H1944 Cells Research Articles

Induction of STING/IL-29 signaling in human lung adenocarcinoma/tumor-associated neutrophil cocultures

Abstract LKB1-mutant lung adenocarcinoma (LUAD), is an intractable form of lung cancer. LKB1-mutant LUAD is poor in T-cells and does not respond to checkpoint inhibitor therapy, and instead is enriched in tumor-infiltrating neutrophils (TINs). STING is a key innate sensor mediating anti-tumor responses that is inhibited in LKB1-mutant LUAD. However, the status of STING signaling and if it can be induced in TINs to antagonize tumors is unknown. Here, we developed a novel human LKB1-mutant LUAD/TIN model to tackle this question. Based on prior work from our lab (Dobosh, STAR Protoc. 2021), we grew the H1944 LKB1-mutant LUAD line at air-liquid interface on Alvetex scaffolds (Reprocell), followed by the transmigration of human blood neutrophils. The effect of the STING agonist MSA-2 was assessed in the model by a 20-plex immune mediator assay(Mesoscale). We successfully manufactured a LKB1-mutant LUAD/TIN model from H1944 cells and human blood neutrophils. MSA-2 treatment resulted in significant increases in the STING-regulated mediators IL-6, IP-10 and MIP-1beta. Most strikingly, MSA-2 treatment induced IL-29 (type III interferon) levels by 27-fold, while neither type I nor type II interferons were induced. The model showcased here mimics LKB1-mutant LUAD/TIN interplay and shows responsiveness to STING agonist treatment through induction of type III (but not type I and II) interferon and other immune mediators. Since IL-29 receptor expression is limited to epithelial and myeloid cells (including neutrophils) and IL-29 is known to mediate immunomodulatory and anti-tumor responses, future work will address if and how the large IL-29 increase observed upon MSA-2 treatment affects LUAD proliferation and TIN activation in this context.

ACAA1 Is a Predictive Factor of Survival and Is Correlated With T Cell Infiltration in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancers. KRAS mutation is the second most prevalent mutation in NSCLC. KRAS mutant cancer cells suppress the anti-tumor T cell response. However, the underlying mechanism is still unknown. Here, we analyzed the differential expression of acetyl-CoA acyltransferase 1 (ACAA1) in various types of cancers using the TIMER database and validated the results in the NSCLC cell line H1944. We silenced oncogenic KRAS by siRNA targeting KRASG13D, and employed an MAPK signaling pathway inhibitor to clarify the possible regulatory pathway. Moreover, we analyzed the correlation of ACAA1 expression level with B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. Correlations between expression of ACAA1 and several biomarkers of mutation burden were also tested. Finally, we evaluated the prognostic value of ACAA1 in a wide range of cancers using the Kaplan-Meier Plotter Database. We found lower expression of ACAA1 in tumor tissue than in adjacent normal tissue in various cancers. This result was confirmed using a GEO dataset. Knock-down of mutant KRAS resulted in increased ACAA1 mRNA level in H1944 cells. ACAA1 mRNA level was significantly upregulated in H1944 after treatment with MAPK pathway inhibitor sorafenib, indicating that oncogenic KRAS may downregulate ACAA1 through MAPK signaling. ACAA1 was negatively correlated with biomarkers of tumor mutation burden, including BRCA1, ATM, ATR, CDK1, PMS2, MSH2, and MDH6. Conversely, ACAA1 expression was positively correlated with infiltrating CD4+ cells and with Th1, Th2, Treg cells in the lung tumor microenvironment. Finally, we showed that ACAA1 is a predictive factor for survival in several cancer types. In summary, decreased ACAA1 expression is correlated with poor prognosis and decreases immune infiltration of CD4+ T cells in LUAD and LUSC. ACAA1 also predicts T cell exhaustion in LUSC. The mechanism underlying KRAS/ACAA1 axis-mediated regulation of immune cell infiltration requires further investigation.

The MicroRNA hsa-let-7g Promotes Proliferation and Inhibits Apoptosis in Lung Cancer by Targeting HOXB1.

PurposeThe goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.Materials and MethodsThe expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.ResultsWe observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.ConclusionThese data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.

Regulation of UGT2A1 by miR-196a-5p and miR-196b-5p.

