Pathway In Non-small Cell Lung Cancer Cells Research Articles

MiR-320a-3p/ELF3 axis regulates cell metastasis and invasion in non-small cell lung cancer via PI3K/Akt pathway

MicroRNAs (miRNAs) play important roles in tumorigenesis and tumor progression. In this study, we investigated the role of miR-320a-3p in non-small cell lung cancer (NSCLC). Expressions of miR-320a-3p were firstly determined in 80 NSCLC patients' cancer tissues and adjacent normal lung tissues by qRT-PCR. Then MTT assay, cell migration and invasion assays were performed in vitro. Potential binding sites on target gene of miR-320a-3p were predicted and luciferase reporter assay was used to identify the potential binding sites. Tumorigenesis assay were performed in nude mice by injecting A549 cells which stably express miR-320a-3p. Results indicated that high expression of miR-320a-3p suppresses cell proliferation, migration and invasion through the inactivation of PI3K/Akt signaling pathway in NSCLC cells. Smaller tumor size and lighter weight were also found in nude mice which had miR-320a-3p higher expressed. Furthermore, data from luciferase reporter assay proved the direct binding of miR-320a-3p on the 3′UTR region of ELF3 mRNA, this could further decrease ELF3 expression transcriptionally. We provided evidence that miR-320a-3p might work as a tumor suppressor in NSCLC both in vivo and in vitro.

Down-regulation of FOXR2 inhibits non-small cell lung cancer cell proliferation and invasion through the Wnt/β-catenin signaling pathway

Forkhead box R2 (FOXR2), a new member of the FOX family, is an important player in a wide range of cellular processes such as proliferation, migration, differentiation and apoptosis. Recently, FOXR2 has been reported to be implicated in cancer development. However, the biological functions of FOXR2 in non-small cell lung cancer (NSCLC) remain unclear. In this study, we investigated the specific role of FOXR2 in NSCLC. The results showed that down-regulation of FOXR2 significantly inhibited NSCLC cell proliferation and invasion in vitro and suppressed NSCLC cell growth and metastasis in vivo. In addition, the decrease in FOXR2 expression markedly reduced the protein levels of β-catenin, cyclinD1 and c-Myc and hence inactivated the Wnt/β-catenin pathway in NSCLC cells. Taken together, we concluded that FOXR2 might be considered as a promising therapeutic target for NSCLC treatment.

178P Smo inhibitor LDE225 reverses epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells

Background: LDE225 is a potent and selective oral inhibitor of Smo, a key component of the hedgehog (Hh) signaling pathway. It has been reported that LDE225 can inhibit the invasion in many solid tumors, and thus attenuate tumor metastasis, but its mechanism of action has been unclear in non-small cell lung cancers(NSCLCs). The aim of the present study was to evaluate whether LDE225 can reverse the epithelial-mesenchymal transition(EMT) induced by co-cultured with macrophage (THP-1) cell lines in NSCLC cells, and its possible mechanisms. Methods: The NSCLC cells (H460 and H1299) were co-cultured with THP-1 cells for 72h to obsreve EMT formation, and then were treated with 5μm LDE225 for 24h. Wound healing assays and transwell assays were used to detect the migratory and invasive activity of lung cancer cells. The EMT-related factors (E-cadherin and vimentin) were detected by western bloting analysis. SHH and GLI1, as the Hh pathway-related factors, were also detected by western bloting analysis. ELISA assays used to detect the concentration of TGF-β1 in the conditioned medium (CM) obtained from THP-1 cells before and after co-culture with NSCLC cells. Results: Our data showed that the expressions of E-cadherin and vimentin could significantly modulate after NSCLC cells co-culture with THP-1 cells, which further enhanced the capacity of the NSCLC cells to migrate and invade the extracellular matrix. LDE225 was able to compromise these effects. Additionally, LDE225 remarkably inhibited the activation of Hh signaling pathway in NSCLC cells after co-cultured with THP-1 cells. Further analysis showed that the concentration of TGF-β1 in the CM from co-culture of NSCLC cells & THP-1 cells was significantly lower in the presence of LDE225. It can suppresse the EMT and mimick the effect of LDE225 on NSCLC cells when administration of anti-TGF-β1 neutralizing antibody during co-culture. Conclusions: Our study confirmd that LDE225 can reverse lung cancer cells EMT and migration via downregulating the concentration of TGF-β1 in co-culture with THP-1 cells. Legal entity responsible for the study: 3rd Affiliated Hospital of Harbin Medical University Funding: National Natural Scientific Foundation of China (No: 81572824, 81673007 and 81773133), postdoctoral scientific research developmental fund of Heilongjiang Province, Health Department of Heilongjiang Provincial of China (No 2011-124) and Harbin Medical University Cancer Hospital major project Foundation (No: JJZ-2010-01). Disclosure: All authors have declared no conflicts of interest.

Jolkinolide B inhibits glycolysis by downregulating hexokinase 2 expression through inactivating the Akt/mTOR pathway in non-small cell lung cancer cells.

