Protects Lung Cancer Cells Research Articles

Abstract 3745: GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage

Abstract Long non-coding RNAs (lncRNAs) control numerous biological processes, including epigenetic regulation, gene expression, proliferation and metabolism. However, the detailed biological mechanisms by which lncRNAs regulate cellular processes are still poorly understood. We identified a set of growth regulator non-coding RNAs based on data mining of CRISPR/Cas9 screens and high-throughput sequencing studies from patient normal and tumor cells. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. In our screen, the Growth Regulator Antisense 1 RNA (GRAS1) was identified as a key factor controlling growth in non-small cell lung cancer cells. Loss of GRAS1 resulted in altered expression of genes involved in DNA repair, cell cycle progression, and p53 and NF-ΚB signaling pathways. Extensive DNA damage occurred within 48 hours after GRAS1 knockdown, with a significant increase in the number of γH2AX loci visualized by immunofluorescence assay. We used RNA antisense purification combined with mass spectrometry to identify the NF-ΚB activating protein (NKAP) as the top directly interacting partner of GRAS1 lncRNA. NKAP stability decreased after GRAS1 knockdown, and this phenotype could be reversed with proteasome inhibition. Further, RelA levels were decreased in GRAS1 knockdown cells. We found that GRAS1 lncRNA binds and stabilizes NKAP to enable survival and protect against DNA damage in lung cancer cells. Citation Format: Tong Su, Bobby Kong, Jonathan Zhu, Colleen A. McHugh. GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3745.

Improvement of ACK1-targeted therapy efficacy in lung adenocarcinoma using chloroquine or bafilomycin A1

BackgroundActivated Cdc42-associated kinase 1 (ACK1) is a promising druggable target for cancer, but its inhibitors only showed moderate effects in clinical trials. The study aimed to investigate the underlying mechanisms and improve the antitumor efficacy of ACK1 inhibitors.MethodsRNA-seq was performed to determine the downstream pathways of ACK. Using Lasso Cox regression analysis, we built a risk signature with ACK1-related autophagy genes in the lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) project. The performance of the signature in predicting the tumor immune environment and response to immunotherapy and chemotherapy were assessed in LUAD. CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study.ResultsRNA-seq revealed the regulatory role of ACK1 in autophagy. Furthermore, the risk signature separated LUAD patients into low- and high-risk groups with significantly different prognoses. The two groups displayed different tumor immune environments regarding 28 immune cell subsets. The low-risk groups showed high immune scores, high CTLA4 expression levels, high immunophenoscore, and low DNA mismatch repair capacity, suggesting a better response to immunotherapy. This signature also predicted sensitivity to commonly used chemotherapy and targeted drugs. In vitro, the ACK1 inhibitors (AIM-100 and Dasatinib) appeared to trigger adaptive autophagy-like response to protect lung cancer cells from apoptosis and activated the AMPK/mTOR signaling pathway, partially explaining its moderate antitumor efficacy. However, blocking lysosomal degradation with chloroquine/Bafilamycine A1 or inhibiting AMPK signaling with compound C/shPRKAA1 enhanced the ACK1 inhibitor’s cytotoxic effects on lung cancer cells. The efficacy of the combined therapy was also verified using a mouse xenograft model.ConclusionsThe resulting signature from ACK1-related autophagy genes robustly predicted survival and drug sensitivity in LUAD. The lysosomal degradation inhibition improved the therapeutic effects of the ACK1 inhibitor, suggesting a potential role for autophagy in therapy evasion.