UDP-glucuronosyltransferase (UGT) 2A1 is an important enzyme in the detoxification of polycyclic aromatic hydrocarbons found in cigarette smoke. This enzyme is expressed in aerodigestive tract tissues including lung as both its wild-type and exon 4-deleted splice variant isoforms, with the latter acting as a negative regulator of wild-type UGT2A1 activity. UGT2A1 regulation may also be mediated by microRNA (miRNA). To identify miRNA important in the regulation of UGT2A1, expression analysis in tandem with in silico analysis suggested miR-196a-5p and miR-196b-5p as potential top candidates. Significant reductions in firefly luciferase activity were observed in human embryonic kidney cell line 293 cells cotransfected with the wild-type UGT2A1 3′-untranslated region (UTR)-containing luciferase plasmid and either miR-196a-5p (62%, P = 0.00080) or miR-196b-5p (60%, P = 0.00030) mimics. In pull-down assays, there was a 3.4- and 5.2-fold increase in miR-196a-5p (P = 0.054) and miR-196b-5p (P = 0.035), respectively, using the UGT2A1 3′-UTR biotinylated mRNA probe as compared with the β-actin coding region control mRNA probe. UGT2A1 mRNA was reduced by 25% (P = 0.058) and 35% (P = 0.023) in H146 and H1944 cells, respectively, after overexpression of the miR196a-5p mimic. A similar 32% (P = 0.030) and 41% (P = 0.016) reduction was observed after over-expression of the miR-196b-5p mimic. In H146 cells transfected with miRNA mimic together with a small interfering RNA (siRNA) specific for the UGT2A1 splice variant, a significant reduction in 3-hydroxy-benzo[a]pyrene-glucuronide formation was observed. The miR-196a-5p- and miR-196b-55p-treated cells exhibited reductions of 35% (P = 0.047) and 44% (P = 0.0063), respectively. These data suggest that miR-196a-5p and miR-196b-5p play an important role in UGT2A1 regulation within the lung and potentially other aerodigestive tract tissues.

PO-019 PTPN11 is a therapeutic target in KRAS mutant lung cancer

IntroductionNon-Small-Cell Lung Cancer (NSCLC) accounts for approximately 85% of lung cancers, and mutations in the KRAS oncogene represent the most common driving event (Molina et al., Mayo Clin Proc 2008). So far, inhibition of the oncoprotein itself has proven difficult, and attempts to target downstream effectors have been hampered by the activation of compensatory resistance mechanisms (Bernards et al., Cell 2012). Notably, while MEK inhibition initially leads to ERK inactivation, P-ERK levels are quickly restored thanks to the upregulation of multiple growth factors receptors (Sun et al., Cell Reports 2014). Here we explored the possibility of targeting PTPN11 as a key mediator of receptor tyrosine kinase signalling in order to overcome intrinsic resistance to MEK inhibition.Material and methodsUsing a panel of 6 KRAS mutant NSCLC cell lines, we studied the biochemical and cell proliferation effect of combining the PTPN11 inhibitor SHP099 (Chen et al., Nature 2016) with the MEK inhibitor AZD6244. We confirmed our findings in CRISPR-based PTPN11 knockout clones of H2122 and H1944 cells. Importantly, we studied the effect of SHP099 in vivo in xenograft and PDX models, as well as in KRASLSLG12D p53fl/fl genetically engineered mouse model of NSCLC. To gain mechanistic insights into the effect of PTPN11 inhibition on mutant RAS activity, we used a panel of isogenic Rasless murine embryonic fibroblasts (Esposito et al., Semin Cancer Biol. 2018; NCI RAS Initiative) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes.Results and discussionsOur data show that depletion or inhibition of PTPN11 in KRAS mutant NSCLC cell lines invariably induces sensitivity to AZD6244. Surprisingly, in vivo models suggest that targeting PTPN11 is by itself sufficient to impair tumour growth, mainly due decreased RAS-GTP loading and induction of cellular senescence under growth factor-limiting conditions. Of note, our data suggest a correlation between the residual GTP hydrolysis capacity of the distinct KRAS mutants and their responsiveness to upstream regulators like PTPN11.ConclusionWhile in the past efforts have been focused on targeting KRAS downstream effectors, our data suggest that inhibiting upstream mediators like PTPN11 could be effective either alone or in combination with downstream inhibition. Our results suggest that targeting PTPN11 could be of clinical utility especially for tumours driven by KRAS mutants with high residual GTP hydrolysis capacity.

Arsenite and Cadmium Activate MAPK/ERK via Membrane Estrogen Receptors and G-Protein Coupled Estrogen Receptor Signaling in Human Lung Adenocarcinoma Cells.

