Chemoresistance In Non-small Cell Lung Cancer Research Articles

Abstract 4393: A pooled shRNA screen in 3D cultures of primary tumor propagating cells identifies regulators of innate chemoresistance in KRAS-driven NSCLC

Abstract Despite advances developing targeted therapies for non-small cell lung cancer (NSCLC), patients carrying activating KRAS mutations still have no effective targeted therapeutics and respond poorly to chemotherapy. Discovering drug targets that work in combination with existing chemotherapeutics to inhibit innate chemoresistance and prevent re-initiation of tumor growth could reduce the likelihood of relapse and extend patient survival. Heterogeneous tumor propagating cell (TPC) populations in NSCLC have been shown to have increased chemoresistance, an enhanced ability to re-seed tumors in immunocompromised mice, and express genes associated with poor prognosis. To target chemoresistance in TPCs, we have developed a 3D culture model for functional genomic screens using primary tumor cells from a mouse model of NSCLC driven by activation of KRAS and p53 loss. Using pooled shRNA libraries, we have performed a focused screen in 3D TPC cultures of over 3,000 shRNAs targeting 600 genes associated with chemotherapeutic response in TPCs, TPC gene expression, DNA damage response, or bulk tumor resistance. We selected targets for validation, characterization, and in vivo studies if knockdown of a gene selectively sensitized TPCs to platinum-based chemotherapy or inhibited growth of TPCs. Multiple DNA damage response pathways were implicated in chemoresistance including known regulators, such as ATR and the translesion synthesis pathway. Many genes expressed selectively by TPCs alone or in response to chemotherapy were required for chemoresistance, including regulators of mitochondrial transport, RNA export, and DNA replication. A parallel screen using the KRAS-mutant LKR10 cell line was used as a control for shRNA representation and to determine which targets are required for TPCs to survive in 3D but not in 2D cultures. 3D-specific vulnerabilities include genes that are expressed highly in TPCs or in chemoresistant tumors and represent potential KRAS vulnerabilities that exist in the TPC population. Most chemoresistance targets and KRAS vulnerabilities are not shared by LKR10 cells grown in 2D suggesting growth in a multidimensional matrix exposes therapeutic opportunities that are distinct from cells grown as monolayers. Although functional genomic screens using primary tumor cells in 3D have limited throughput, focused screens utilizing primary tumor cells have the potential to discover therapeutic opportunities that exist in vivo that have not been uncovered in screens utilizing cell lines in 2D. Citation Format: David Richard Simpson, Dedeepya Vaka, Yanyan Zheng, John Tamaresis, Alejandro Sweet-Cordero. A pooled shRNA screen in 3D cultures of primary tumor propagating cells identifies regulators of innate chemoresistance in KRAS-driven NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4393.

Chemoresistance factors in non-small cell lung cancer

Lung cancer takes the leading position in cancer morbidity and mortality structure in the world and Ukraine. Advanced stages are diagnosed in more than 70% of lung cancer patients. Chemotherapy remains the main method of treatment for advanced lung cancer. Currently, the standard of chemotherapy in patients with III–IV stages of non-small cell lung cancer (NSCLC) is a combination of cisplatin/carboplatin with paclitaxel, etoposide, gemcitabine and vinorelbine. Although, the objective response rate after chemotherapy treatment is only about 20–50%. Low rates of chemotherapy efficacy are connected with chemoresistance of tumor to definite chemotherapeutic agents. Tumor chemoresistance is the result of different tumor intracellular mechanisms activity. Basing on current data we tried to analyze prognostic and predictive value of main chemoresistance factors in NSCLC. Conclusions . Nowadays we can certainly affirm that chemoresistance to cisplatin in NSCLC is due to increased expression of ERCC1 (Excision repair cross-complementation group 1 protein), BCRP; and resistance to taxanes is connected with elevated expression of β-tubuline III class; and tumoral chemoresistance to gemcitabine is due to high RRM1 (Ribinucleotide reductase M1) expression. High expression of BRCA1 (Breast Cancer 1) protein in tumors leads to increase of tumor cells resistance to cisplatin, but at the same time it increases the sensitivity to taxanes. Therefore, investigation of tumor chemoresistance plays an important role in algorithms’ creation for optimal chemotherapy schemes selection in patients with different stages of NSCLC in order to achieve the best tumor response to treatment and improve overall and disease-free survival. The information of main NSCLC chemoresistance factors, its prognostic and predictive value are presented in this overview.