Novel function of PERP-428 variants impacts lung cancer risk through the differential regulation of PTEN/MDM2/p53-mediated antioxidant activity

Lung cancer is the leading cause of cancer-related deaths worldwide. Identifying genetic risk factors and understanding their mechanisms will help reduce lung cancer incidence. The p53 apoptosis effect is related to PMP-22 (PERP), a tetraspan membrane protein, and an apoptotic effector protein downstream of p53. Although historically considered a tumor suppressor, PERP is highly expressed in lung cancers. Stable knockdown of PERP expression induces CL1-5 and A549 lung cancer cell death, but transient knockdown has no effect. Interestingly, relative to the PERP-428GG genotype, PERP-428CC was associated with the highest lung cancer risk (OR = 5.38; 95% CI = 2.12–13.65, p < 0.001), followed by the PERP-428CG genotype (OR = 2.34; 95% CI = 1.55–3.55, p < 0.001). Ectopic expression of PERP-428G, but not PERP-428C, protects lung cancer cells against ROS-induced DNA damage. Mechanistically, PERP-428 SNPs differentially regulate p53 protein stability. p53 negatively regulates the expression of the antioxidant enzymes catalase (CAT) and glutathione reductase (GR), thereby modulating redox status. p53 protein stability is higher in PERP-428C-expressing cells than in PERP-428G-expressing cells because MDM2 expression is decreased and p53 Ser20 phosphorylation is enhanced in PERP-428C-expressing cells. The MDM2 mRNA level is decreased in PERP-428C-expressing cells via PTEN-mediated downregulation of the MDM2 constitutive p1 promoter. This study reveals that in individuals with PERP-428CC, CAT/GR expression is decreased via the PTEN/MDM2/p53 pathway. These individuals have an increased lung cancer risk. Preventive antioxidants and avoidance of ROS stressors are recommended to prevent lung cancer or other ROS-related chronic diseases.

Induction of NEDD8-conjugating enzyme E2 UBE2F by platinum protects lung cancer cells from apoptosis and confers to platinum-insensitivity

Platinum is a widely used first-line chemotherapy in treating non-small cell lung cancer of adenocarcinoma. Unfortunately, platinum resistance leads to relapse and therapeutic failure, enabling the development of platinum-sensitization strategies to be of great clinical significance. Here, we report that the upregulation of the NEDD8-conjugating enzyme UBE2F is an important way for lung cancer cells to escape platinum-induced cell apoptosis, which confers to insensitivity to platinum-based chemotherapy. Mechanistically, platinum treatment impairs the complex formation for proteasome-mediated UBE2F degradation, evidenced by the weaker association between UBE2F and Ring-box protein 1 (RBX1), an essential component of Cullin-Ring E3 ligases (CRLs), thus leading to the accumulation of UBE2F. The accumulated UBE2F promotes the neddylation levels and activity of Cullin5, in accord with the lower expression of pro-apoptotic protein NOXA, a well-known substrate of Cullin-Ring E3 ligase 5 (CRL5). Additionally, knockout of UBE2F significantly sensitizes lung cancer cells to platinum treatment by enhancing the protein levels of NOXA and subsequently promoting cell apoptosis. Our observations uncover a previously unknown regulatory mechanism of UBE2F stability upon platinum chemotherapy and suggest that UBE2F might be a novel therapy target for sensitizing lung cancer cells to platinum-based chemotherapy.

Alpha1-antitrypsin protects lung cancer cells from staurosporine-induced apoptosis: the role of bacterial lipopolysaccharide

Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung cancer cell lines with high (H1975) and very low (H661) baseline expression of SERPINA1 gene encoding AAT protein. Comparison of cells grown for 3 weeks in a regular medium versus medium supplemented with 2 mg/ml of AAT revealed that in the presence of AAT cells acquire better proliferative properties, resistance to staurosporine (STS)-induced apoptosis, and show higher expression of CLU, a pro-tumorigenic gene coding clusterin protein. Similarly, the co-administration of STS with AAT or addition of AAT to the cells pre-treated with STS abrogated effects of STS in both cell lines. Following experiments with H1975 cells have shown that AAT blocks critical steps in STS-induced cell death: inhibition of AKT/MAPK pathways, and activation of caspase 3 and autophagy. AAT does not inhibit apoptosis-triggered by chloroquine (inhibitor of autophagy) or streptonigrin (inducer of p53 pathway). The anti-apoptotic effects of AAT were unaffected by lipopolysaccharide (LPS). However, AAT induced TLR4 levels and enhanced LPS effects on the production of IL-6, a tumor-promoting cytokine. Our data provide further evidence that AAT plays a significant role in the tumorigenesis.