Jolkinolide B (JB), a bioactive compound isolated from herbal medicine, has been found to inhibit tumor growth by altering glycolysis. However, whether glycolysis is influenced by JB in non-small cell lung cancer (NSCLC) cells and the mechanism remain unknown. The aim of the present study was to evaluate the effect of JB on the glycolysis in NSCLC cells and the underlying molecular mechanism. The results showed that JB treatment inhibited cell viability of A549 and H1299 cells in a concentration-dependent manner. JB reduced the glucose consumption, lactate production, and HK2 expression. The expressions of p-Akt and p-mTOR were also decreased by JB treatment. Knockdown of HK2 reduced glucose consumption and lactate production. Inhibition of the Akt/mTOR pathway decreased HK2 expression and inhibited glycolysis. In conclusion, the results indicated that JB inhibits glycolysis by down-regulating HK2 expression through inactivating the Akt/mTOR pathway in NSCLC cells, suggesting that JB might be a potential therapeutic agent for the treatment of NSCLC.

Interferon regulatory factor3 mediates Poly(I:C)-induced innate immune response and apoptosis in non‑small cell lung cancer.

Immunotherapy is considered one of the most promising treatments for lung cancer. The cell signalling molecules melanoma differentiation-associated protein5 (MDA5) and retinoic acid-inducible geneI protein (RIG‑I) are essential receptors that recognise intracellular pathogen-associated nucleic acids, whereas interferon regulatory factor3 (IRF3) controls the expression of innate immunity-associated genes in macrophages. However, the innate immune response to polyinosinic:polycytidylic acid [Poly(I:C)] in lung cancer remains to be elucidated. In the present study, western blot analysis, reverse transcription-quantitative polymerase chain reaction, RNA interference, IRF3 plasmid construction, ELISA and apoptosis analysis were employed to study the innate immune response and apoptosis of non‑small cell lung cancer (NSCLC) cells. Poly(I:C) transfection in NSCLC cells triggered apoptosis via the extrinsic apoptotic pathway, and activated the innate immune response by promoting interferon-β and C-X-C motif chemokine ligand10 expression. Treatment with the IκB kinase ε/tumour necrosis factor receptor-associated factor family member-associated nuclear factor-κB activator-binding kinase1 inhibitor BX795, which inhibits IRF3 phosphorylation, or transfection with small interfering RNA/short hairpin RNA to downregulate MDA5, RIG‑I or IRF3, prior to Poly(I:C) transfection inhibited the innate immune response and apoptotic pathway. Conversely, IRF3 overexpression promoted activation of the apoptotic pathway, thus indicating that the MDA5/RIG‑I/IRF3 axis may mediate responses to Poly(I:C) transfection. Furthermore, phosphorylation of the transcription factor signal transducer and activator of transcription 1 (STAT1) was associated with the alterations in IRF3 phosphorylation and apoptosis, thus suggesting that STAT1 may be involved in Poly(I:C)-induced apoptosis. In NSCLC surgical samples, MDA5, RIG‑I and IRF3 were highly expressed, whereas the expression levels of phosphorylated‑IRF3 were reduced. These findings indicated that the function of the MDA5/RIG‑I/IRF3 axis may be impaired in some lung cancers. In conclusion, the present findings suggested that the MDA5/RIG‑I/IRF3 axis, which is associated with innate immunity, is intact in NSCLC cells, and IRF3 is involved in regulating the apoptotic pathway in NSCLC cells.

Targeting EHMT2 reverses EGFR-TKI resistance in NSCLC by epigenetically regulating the PTEN/AKT signaling pathway

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Epigenetic alterations have been shown to be involved in NSCLC oncogenesis; however, their function in EGFR-TKI resistance remains uncharacterized. Here, we found that an EHMT2 inhibitor, UNC0638, can significantly inhibit cell growth and induce apoptosis in EGFR-TKI-resistant NSCLC cells. Additionally, we also found that EHMT2 expression and enzymatic activity levels were elevated in EGFR-TKI-resistant NSCLC cells. Moreover, we determined that genetic or pharmacological inhibition of EHMT2 expression enhanced TKI sensitivity and suppressed migration and tumor sphere formation in EGFR-TKI-resistant NSCLC cells. Further investigation revealed that EHMT2 contributed to PTEN transcriptional repression and thus facilitated AKT pathway activation. The negative relationship between EHMT2 and PTEN was confirmed by our clinical study. Furthermore, we determined that combination treatment with the EHMT2 inhibitor and Erlotinib resulted in enhanced antitumor effects in a preclinical EGFR-TKI-resistance model. We also found that high EHMT2 expression along with low PTEN expression can predict poor overall survival in patients with NSCLC. In summary, our findings showed that EHMT2 facilitated EGFR-TKI resistance by regulating the PTEN/AKT pathway in NSCLC cells, suggesting that EHMT2 may be a target in the clinical treatment of EGFR-TKI-resistant NSCLC.