MiR-96 induces cisplatin chemoresistance in non-small cell lung cancer cells by downregulating SAMD9.

Cisplatin is effective as a single agent or in combination with other drugs for the treatment of non-small cell lung cancer (NSCLC). A concerning clinical challenge with cisplatin-based NSCLC chemotherapy is the intrinsic and acquired chemoresistance to cisplatin. The sterile α motif domain-containing (SAMD9) gene has been reported as a potent tumor suppressor gene that inhibits tumorigenesis and progression of NSCLC. microRNAs (miRNA) have been revealed to play important roles in the regulation of cancer chemoresistance. To the best of our knowledge the present study explored the role of miRNA/SAMD9 signaling in regulating cisplatin chemoresistance in NSCLC for the first time. Out of the several candidate miRNAs predicted to bind the 3′-untranslated region (UTR) of the SAMD9 gene, miRNA-96 (miR-96) demonstrated significant target-sequence-specific inhibition of the SAMD9 3′-UTR luciferase reporter activity in NSCLC cells. In addition, while NSCLC tumor samples exhibited significantly higher expression levels of miR-96 compared with adjacent normal tissues, the expression levels of SAMD9 were significantly lower than those in adjacent normal tissues. miR-96 and SAMD9 were overexpressed and knocked down in the human NSCLC H358 and H23 cell lines and the half maximal inhibitory concentration (IC50) of cisplatin and cell apoptosis rate under cisplatin treatment were used as measures of cisplatin chemoresistance. The present results identified that overexpression of miR-96 in NSCLC cells markedly decreased SAMD9 expression and cisplatin-induced apoptosis, and increased the cisplatin IC50, which could be eliminated by overexpression of SAMD9. By contrast, knocking down miR-96 in NSCLC cells using antagomir-96 significantly increased SAMD9 expression and the cisplatin-induced apoptosis and decreased cisplatin IC50, which could be completely reversed by a knockdown of SAMD9. In conclusion, the current study demonstrates that miR-96 targets and downregulates SAMD9 in NSCLC, which decreases cisplatin-induced apoptosis and induces cisplatin chemoresistance in NSCLC cells. The findings of the present study add novel insights into the function of miR-96 and SAMD9 in cancer, as well as into the molecular mechanisms underlying NSCLC chemoresistance.

Overexpression of Sphk2 is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer is the major cause of cancer-related death worldwide, and 80% of them are non-small cell lung cancer (NSCLC) cases. Recent studies have shown that sphingosine kinase 2 (SphK2) could promote tumor progression; however, whether SphK2 could affect the chemoresistance of NSCLC to chemotherapy remains unclear. To determine whether SphK2 serves as a potential therapeutic target of NSCLC, we utilized small interference RNA (siRNA) to knock down SphK2 expression in human NSCLC cells and analyzed their phenotypic changes. The data demonstrated that knockdown of SphK2 led to decreased proliferation and enhanced chemosensitivity and apoptosis to gefitinib in NSCLC cells. In this study, we describe the findings that overexpression of SphK2 promotes chemoresistance in NSCLC cells. Inhibition of SphK2 might be considered as a strategy in NSCLC treatment with gefitinib.

Curcumin-induced downregulation of Axl receptor tyrosine kinase inhibits cell proliferation and circumvents chemoresistance in non-small lung cancer cells.

Lung cancer is still in the first place in terms of both incidence and mortality. In the present study, we demonstrated the effect of curcumin, a phytochemical of the plant Curcuma longa, on expression and activation of Axl receptor tyrosine kinase (RTK) which plays an important role in cell survival, proliferation and anti-apoptosis. Curcumin treatment of non-small cell lung cancer (NSCLC) A549 and H460 cells, was found to decrease Axl protein as well as mRNA levels in a dose- and time-dependent manner. Axl promoter activity was also reduced by curcumin, indicating that curcumin downregulates Axl expression at the transcriptional level. Moreover, Axl phosphorylation in response to binding of its ligand, Gas6, was abrogated by curcumin, suggesting the inhibitory effect of curcumin on Gas6-induced Axl activation. We next found cytotoxic effect of cucumin on both the parental A549 and H460 cells, and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) and paclitaxel (A549/TR and H460/TR). Exposure of these cells to curcumin resulted in dose-dependent decline of cell viability and clonogenic ability. It is further observed that the anti-proliferative effect of curcumin on A549 cells overexpressing Axl protein was reduced, while that on H460 cells transfected Axl specific siRNA was augmented, confirming that curcumin inhibits cell proliferation via downregulation of Axl expression. In addition, curcumin was found to cause the induction of p21, a cyclin-dependent kinase inhibitor, and reduction of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic molecule, in parental H460 cells as well as chemoresistant cells, H460/CisR and H460/TR. Taken together, our data imply that Axl RTK is a novel target of curcumin through which it exerts anti-proliferative effect in both parental and chemoresistant NSCLC cells.