Human non‑small cell lung cancer cells can be sensitized to camptothecin by modulating autophagy.

Lung cancer is a prevalent disease and is one of the leading causes of mortality worldwide. Despite the development of various anticancer drugs, the prognosis of lung cancer is relatively poor. Metastasis of lung cancer, as well as chemoresistance, is associated with a high mortality rate for patients with lung cancer. Camptothecin (CPT) is a well-known anticancer drug, which causes cancer cell apoptosis via the induction of DNA damage; however, the cytotoxicity of CPT easily reaches a plateau at a relatively high dose in lung cancer cells, thus limiting its efficacy. The present study demonstrated that CPT may induce autophagy in two human non-small cell lung cancer cell lines, H1299 and H460. In addition, the results of a viability assay and Annexin V staining revealed that CPT-induced autophagy could protect lung cancer cells from programmed cell death. Conversely, the cytotoxicity of CPT was increased when autophagy was blocked by 3-methyladenine treatment. Furthermore, inhibition of autophagy enhanced the levels of CPT-induced DNA damage in the lung cancer cell lines. Accordingly, these findings suggested that autophagy exerts a protective role in CPT-treated lung cancer cells, and the combination of CPT with a specific inhibitor of autophagy may be considered a promising strategy for the future treatment of lung cancer.

Fibronectin protects lung cancer cells against docetaxel-induced apoptosis by promoting Src and caspase-8 phosphorylation.

Fibronectin is involved in orchestrating many diverse cellular behaviors, including adhesion, invasion, differentiation, and proliferation and recently has also been shown to participate in the development of chemoresistance. In this study, we found that fibronectin expression was inversely correlated with clinical responses to docetaxel treatment in non-small cell lung cancer patients. Subsequently, we showed that fibronectin pretreatment could enhance cell viability and reduce apoptosis in docetaxel-treated lung cancer cells because fibronectin induced phosphorylated Src and caspase-8, rendering the later inactive, thus inhibiting docetaxel-induced apoptosis. The inhibition of apoptosis by fibronectin was found to be enhanced by Src overexpression and reversed by Src knockdown in lung cancer cells. Further investigation revealed that a downregulation of phospho-Src via treatment with a Src kinase inhibitor could also abolish fibronectin activity and recover docetaxel-induced apoptosis. Molecular studies revealed that this reversion was due to decreased phospho-Src levels rather than a reduction in total Src expression. Inhibition of phospho-Src reduced phospho-caspase-8 and promoted caspase-8 activity, restoring apoptosis following docetaxel and fibronectin co-treatment. Finally, xenografts experiments demonstrated that fibronectin promoted lung cancer cell proliferation during docetaxel treatment in vivo. Our findings indicate that fibronectin promotes Src and caspase-8 phosphorylation in lung cancer cells, which decreases caspase-8 activation and protects tumor cells from docetaxel-induced apoptosis. Therefore, the fibronectin/Src/caspase-8 pathway may play a crucial role in docetaxel resistance in lung cancer.

EMT or EMT-Promoting Transcription Factors, Where to Focus the Light?

