrigel invasion and xenograft experiment were performed to evaluate the malignant biological behavior of lung cancer cells with irradiation.ResultsThe result of immunostaining of Dab2 in lung cancer tissues showed that decreased Dab2 expression was positively correlated with poor differentiation, lymph node metastasis, advanced TNM stage and poor prognosis. X-ray treatment significantly up-regulated Dab2 expression and inhibited Wnt factors in LK2 cells (with hypermethylation of the Dab2 gene promoter, P < 0.05), but not in SPC-A-1 cells (with hypomethylation of the Dab2 gene promoter); however, the effect could be reversed by Dab2 or Axin knockdown (P < 0.05). Decreased expression of DNMT1, DNMT3b and MeCP2 could be detected in both LK2 and SPC-A-1 cells compared to non-irradiated cells (P < 0.05). Both in vitro studies and in vivo xenograft tumor growth demonstrated that X-ray could significantly inhibit the proliferation and invasion of LK2 but not SPC-A-1 cells (P < 0.05).ConclusionIn general, X-ray-induced up-regulation of Dab2 and inhibition of the Wnt pathway may be mediated by de-methylation of a hypermethylated Dab2 gene promoter. X-ray treatment significantly inhibits proliferation and invasion of lung cancer cells with hypermethylation of the Dab2 gene promoter, but is less effective in lung cancer cells with hypomethylation of the Dab2 gene promoter. These results indicate that the methylation status of the Dab2 gene promoter might be a potential predictor of the radiosensitivity of lung cancer cells.

P2.13-44 Targeting NFE2L2 Mutations in Advanced Squamous Cell Lung Cancers with the TORC1/2 Inhibitor TAK-228

Despite research efforts over the past decade, no targeted therapy options exist for patients with SQCLC. Analyses by TCGA and others (Paik Cancer Disc 2015) have identified a heretofore untargeted, frequently mutated oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ∼20% of patients with SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (∼aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through regulation of RagD (Shibata Cancer Res 2010). We now report translational studies and preliminary results from a phase 2 trial of the oral TORC1/2 inhibitor TAK-228 in SQCLC patients with these mutations. Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells harboring an NFE2L2 E79K mutation treated with TAK-228, everolimus, rapamycin, or deforolimus with requisite vehicle controls. Patients with stage IV SQCLC harboring NFE2L2 mutations were treated on an NCI CTEP- single-institution phase 2 study of TAK228 3mg po qd (continuous, 28 day cycles; NCT02417701). Primary endpoint: ORR. Secondary endpoints: PFS/OS. The study utilizes a Simon 2-stage design with H0=5% (N≥1/5 responses), HA=40% (N≥2/10 responses). TAK-228 exhibited significantly increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK-228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs exhibited IC50s >10μM. This was associated with an 80% decrease in downstream S6 phosphorylation. Tumor response (-55% shrinkage) was also seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog tested. N=4 patients have been treated on study (exon 2 del, D29H, F37V, W24C). 3 are evaluable for response. 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; no other G3 AEs reported. ORR=67% (2 PR, 1 SD, 0 PD). Prolonged disease response is present, with DOR= 12mo, 10mo (ongoing), 8mo (ongoing). TAK228 is tolerable with evidence of pre-clinical and clinical activity in this biomarker-selected population. The trial has met its first-stage endpoint and has expanded to N=10 patients. The trial has also expanded to included patients with KRAS mutant lung cancers harboring co-alterations in NFE2L2 or KEAP1.

Matrine induces apoptosis via targeting CCR7 and enhances the effect of anticancer drugs in non-small cell lung cancer in vitro.

This study mainly investigated the effects of matrine on cell apoptosis and the effects of anticancer drugs in non-small cell lung cancer (NSCLC) cell lines (A549 and LK2 cells). The results showed that matrine (≥10 μM) caused a significant inhibition on cell viability and 10 and 100 μM matrine induced cell apoptosis via influencing p53, bax, casp3, and bcl-2 expressions in A549 cells. In addition, matrine significantly down-regulated C-C chemokine receptor type 7 (CCR7) expression, and blocking the down-regulation of CCR7 by exogenous chemokine ligand 21 (CCL21) treatment alleviated matrine-caused effects of apoptosis genes in A549 cells. The results were further validated in LK2 cells that matrine regulated apoptosis gene expressions, which were reversed by CCL21 treatment. Furthermore, matrine enhances the effects of cisplatin, 5-fluorouracil, and paclitaxel in A549 cells, and the anticancer effects exhibit a dosage-dependent manner. In summary, matrine induced cell apoptosis and enhanced the effects of anticancer drugs in NSCLC cells; the mechanism might be associated with the CCR7 signal.

More expression of BDNF associates with lung squamous cell carcinoma and is critical to the proliferation and invasion of lung cancer cells.

BackgroundBrain-derived neurotrophic factor (BDNF) has been reported to promote tumorigenesis and progression in several human malignancies. The purpose of this study was to explore the function of BDNF in lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC).MethodsThe expression of BDNF was examined in 110 samples of lung SCC and ADC by immunohistochemistry. The protein level of BDNF was examined in 25 lung SCC or ADC samples and paired non-tumors by western blot. BDNF expression was also evaluated in human bronchial epithelial cells (HBE) and 4 lung cancer cell lines using western blot. Three BDNF mRNA variants containing exons IV, VI and IX were evaluated in HBE, two SCC (SK, LK2) and two ADC (A549, LTE) cell lines by RT-PCR. The expression and secretion of BDNF were also determined in cells using western blot and ELISA. Then the shRNA specific for BDNF was transfected into LK2 or A549 cells to further elucidate the BDNF knockdown on cell proliferation, apoptosis and invasion, which were confirmed by MTT, flow cytometry and transwell examinations.Results71.8 % (79 out of 110) of lung SCC and ADC samples were detected positive BDNF, and high expression of BDNF was significantly correlated with histological type and T stage. Compared with non-tumorous counterparts, BDNF was apparently overexpressed in SCC and ADC tissues. In cell studies, the extensive expression and secretion of BDNF were demonstrated in lung cancer cells compared with HBE cells. Interestingly, the expressions of BDNF mRNA variant IV and VI were identical in all cells examined. However, more expression of BDNF mRNA variant IX was found in SK and LK2 cells. The apoptotic cells were increased, and the cell proliferation and invasion were both attenuated once the expression of BDNF was inhibited. When retreated by rhBDNF, BDNF knockdown cells showed less apoptotic or more proliferative and invasive.ConclusionsOur data show that BDNF probably facilitates the tumorigenesis of lung SCC and ADC. The expression of BDNF mRNA variant IX is probably more helpful to the upregulation of BDNF in SCC, and intervening the production of BDNF could be a possible strategy to lung cancer therapy.