TAB3 overexpression promotes cell proliferation in non-small cell lung cancer and mediates chemoresistance to CDDP in A549 cells via the NF-κB pathway.

Transforming growth factor-activated kinase 1 (TAK1)-binding protein 3 (TAB3) is essential for the activation of the nuclear factor kappa B (NF-κB) pathway and has important roles in cell survival. However, the contribution of TAB3 to non-small cell lung cancer (NSCLC) remains elusive. In the present study, Western blotting and immunohistochemistry assays demonstrated that TAB3 expression was frequently increased in NSCLC tissues and cells. In addition, chi-square test and Kaplan-Meier analysis revealed that upregulation of TAB3 expression correlated with a more invasive tumor phenotype and poor prognosis. In addition, a series of experiments, including serum starvation-refeeding experiment and TAB3-siRNA transfection assay, showed that TAB3 expression promoted NSCLC cell proliferation. Furthermore, the effect of TAB3 expression on the sensitivity to cis-diamminedichloroplatinum (CDDP) and possible signaling transduction pathways was investigated. When the expression of TAB3 was inhibited by siRNA transfection, the sensitivity to CDDP was enhanced. Moreover, it showed that downregulation of TAB3 enhanced CDDP-induced A549 cell apoptosis through the inhibition of the NF-κB pathway. These results suggest that TAB3 plays a critical role in NSCLC progression and chemoresistance and that TAB3 depletion may be a promising approach to lung cancer therapy.

Abstract 3660: Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer

Abstract Background: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. βIII-tubulin (encoded by TUBB3 gene) is aberrantly expressed and is associated with chemoresistance and tumor aggressiveness in NSCLC (1), where it has been identified as a bona fide target for chemosensitisation (2,3). In order to understand how TUBB3/βIII-tubulin is regulated in NSCLC cells we sought to identify chemical small molecules that can modulate its expression. Methods: H460 cells expressing a TUBB3 or GAPDH promoter-luciferase reporter construct were generated and used in drug screening and promoter activity testing. To isolate modulators of TUBB3 promoter activity, a cell-based screen of libraries of diverse chemical small molecules (30K) and FDA-approved bioactives (3680) was performed. Cell viability, growth and proliferation were measured using standard methods. Cell cycle was assessed using flow cytometry, and gene and protein expression by RT-PCR and Western blotting, respectively. Microtubule morphology was assessed using immunostaining and confocal microscopy. Drug-treated clonogenic assays were used to quantitate changes in drug sensitivity. Results: Based upon their ability to modulate TUBB3 promoter activity, we identified two hit compounds, CCI01 and CCI02, as well as the bioactive compound, RITA. For all three leads we observed: 1) repression in TUBB3 promoter activity which was not a result of cell cytotoxicity; 2) no effects on cell cycle or viability, but instead cytostatic effects; 3) significantly enhanced TUBB3 expression in a time and dose-dependent manner. TUBB3 gene enhancement was translated at the protein level in CCI01 treated H460, but not in CCI02 or RITA treated cells. Additionally, CCI01 did not alter microtubule morphology but enhanced βIII-tubulin immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, CCI01 enhanced βIII-tubulin expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents in H460 cells. Conclusion: A novel lead small molecule TUBB3/βIII-tubulin enhancer has been identified that is able to increase expression of βIII-tubulin in NSCLC and significantly reduce sensitivity to tubulin binding and DNA-damaging agents. Implications: Identification of a modulator of TUBB3/βIII-tubulin expression will provide a valuable research tool to probe βIII-tubulin regulation. (1) Kavallaris. Nature Rev Cancer, 10:194-204, 2010 (2) McCarroll et al., Cancer Res 70:4995-5003, 2010 (3) Gan et al., Cancer Res. 67:9356-9363, 2007 Citation Format: Felicity Chao Lin Kao, Tim Failes, Greg M. Arndt, Murray Norris, Maria Kavallaris. Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2015-3660

Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer.

Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer.

The Clinical Relevance of the miR-197/CKS1B/STAT3-mediated PD-L1 Network in Chemoresistant Non-small-cell Lung Cancer

Programmed cell death ligand-1 (PD-L1) has recently gained considerable attention for its role in tumor immune escape. Here, we identify a miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer (NSCLC), independent of immunoinhibitory signals. miR-197 is downregulated in platinum-resistant NSCLC specimens, resulting in the promotion of chemoresistance, tumorigenicity, and pulmonary metastasis in vitro and in vivo. Mechanistic investigations reveal that a miR-197-mediated CKS1B/STAT3 axis exerts tumor progression regulated by various oncogenic genes (Bcl-2, c-Myc, and cyclin D1), and PD-L1 is a putative biomarker of this axis. Furthermore, we demonstrate that a miR-197 mimic sensitizes PD-L1high drug-resistant cells to chemotherapy. These results indicate that the biological interaction between PD-L1 and chemoresistance occurs through the microRNA regulatory cascade. More importantly, expression levels of miR-197 are inversely correlated with PD-L1 expression (n = 177; P = 0.026) and are associated with worse overall survival (P = 0.015). Our discoveries suggest that the miR-197/CKS1B/STAT3-mediated network can drive tumor PD-L1 expression as a biomarker of this cascade, and miR-197 replacement therapy may be a potential treatment strategy for chemoresistant NSCLC.

Tudor staphylococcal nuclease drives chemoresistance of non-small cell lung carcinoma cells by regulating S100A11.

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC), the major lung cancer subtype, is characterized by high resistance to chemotherapy. Here we demonstrate that Tudor staphylococcal nuclease (SND1 or TSN) is overexpressed in NSCLC cell lines and tissues, and is important for maintaining NSCLC chemoresistance. Downregulation of TSN by RNAi in NSCLC cells led to strong potentiation of cell death in response to cisplatin. Silencing of TSN was accompanied by a significant decrease in S100A11 expression at both mRNA and protein level. Downregulation of S100A11 by RNAi resulted in enhanced sensitivity of NSCLC cells to cisplatin, oxaliplatin and 5-fluouracil. AACOCF(3), a phospholipase A(2) (PLA(2)) inhibitor, strongly abrogated chemosensitization upon silencing of S100A11 suggesting that PLA(2) inhibition by S100A11 governs the chemoresistance of NSCLC. Moreover, silencing of S100A11 stimulated mitochondrial superoxide production, which was decreased by AACOCF(3), as well as N-acetyl-L-cysteine, which also mimicked the effect of PLA(2) inhibitor on NSCLC chemosensitization upon S100A11 silencing. Thus, we present the novel TSN-S100A11-PLA(2) axis regulating superoxide-dependent apoptosis, triggered by platinum-based chemotherapeutic agents in NSCLC that may be targeted by innovative cancer therapies.

Critical contribution of MCL-1 in EMT-associated chemo-resistance in A549 non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of death in all lung cancer patients due to its metastatic spread. Even though cisplatin treatment after surgical resection of the primary tumor has been established as a standard chemotherapy for residual disease including metastatic spread, NSCLC often acquires a resistance against chemotherapy, and metastatic disease is often observed. Amongst many potential mechanisms, epithelial-to-mesenchymal transition (EMT) has been considered as an important process in acquiring both metastatic spread and chemo-resistance of NSCLC. In this study, we identified MCL-1 as a critical molecule for chemo-resistance in A549 cells associated with TGF-β-induced EMT. Importantly, downregulation of MCL-1 by siRNA or inhibition of MCL-1 with pan-BCL2 inhibitor to inhibit MCL-1 was able to overcome the EMT-associated chemo-resistance in A549 cells. Collectively, MCL-1 can be a new therapeutic target for overcoming EMT-associated chemo-resistance in NSCLC patients in the context of post-operative chemotherapies.