The epithelial-to-mesenchymal transition (EMT) is commonly considered as a main driving force of the metastatic cascade. An impressive series of experiments and observations worldwide supports its pivotal role in promoting cancer cell dissemination at the invasive fronts of tumors, intravasation, cell survival in fluids, and extravasation, with secondary site colonization being the only step requiring a return to an epithelial phenotype (1). Beyond this, commitment of epithelial cells into an EMT program has been associated with resistance to chemo, radio-, and hormono-therapies, definitively extending the interest of studying EMT from the embryologist community to the oncology and medical fields. Obviously, EMT turns out to constitute a transdifferentiation program, allowing epithelial cells to escape from numerous stresses, including mechanical constrains, hypoxia, nutrient depletion, and unfortunately therapeutic treatments (1). EMT is orchestrated by a restricted number of transcription factors mainly the three Snail, Twist, and Zeb families (EMT-TFs). With inclusion of miRNAs, these factors constitute a complex interactome, able to sensor multiple signals received from the proximal microenvironment and relay them onto gene expression (2, 3). Generally undetectable in adult epithelial cells in homeostatic conditions, EMTTFs were found to be recurrently expressed in various types of cancers including multiple carcinomas, an expression often associated with a high metastatic risk. An outstanding amount of work has been performed over the last years evaluating their relative contribution to the initiation and maintenance of the EMT process, with a significant impact on the prognostic value occurring following to their detection in primary tumors or in disseminated cancer cells (4, 5). Does this mean that the oncogenic potential of these factors in epithelial cells only rely on their ability to promote EMT or does their oncogenic activity extend further than EMT induction? By performing in vitro functional assays, we and others firstly demonstrated that several of these embryonic transcription factors, including the TWIST proteins, alleviated fail-safe program induction. Through this, they were shown to cooperate with mitogenic oncoproteins such as RAS and N-MYC in promoting cell transformation in vitro, as well as in lung and breast carcinoma development in transgenic mouse models (4, 6–9). The underlying mechanisms have been largely explored. The TWIST1 protein was found to directly interact with p53 and to destabilize the oncosuppressive protein by altering specific post-translational modifications (10). Furthermore, TWIST1 alleviates induction of cyclin-dependent kinase inhibitors (CDKN1A,CDKN2A, and CDKN2B), thereby sustaining cell proliferation (8, 11). These pleiotropic properties probably provide TWIST proteins unique properties. As TWIST proteins experimentally are inefficient in triggering EMT as compared with SNAIL and ZEB proteins, their main functions may consist in protecting cells from fail-safe programs during the EMT-associated genetic reprograming. In this respect, they might be considered as survival factors rather than EMT inducers. Although SNAIL and ZEB proteins, unlike the TWIST transcription factors, fail to prevent HRASG12Vinduced senescence in murine embryonic fibroblasts (B. Gras, unpublished data), we obviously cannot exclude that they facilitate the escape from oncogene-induced fail-safe programs in other cellular settings and/or experimental conditions. In support of this assumption, ZEB proteins were indeed reported to protect lung cancer cells from EGFR-induced senescence through their ability to down-modulate CDKN1A and CDKN2B (12). In line with this observation, ZEB1 was demonstrated to be positioned downstream of RB and to contribute to fibroblast immortalization induced by RB and RB-like protein depletion (13). Enforced expression of SNAIL or ZEB proteins in non-transformed mammary epithelial cells, and the consequent activation of RAS-downstream pathways, predominantly triggers EMT. Nonetheless, it also accidentally promotes the commitment of cells into a senescence program, unveiled by their SA-β-galactosidase activity (B Gras and SA, unpublished data). This observation is consistent with the reported anti-proliferative properties of SNAIL and ZEB proteins in epithelial cells (14) and with the recognized antagonism between cell proliferation and dissemination. It additionally gives a rationale to the restricted staining of these transcription factors to the tumor-stromal interface, stabilized by microenvironmental EMTpermissive conditions (15–17). The need to maintain ZEB and SNAIL proteins at

Tankyrase and the Canonical Wnt Pathway Protect Lung Cancer Cells from EGFR Inhibition

Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC.