Epidemiological evidence indicates that cadmium and arsenic exposure increase lung cancer risk. Cadmium and arsenic are environmental contaminants that act as endocrine disruptors (EDs) by activating estrogen receptors (ERs) in breast and other cancer cell lines but their activity as EDs in lung cancer is untested. Here, we examined the effect of cadmium chloride (CdCl2) and sodium arsenite (NaAsO2) on the proliferation of human lung adenocarcinoma cell lines. Results demonstrated that both CdCl2 and NaAsO2 stimulated cell proliferation at environmentally relevant nM concentrations in a similar manner to 17β-estradiol (E2) in H1793, H2073, and H1944 cells but not in H1792 or H1299 cells. Further studies in H1793 cells showed that 100 nM CdCl2 and NaAsO2 rapidly stimulated mitogen-activated protein kinase (MAPK, extracellular-signal-regulated kinases) phosphorylation with a peak detected at 15 min. Inhibitor studies suggest that rapid MAPK phosphorylation by NaAsO2, CdCl2, and E2 involves ER, Src, epidermal growth factor receptor, and G-protein coupled ER (GPER) in a pertussis toxin-sensitive pathway. CdCl2 and E2 activation of MAPK may also involve ERβ. This study supports the involvement of membrane ER and GPER signaling in mediating cellular responses to environmentally relevant nM concentrations of CdCl2 and NaAsO2 in lung adenocarcinoma cells.

Abstract B90: HSP90 inhibitor NVP-AUY922 sensitizes intrinsic MEK inhibitor Trametinib-resistant NSCLC cells harboring KRAS mutation

Abstract Introduction: Mutant KRAS is feature of more than 25% of non-small cell lung cancers (NSCLC) and exemplifies one of the most popular oncogenic drivers. Ras-driven tumors are often hard to cure with conventional therapies and are necessary novel treatment strategies. Experiment: Using the HSP90 inhibitor NVP-AUY922, we investigated the potential of HSP90 inhibition to overcome the resistance to Trametinib, MEK inhibitor, in lung cancers harboring K-RAS mutant cell lines (H23, H358, H647, H1944, and A549). The synergistic effect of AUY-922 and trametinib was determined by analysis of the combination index (CI). After performing western blotting, we underwent following xenograft study with 6-week-old Balb/c-nu/nu female using H649 and H1944 cell lines. The mice were randomized into groups of 5, and NVP-AUY922 (5 mg/kg) was administered 3 days/week and GSK1120212 (300 μg/kg) was administered 5 days/week for up to 21 days in total. Result: NSCLC cells harboring G13D KRAS mutation showed intrinsic MEK inhibitor resistance via PI3K activation by highly expressed AKT as a bypass pathway. And then, HSP90 inhibitor AUY-922 suppresses both PI3K-AKT-mTOR and RAF-MEK-ERK reactivated by MEK inhibitor GSK1120212 and finally AUY-922 renders intrinsic MEK inhibitor resistant NSCLC cells sensitive to GSK1120212 with significant synergy. Furthermore, we showed this synergistic effect is occurred on sub-effective dose of either drug. Conclusion: The dual inhibition of HSP90 and MEK signaling pathways on sub-effective dose may constitute a potent therapeutic strategy to treat intrinsic MEK inhibitor resistant G13DKRAS mutant NSCLCs for resolving toxicity problem of dual inhibition of AKT and MEK in clinical trials. Citation Format: Dae Ho Lee, Kang-Seo Park, Bora Oh, Mi-Hee Lee, Hannah Yang. HSP90 inhibitor NVP-AUY922 sensitizes intrinsic MEK inhibitor Trametinib-resistant NSCLC cells harboring KRAS mutation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B90.

An integrated genome-wide approach to discover deregulated microRNAs in non-small cell lung cancer: Clinical significance of miR-23b-3p deregulation.

In spite of significant technical advances, genesis and progression of non-small cell lung cancer (NSCLC) remain poorly understood. We undertook an integrated genetic approach to discover novel microRNAs that were deregulated in NSCLCs. A total 119 primary NSCLCs with matched normal were analyzed for genome-wide copy number changes. We also tested a subset of matched samples by microRNA expression array, and integrated them to identify microRNAs positioned in allelic imbalance area. Our findings support that most of the identified deregulated microRNAs (miR-21, miR-23b, miR-31, miR-126, miR-150, and miR-205) were positioned in allelic imbalance areas. Among microRNAs tested in independent 114 NSCLCs, overexpression of miR-23b was revealed to be a significantly poor prognostic factor of recurrence free survival (HR = 2.40, P = 0.005, 95%CI: 1.32–4.29) and overall survival (HR = 2.35, P = 0.005, 95%CI: 1.30–4.19) in multivariable analysis. In addition, overexpression of miR-23b in H1838 cell line significantly increased cell proliferation, while inhibition of miR-23b in H1437 and H1944 cell lines significantly decreased cell doubling time. In summary, integration of genomic analysis and microRNA expression profiling could identify novel cancer-related microRNAs, and miR-23b could be a potential prognostic marker for early stage NSCLCs. Further biological studies of miR-23b are warranted for the potential development of targeted therapy.

Parathyroid-hormone-related protein signaling mechanisms in lung carcinoma growth inhibition.