Evaluation of Antitumor Activity Using Change in Tumor Size of the Survivin Antisense Oligonucleotide LY2181308 in Combination with Docetaxel for Second-Line Treatment of Patients with Non–Small-Cell Lung Cancer: A Randomized Open-Label Phase II Study

Chemoresistance is mediated, in part, by the inhibition of apoptosis in tumor cells. Survivin is an antiapoptotic protein that blocks chemotherapy-induced apoptosis. To investigate whether blocking survivin expression enhances docetaxel-induced apoptosis in patients with non-small-cell lung cancer (NSCLC), we compared the antitumor activity of the survivin inhibitor LY2181308 plus docetaxel with docetaxel alone. We used change in tumor size (CTS) as a primary endpoint to assess its use in early decision-making for this and future studies of novel agents in NSCLC. Patients (N = 162) eligible for second-line NSCLC treatment (stage IIIB/IV) with an Eastern Cooperative Oncology Group performance status of 0 to 1 were randomized 2:1 to receive LY2181308 (750 mg intravenously, weekly) and docetaxel (75 mg/m intravenously, day 1) or docetaxel alone every 21 days. CTS from baseline to the end of cycle 2 was compared between the two treatment arms. The mean (SD) tumor size ratio for LY2181308/docetaxel and docetaxel was 1.05 (0.21) and 1.00 (0.15) (p = 0.200), respectively, suggesting no significant improvement in antitumor activity between the arms. Because there was also no significant difference between the two arms for progression-free survival (PFS) (2.83 months with LY2181308/docetaxel and 3.35 months with docetaxel [p = 0.191]), both arms were combined. Using the combined arms, CTS correlated with PFS (PFS = 4.63 months in patients with decreased CTS compared with 2.66 months in patients with increased CTS), supporting its use in early decision-making in phase II studies.

Redesigning the DNA-targeted chromophore in platinum-acridine anticancer agents: a structure-activity relationship study.

Platinum-acridine hybrid agents show low-nanomolar potency in chemoresistant non-small cell lung cancer (NSCLC), but high systemic toxicity in vivo. To reduce the promiscuous genotoxicity of these agents and improve their pharmacological properties, a modular build-click-screen approach was used to evaluate a small library of twenty hybrid agents containing truncated and extended chromophores of varying basicities. Selected derivatives were resynthesized and tested in five NSCLC cell lines representing large cell, squamous cell, and adenocarcinomas. 7-Aminobenz[c]acridine was identified as a promising scaffold in a hybrid agent (P1-B1) that maintained submicromolar activity in several of the DNA-repair proficient and p53-mutant cancer models, while showing improved tolerability in mice by 32-fold compared to the parent platinum-acridine (P1-A1). The distribution and DNA/RNA adduct levels produced by the acridine- and benz[c]acridine-based analogues in NCI-H460 cells (confocal microscopy, ICP-MS), and their ability to bind G-quadruplex forming DNA sequences (CD spectroscopy, HR-ESMS) were studied. P1-B1 emerges as a less genotoxic, more tolerable, and potentially more target-selective hybrid agent than P1-A1.

Chapter Five - Gefitinib

Gefitinib (Iressa®) is a selective inhibitor of epidermal growth factor, a growth factor that plays a pivotal role in the control of cell growth, apoptosis, and angiogenesis. Gefitinib is clinically used for the treatment of chemoresistant non-small cell lung cancer patients. Gefitinib is freely soluble in dimethylsulphoxide but slightly soluble in methanol and ethanol. Several methods of gefitinib synthesis are included in this review. UV spectroscopy of gefitinib showed a λmax of approximately 331nm, whereas IR spectroscopy principal peaks were observed at 3400cm(-1) (NH), 2956cm(-1) (CH2, CH, alkyl), 1625cm(-1) (CC, CN), 1500cm(-1) (HCCH, aryl), 1110cm(-1) (CO), 1028cm(-1) (CF). In addition, different analytical methods for determination of gefitinib are also described in this review. Pharmacokinetically, after oral administration, gefitinib is slowly absorbed with bioavailability of approximately 60% in human. Gefitinib is metabolized extensively in the liver into five metabolites by cytochrome P450s, primarily by CYP3A4 and to a lesser extent by CYP3A5 and CYP2D6. Gefitinib is eliminated mainly hepatically with total plasma clearance of 595mL/min after intravenous administration. Most of the adverse effects associated with gefitinib therapy are mild to moderate in severity and are usually reversible and manageable with appropriate intervention, such as diarrhea, dry skin, rash, nausea, and vomiting.