Abstract 5135: Functional genomic shRNA screens reveal that the canonical Wnt pathway protects lung cancer cells from EGFR inhibition

Abstract Lung cancers are the leading cause of cancer deaths globally. In particular, non-small cell lung cancer (NSCLC) is a devastating disease, and is rarely curable. Hyperactivation of mitogenic signaling cascades is a hallmark of cancer development, including for lung cancers. Targeted therapies inhibiting specific receptor tyrosine kinases have shown clinical promise, but rarely produce complete responses and are not curative, suggesting the existence of escape mechanisms promoting cell survival. Using a loss-of-function, whole genome shRNA screen, we identified various components and mediators of the canonical Wnt pathway that contribute to the maintenance of NSCLC cells during inhibition of the epidermal growth factor receptor (EGFR). Modulation of tankyrases, casein kinases and different components of the Wnt pathway with shRNAs or small molecules, in vitro and in vivo, significantly increased the efficacy of EGFR inhibitors, thus revealing a critical role for the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt/β-catenin pathway together with EGFR inhibition in the clinic could lead to more durable remissions for non-small cell lung cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5135. doi:1538-7445.AM2012-5135

MiR-34c may protect lung cancer cells from paclitaxel-induced apoptosis

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that negatively regulate the expression of their target genes. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and are considered as promising new therapeutic targets for cancer. However, the identity of miRNAs involved in apoptosis and their respective targets remain largely unknown. Given the elevated complexity of miRNA regulation of gene expression, we performed a functional screening as an alternative strategy to identify those miRNAs that in lung cancer cells may interfere with the apoptotic process. To this aim, we generated a derivative of the non-small cell lung carcinoma A549 cell line in which caspase-8, a critical upstream initiator of apoptosis, can be activated by administration of the small dimerizer drug AP20187. We found a number of miRNAs that may rescue cell viability from caspase-8 activation. They included miRNAs already described as oncogenic such as miR-17, miR-135 and miR-520, but also some miRNAs such as miR-124-1 and miR-34c for which a tumor-suppressive role has instead been described or expected. Among them, miR-34c-5p markedly increased resistance to paclitaxel-induced apoptosis. We demonstrate that Bmf (Bcl-2-modifying factor) is a target of miR-34c-5p, and that its silencing, together with that of c-myc, a known target of miR-34c-5p, contributes to resistance to apoptosis induced by paclitaxel through p53 downregulation.

Aldo-Keto Reductases Protect Lung Adenocarcinoma Cells from the Acute Toxicity of B[a]P-7,8-trans-Dihydrodiol

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC(50) of ∼2 μM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC(50) > 20 μM. Similar differences in cytoxicity were observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ∼6 h post-treatment at concentrations as low as 1 μM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10-fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

P53 protects lung cancer cells against metabolic stress

The preferential use of aerobic glycolysis for energy production by cancer cells, a phenomenon known as the 'Warburg effect', is well recognized and is being considered for therapeutic applications. However, whether inhibition of glycolysis will be effective in all types of cancer is unclear. The current study shows that a glycolytic inhibitor, 2-deoxy-D-glucose (2DG), exhibits the cytotoxic effect on non-small cell lung cancer in a p53-dependent manner. 2DG significantly inhibits ATP production in p53-deficient lung cancer cells (H358) but not in p53-wt cells (A549). In contrast to p53-wt cells, p53-defective cells are unable to compensate for their need of energy via oxidative phosphorylation (OXPHOS) when glycolysis is inhibited. In the presence of p53, increased ROS from OXPHOS increases the expression of p53 target genes known to modulate metabolism, including synthesis of cytochrome c oxidase 2 (SCO2) and TP53-induced glycolysis and apoptosis regulator (TIGAR). Importantly, 2DG selectively induces the expression of the antioxidant enzymes manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx1) in a p53-dependent manner. The results demonstrate that the killing of cancer cells by the inhibitor of glycolysis is more efficient in cancer cells without functional p53 and that p53 protects against metabolic stress by up-regulation of oxidative phosphorylation and modulation of antioxidants.