Parathyroid hormone-related protein (PTHrP) inhibits proliferation of several lung cancer cell lines, but the signaling mechanism has not been established. This study tested the hypotheses that growth inhibition is mediated through the PTHrP receptor, PTH1R, and that the process is modified by ERK activation. PTHrP-positive and negative clones of H1944 lung adenocarcinoma cells underwent stable PTH1R knockdown with lentiviral shRNA or transient transfection with ERK1 and ERK2 siRNA. Alternatively, cells were treated with 8-CPT cAMP, 8-CPT 2′-O-methyl cAMP, and N-6-phenyl cAMP analogs. H1944 cells expressing ectopic PTHrP showed 20–40% decrease in proliferation compared to the PTHrP-negative cells in the presence of normal levels of PTH1R (P < 0.01). PTH1R knockdown eliminated this difference and increased cell proliferation regardless of PTHrP status. The three cAMP analogs each inhibited proliferation over 5 days by 30–40%. ERK2 knockdown inhibited proliferation of PTHrP-positive cells alone and in combination with ERK1 knockdown. The growth inhibition mediated by cAMP analogs was unaffected by ERK1 knockdown. In conclusion, ectopic expression of PTHrP 1–87 inhibits H1944 cell proliferation. PTH1R knockdown blocks this effect and stimulates proliferation, indicating that the ligand exerts anti-mitogenic effects. cAMP, the second messenger for PTH1R also inhibits proliferation and activates ERK. PTHrP growth inhibition may be opposed by concomitant ERK activation.

Cell cycle actions of parathyroid hormone-related protein in non-small cell lung carcinoma

Parathyroid hormone-related protein (PTHrP), a paraneoplastic protein expressed by two-thirds of human non-small cell lung cancers, has been reported to slow progression of lung carcinomas in mouse models and to lengthen survival of patients with lung cancer. This study investigated the effects of ectopic expression of PTHrP on proliferation and cell cycle progression of two human lung adenocarcinoma cell lines that are normally PTHrP negative. Stable transfection with PTHrP decreased H1944 cell DNA synthesis, measured by thymidine incorporation, bromodeoxyuridine uptake, and MTT proliferation assay. A substantial fraction of PTHrP-positive cells was arrested in or slowly progressing through G1. Cyclin D2 and cyclin A2 protein levels were 60-70% lower in PTHrP-expressing cells compared with control cells (P < 0.05, N = 3 independent clones per group), while expression of p27(Kip1), a cyclin-dependent kinase inhibitor, was increased by 35 +/- 9% (mean +/- SE, P < 0.05) in the presence of PTHrP. Expression of other cyclins, including cyclins D1 and D3, and cyclin-dependent kinases was unaffected by PTHrP. PTHrP did not alter the phosphorylation state of Rb, but decreased cyclin-dependent kinase (CDK) 2-cyclin A2 complex formation. Ectopic expression of PTHrP stimulated ERK phosphorylation. In MV522 cells, PTHrP had similar effects on DNA synthesis, cyclin A2 expression, pRb levels, CDK2-cyclin A2 association, and ERK activation. In summary, PTHrP appears to slow progression of lung cancer cells into S phase, possibly by decreasing activation of CDK2. Slower cancer cell proliferation could contribute to slower tumor progression and increased survival of patients with PTHrP-positive lung cancer.

Effects of selenium supplementation on expression of glutathione peroxidase isoforms in cultured human lung adenocarcinoma cell lines

Selenium is an essential nutrient, a component of several anti-oxidant enzymes, and a possible factor in cancer risk, including lung cancer. We determined the subtoxic range of selenium concentration (as sodium selenite) required to increase and maintain the expression of anti-oxidant selenoproteins gluthathione peroxidases GPX1 and GPX4 at a constant level in cultures of human lung adenocarcinoma cell lines (H460, H1703 and H1944) and in HPL1D, a non-transformed lung epithelial cell line. Selenium dose-dependently increased GPX1 protein expression 1.8-fold in HPL1D cells and approximately 40-fold in H460 and H1944 cancer cells, with maximum effects at 20-40 nM. GPX4 protein was also increased, but more so in HPL1D (five-fold) than in H460 or H1944 cells (two- to three-fold). GPX1 mRNA showed similar patterns but differences of lesser magnitude. GPX1 protein and activity level was not consistently detectable in H1703 cells, with or without Se supplementation; its mRNA was present but very low. GPX4 protein level was also low in H1703 cells, but was markedly increased by selenium supplementation (48-fold). These results confirm a role for selenium in risk of lung cancer and the independent regulation of GPX1 and GPX4. Characterization of individual tumors with regard to GPX1 and GPX4 levels and regulation might be useful for interpretation of clinical studies on effects of selenium in lung cancer risk.