MiR-205 promotes the growth, metastasis and chemoresistance of NSCLC cells by targeting PTEN

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related mortality worldwide. microRNAs (miRNAs) play critical roles in carcinogenesis. miR-205 has been shown to be upregulated in NSCLC. In the present study, we identified the promotive effects of miR-205 on various significant biological properties of NSCLC cells, and confirmed the regulation of PTEN by miR-205. The expression of miR-205 was examined by quantitative real-time PCR both in NSCLC cell lines and tissues. The effect of miR-205 on PTEN expression was assessed in NSCLC cell lines with miR-205 mimics/inhibitor to elevate/decrease miR-205 expression. Furthermore, the roles of miR-205 in regulating the biological properties of NSCLC cells, including growth, invasion and chemoresistance, were assayed using miR-205 mimic/inhibitor-transfected cells. The 3'-untranslated region (3'-UTR) of PTEN combined with miR-205 and this was confirmed by luciferase reporter assay and western blotting. miR-205 expression was increased in NSCLC cell lines as well as in tissues. Overexpression of miR-205 promoted growth, migration and invasion, and enhanced the chemoresistance of NSCLC cells. Luciferase activity and western blotting demonstrated that miR-205 negatively regulated PTEN at a posttranscriptional level. However, miR-205 knockdown suppressed these processes in A549 cells and increased the expression of PTEN protein. Furthermore, overexpression of PTEN exhibited effects identical with those of the miR-205 inhibitor in NSCLC cells. Our results demonstrated that miR-205 is involved in the tumorigenesis of NSCLC through modulation of the PTEN signaling pathway.

Elevated Expression of BIRC6 Protein in Non–Small-Cell Lung Cancers is Associated with Cancer Recurrence and Chemoresistance

Non-small-cell lung cancer (NSCLC) is an aggressive, highly chemoresistant disease. Reliable prognostic assays and more effective treatments are critically required. BIRC6 (baculoviral inhibitors of apoptosis proteins repeat-containing 6) protein is a member of the inhibitors of apoptosis protein family thought to play an important role in the progression or chemoresistance of many cancers. In this study, we investigated whether BIRC6 expression can be used as a prognostic marker or potential therapeutic target for NSCLC. In a retrospective analysis, BIRC6 protein expression was determined for 78 resected primary NSCLCs and nine benign lung tissues. Twenty-nine chemoresistant or chemosensitive subrenal capsule NSCLC tissue xenografts were assessed for BIRC6 expression, using immunohistochemistry, and 13 of them for BIRC6 gene copy number, using array comparative genomic hybridization analysis. The effect of small interfering RNA-induced BIRC6 knockdown on the growth of human NSCLC cell cultures and apoptosis (in combination with cisplatin) was investigated. Elevated BIRC6 protein expression in NSCLC tissues was associated with poor 3-year relapse-free patient survival, lymph node involvement, and advanced pathological tumor, node, metastasis stage. In patient-derived lung squamous cell carcinoma xenografts, chemoresistance was associated with elevated BIRC6 expression and increased gene copy number. Small interfering RNA-induced BIRC6 down-regulation inhibited growth of the NSCLC cells and sensitized the cells to cisplatin. BIRC6 may play an important role in the malignant progression and chemoresistance of NSCLC. Elevated BIRC6 protein expression may serve as a predictive marker for chemoresistance of NSCLCs and a poor prognostic factor for NSCLC patients. Down-regulation of the BIRC6 gene as a therapeutic approach may be effective, especially in combination with conventional chemotherapeutics.

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines

Five-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance.

Increased expression and function of P-glycoprotein in peripheral blood CD56+ cells is associated with the chemoresistance of non-small-cell lung cancer

Chemoresistance is common among non-small-cell lung cancer (NSCLC), P-glycoprotein (P-gp), encoded by the human multi-drug-resistant MDR1 gene, and multidrug-resistance protein 1 (MRP1) might be major contributors. The aim of the present study was to develop an effective method to investigate the expression and function of P-gp in the peripheral CD56+ cells in order to clarify their correlation with the chemoresistance in NSCLC. Using microbead technology and a RT-qPCR methodology, we evaluated the expression levels of P-gp and MRP1 in the purified CD56+ cells in the chemoresistance and chemo-naive NSCLC patients compared with that in the healthy volunteers. Flow cytometric analysis was used to investigate the changes of P-gp function in the CD56+ cells between the three cohorts. The MDR1 gene expression was elevated markedly (twofold-tenfold), and P-gp function was increased in the chemoresistance cohort compared with the chemo-naive and the healthy cohorts; whereas there was only about two times averagely elevated for the MRP1 gene expression. No statistical significance (p > 0.05) was seen with respect to the expression of MDR1 and MRP1, the function of P-gp between the chemo-naive and the healthy cohorts. P-gp in peripheral CD56+ cells demonstrated possible clinical relevance as predictive biomarkers for the identification of chemoresistance in NSCLC, while MRP1 may not play a significant role in the drug resistance in NSCLC. The potential applications for this finding are provided evidence to screen the potential P-gp reversors and to diagnose and manage the chemoresistance in NSCLC patients.