Abstract 3468: Aldo-keto reductases protect lung epithelial cells from polycyclic aromatic hydrocarbons at the expense of increased oxidative stress

Abstract Tobacco exposure stimulates genes that are likely to be involved in the metabolism of tobacco and its combustion products. Four induced genes are aldo-keto reductases (AKR), a family of enzymes that reduce aldehydes and ketones to alcohols. AKRs act on a range of substrates including polycyclic aromatic hydrocarbons (PAH). AKRs convert PAH to form o-quinones. Alternatively, PAHs are metabolized to (+)-anti-benzo[α]pyrene diol epoxide ((+)-anti-BPDE)) by the combined action of P4501A1/1B1 and epoxide hydrolase. (+)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause oxidative stress through a futile redox cycle. To address the role of AKRs in PAH toxicity, we compared the toxicities of PAH metabolites and the effects of overexpressing AKR 1A1 in lung cells. We find that anti-BPDE and BP,-7,8-diol, an intermediate product, are toxic to A549 cells at concentrations with an IC50 of ∼2 µM. In addition, both anti-BPDE and BP 7,8-diol induce p53 expression in 6 hrs post-treatment at concentrations as low as 1 µM consistent with extensive DNA damage. In contrast, we found that the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC 50 &amp;gt; 20 µM. B[a]P-7,8-dione treatment also did not induce p53 expression in 24 hrs at 20 µM. However, B[a]P-7,8-dione treatment generated reactive oxygen species (ROS) in A549 cells as previously reported. We also observed that overexpression of AKR 1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10 fold from the toxic effects of BP, 7,8-diol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH at the expense of increasing the burden oxidative stress Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3468.

Role of Rad51 Down-Regulation and Extracellular Signal-Regulated Kinases 1 and 2 Inactivation in Emodin and Mitomycin C-Induced Synergistic Cytotoxicity in Human Non–Small-Cell Lung Cancer Cells

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It is a tyrosine kinase inhibitor and has anticancer effects on lung cancer. Rad51 plays a central role in homologous recombination, and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the mitogen-activated protein kinase kinase (MKK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 signal pathway maintains the expression of Rad51. Therefore, in this study, we hypothesized that emodin could enhance the effects of the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing the expression of Rad51 and the phosphorylation of ERK1/2. Exposure of the human non-small-cell lung cancer H1703 or A549 cell lines to emodin decreased the MMC-elicited phosphorylated ERK1/2 and Rad51 levels. Moreover, emodin significantly decreased the MMC-elicited Rad51 mRNA and protein levels by increasing the instability of Rad51 mRNA and protein. In emodin- and MMC-cotreated cells, ERK1/2 phosphorylation was enhanced by constitutively active MKK1/2 (MKK1/2-CA), thus increasing Rad51 protein levels and protein stability. The synergistic cytotoxic effects induced by emodin combined with MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by small interfering Rad51 RNA transfection significantly enhanced MMC-induced cell death and cell growth inhibition. In contrast, overexpression of Rad51 protects lung cancer cells from the synergistic cytotoxic effects induced by emodin and MMC. We conclude that suppression of Rad51 expression or a combination of emodin with chemotherapeutic agents may be considered as potential therapeutic modalities for lung cancer.

Role of repair protein Rad51 in regulating the response to gefitinib in human non-small cell lung cancer cells

Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can block growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) activation. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas. In this study, we examined the role of Rad51 in regulating the response to gefitinib among different human lung cancer cell lines. The H520 line (human squamous cell carcinoma) was less sensitive to gefitinib compared with the H1650 (human adenocarcinoma) or A549 (human bronchioloalveolar carcinoma) lines. In H1650 and A549 cells but not in H520 cells, gefitinib decreased cellular levels of phospho-ERK1/2 and Rad51 protein and message levels. Moreover, gefitinib decreased Rad51 protein levels by enhancing Rad51 protein instability through 26S proteasome-mediated degradation. Inhibition of endogenous Rad51 levels by si-Rad51 RNA transfection significantly enhanced gefitinib-induced cytotoxicity. In contrast, transfection with constitutively active MKK1 vector could restore both Rad51 protein levels and cell survival inhibited by gefitinib. The MKK1/2-ERK1/2 signaling pathway constitutes the upstream signaling for maintaining Rad51 message and protein levels. Rad51 protein can protect lung cancer cells from cytotoxic effects induced by gefitinib. Suppression of Rad51 may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.