Abstract LB-399: Genome-wide expression analysis of subtypes of non-small cell lung carcinoma in highly degraded formalin-fixed paraffin-embedded tissue

Abstract Previous studies demonstrated that gene expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues are largely comparable to fresh frozen matched tissue. This technology can assist in discovering biomarkers of lung cancer prognosis and progression by vastly increasing in the potential resources available for cancer research. In this study, we investigated the potential use of the Whole-Genome DASL HT Assay (Illumina, San Diego, CA) for global expression profiling in FFPE tissues obtained by surgical biopsy from patients with early stage non-small cell lung cancer (NSCLC). We obtained high-quality microarray data on 26 pairs of tumor/non-affected tissues, including 18 pairs of adenocarcinoma and 8 pairs of squamous carcinomas. Among genes differentially expressed between squamous carcinomas and adjacent non-affected lung tissue, we found that the AKR1C1 gene, encoding an aldo-keto reductases, was increased strongly in tumors (fold change: 3.0; p=0.02). This finding was consistent with previous reports that overexpression of AKR1C1 gene as an indicator of poor prognosis and chemoresistance in NSCLC. During pathway-based analysis using GeneGo database, we found that differentially expressed genes of adenocarcinoma were significantly enriched in the pathway of cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling; and differentially expressed genes of squamous carcinomas were significantly enriched in the pathway of Lung Neoplasms_Signal transduction. Our study demonstrates that with DASL expression profiling technology, genome-wide expression profiles can be successfully generated from FFPE lung tissues. Support: CA092824 and CA CA90578, and CA074386 from NCI 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 LB-399. doi:1538-7445.AM2012-LB-399

Molecular mechanisms for the regulation of Nrf2-mediated cell proliferation in non-small-cell lung cancers

We previously demonstrated that the transcription factor NF-E2-related factor2 (Nrf2), expressed abundantly in non-small-cell lung cancer (NSCLC) cells, plays a pivotal role in the proliferation and chemoresistance of NSCLC. Here we show that Nrf2-mediated NSCLC cell proliferation is dually regulated by epidermal growth factor receptor (EGFR) signaling and an Nrf2 repressor protein Keap1 (Kelch-like ECH-associated protein-1). NSCLC cells expressing wild-type EGFR and Keap1 genes show enhanced proliferation on stimulation with EGFR ligand under non-stress conditions. Exposure to cigarette smoke extract (CSE) enhanced cell proliferation by modification of the Nrf2/Keap1 interaction. Although EGFR-tyrosine kinase inhibitor (TKI) inhibited the proliferation of these cells, exposure to CSE attenuated its efficacy. In NSCLC cells with Keap1 gene mutations, Nrf2 was constitutively activated owing to dysfunction of Keap1 and cells proliferated independently of EGFR signaling. Furthermore, EGFR-TKI was unable to inhibit their proliferation. In NSCLC cells with EGFR gene mutations, Nrf2 was constitutively activated by EGFR signaling. In these cells, proliferation was largely dependent on the EGFR signaling pathway. Although these cells were highly sensitive to EGFR-TKI, exposure to CSE or knockdown of Keap1 mRNA reduced sensitivity to EGFR-TKI. We found a case of NSCLC showing resistance to EGFR-TKI despite having EGFR-TKI-sensitive EGFR gene mutation because of dysfunctional mutation in Keap1 gene. Results indicate that oxidative stress reduces the anticancer effects of EGFR-TKI in wild-type Keap1 NSCLC cells. Analysis of Keap1 dysfunction may become a novel molecular marker to predict resistance to EGFR-TKI in NSCLC cells having EGFR-TKI-sensitive EGFR mutations. Finally, as the downstream molecule of both EGFR and Keap1 signaling, Nrf2 is an important molecular target for the treatment of NSCLC, where cells have mutations in EGFR, KRAS or Keap1 genes.