Blockage of NF‐κB by IKKβ‐ or RelA‐siRNA rather than the NF‐κB super‐suppressor IκBα mutant potentiates adriamycin‐induced cytotoxicity in lung cancer cells

Ambiguous roles of genotoxic anticancer therapeutic-induced NF-kappaB activation in regulating gene expression (activation or suppression) and apoptosis (anti- or pro-apoptosis) have recently been suggested. In order to clarify this controversy and determine the usefulness of NF-kappaB blockage for sensitizing anticancer therapy, we have systematically investigated the effect of distinct NF-kappaB-blocking approaches on lung cancer cells' responses to Adriamycin-induced cytotoxicity. The results show that Adriamycin-induced NF-kappaB activation functions as a transcriptional activator triggering the expression of anti-apoptotic genes. Blocking NF-kappaB with IKKbeta- or RelA siRNA substantially sensitized Adriamycin-induced cytotoxicity, suggesting that the NF-kappaB pathway could be a target for sensitizing lung cancer cells to Adriamycin's anticancer effect. Surprisingly, although it effectively blocks NF-kappaB activation, the IkappaBalpha super-suppressor (IkappaBalphaAA) antagonized Adriamycin-induced cell death. Additionally, the induction of death receptor 5 (DR5), which contributes to Adriamycin-induced cytotoxicity, was not affected by NF-kappaB blockage. Thus, our results suggest that Adriamycin-induced NF-kappaB is a transcriptional activator that protects lung cancer cells against apoptosis, and IKKbeta- or RelA siRNA rather than IkappaBalphaAA is an appropriate NF-kappaB blocking approach for sensitizing lung cancer cells to Adriamycin-induced cytotoxicity.

Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells

Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling activation. Rad51 is an essential component of the homologous recombination repair pathway. High level of Rad51 expression has been reported in chemo- or radioresistant carcinomas. We hypothesized that gefitinib may enhance the effects of the alkylating agent cisplatin- or the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing ERK1/2 activation and Rad51 expression. Exposure of human non-small lung cancer cells to gefitinib decreased cisplatin- or MMC-elicited ERK1/2 activation and Rad51 protein induction. Neither cisplatin nor MMC treatment affected Rad51 messenger RNA (mRNA). However, gefitinib cotreatment with cisplatin or MMC significantly decreased Rad51 mRNA levels. In addition, gefitinib decreased cisplatin- or MMC-elicited Rad51 protein levels by increasing Rad51 protein instability. Enhancement of ERK1/2 signaling by constitutively active mitogen-activated protein kinase kinase 1/2 (MKK1/2-CA) increased Rad51 protein levels and protein stability in gefitinib and cisplatin or MMC cotreated cells. Moreover, the synergistic cytotoxic effects induced by gefitinib cotreatment with cisplatin or MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced lung cancer cell death upon treatment with cisplatin or MMC. We conclude that Rad51 protein protects lung cancer cells from synergistic cytotoxic effects induced by gefitinib and chemotherapeutic agents. Suppression of Rad51 expression may be a novel lung cancer therapeutic modality to overcome drug resistance to EGFR inhibitors and chemotherapeutic agents.

The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, Iressa R) and benzo[ a]pyrene in human lung cancer

Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[ a]pyrene (B[ a]P) affects MAPKs transduction pathways. Gefitinib (Iressa R, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[ a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[ a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[ a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[ a]P co-treatment. Gefitinib enhances B[ a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[ a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[ a]P- and gefitinib-induced cytotoxicity, and B[ a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[ a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.

Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in human non-small cell lung cancer cells

Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been observed in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-xL proteins as survival factors in lung cancer cells since reductions in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a critical survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to determine expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Reduction in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-xL antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.