CPNE3 promotes migration and invasion in non-small cell lung cancer by interacting with RACK1 via FAK signaling activation.

Approximately 90% of patients diagnosed with non-small cell lung cancer (NSCLC) die due to distant metastases. However, the complicated molecular and cellular mechanisms involved in lung cancer metastasis remain poorly understood. Copine III (CPNE3), a member of a Ca2+-dependent phospholipid-binding protein family, was identified as a novel metastasis-associated protein in NSCLC in our previous study, however, its function in metastasis remains unclear. Here, we found that CPNE3 was expressed at high levels in NSCLC tissues and advanced TNM stages and was significantly associated with poor prognosis. In addition, CPNE3 interacted with phosphorylated erb-b2 receptor tyrosine kinase 2 (pErbB2) and receptor of activated protein C kinase 1 (RACK1) and activated the focal adhesion (FA) signaling pathway in NSCLC cells. Moreover, knockdown of RACK1 inhibited cell motility in the CPNE3-overexpressed NSCLC cells. These findings offer mechanistic insights into the oncogenic roles of CPNE3 and the pivotal effects of CPNE3 as a biomarker and therapeutic target for NSCLC metastasis.

Identification of Clinically Approved Drugs Indacaterol and Canagliflozin for Repurposing to Treat Epidermal Growth Factor Tyrosine Kinase Inhibitor-Resistant Lung Cancer.

In advanced lung cancer, epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs) have extraordinary clinical efficacy. However, their usefulness is severely compromised by drug resistance mediated by various mechanisms, the most important of which is the secondary EGFR T790M mutation. The mutation blocks the binding of EGFR TKIs to the receptor kinase, thereby abolishing the therapeutic efficacy. In this study, we used our free and open-source protein-ligand docking software idock to screen worldwide approved small-molecule drugs against EGFR T790M. The computationally selected drug candidates were evaluated in vitro in resistant non-small cell lung cancer (NSCLC) cell lines. The specificity of the drugs toward the mutant EGFR was demonstrated by cell-free kinase inhibition assay. The inhibition of EGFR kinase activity and its downstream signaling pathways in NSCLC cells was shown by immunoblot analysis. The positive hints were revealed to be indacaterol, canagliflozin, and cis-flupenthixol, all of which were shown to induce apoptosis in NSCLC cells harboring the EGFR T790M mutation. Moreover, the combination of indacaterol with gefitinib was also found to produce synergistic anticancer effect in NSCLC cells bearing EGFR T790M. The observed synergistic effect was likely contributed by the enhanced inhibition of EGFR and its downstream signaling molecules.

MiR-145-5p inhibits epithelial-mesenchymal transition via the JNK signaling pathway by targeting MAP3K1 in non-small cell lung cancer cells.

Lung cancer is one of the most common types of tumors and the leading cause of cancer-associated mortality in the world. Additionally, non-small cell lung cancer (NSCLC) accounts for ~80% of all lung cancer cases. Epithelial-mesenchymal transition (EMT) is an important cell biological process, which is associated with cancer migration, metastasis, asthma and fibrosis in the lung. In the present study, it was revealed that miR-145-5p was able to suppress EMT by inactivating the c-Jun N-terminal kinase (JNK) signaling pathway in NSCLC cells. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) was predicted and confirmed to be a novel target of miR-145-5p. Overexpression of MAP3K1 was able to reverse the inhibition of EMT induced by miR-145-5p via the JNK signaling pathway. Overall, the results revealed that miR-145-5p inhibits EMT via the JNK signaling pathway by targeting MAP3K1 in NSCLC cells.

CXCL12 suppresses cisplatin-induced apoptosis through activation of JAK2/STAT3 signaling in human non-small-cell lung cancer cells.

AimsPoor efficacy of chemotherapy drugs in non-small-cell lung cancer (NSCLC) is the key reason for the failure of treatment, but the mechanism of this remains largely unknown. Stromal cell-derived factor 1-alpha (SDF-1α/CXCL12) is a small chemotactic cytokine protein that plays an important role in tumor progression. In this study, we investigated the anti-apoptotic mechanism of the CXCL12/CXCR4 axis in response to cisplatin, a commonly used chemotherapeutic drug, in human lung adenocarcinoma A549 cells.MethodsCXCL12 blocks cisplatin-induced apoptosis in A549, and the results were shown by propidium iodide/annexin V staining in vitro. The mechanism of CXCL12 stimulating phosphorylation of STAT3 through CXCR4/JAK2 was demonstrated by immunofluorescence and Western blotting. The expression of CXCL12 and p-STAT3 in clinical specimens was examined by immunohistochemistry.ResultsCXCL12 significantly decreased the ratio of apoptotic cells and stimulation of phospho-signal transducer and activator of transcription (p-STAT)-3 in a time-dependent manner through interaction with CXCR4. Among the signaling molecules downstream of CXCR4, the JAK2/STAT3 pathway plays a predominant role in the anti-apoptotic effect of CXCL12. Analysis of clinical specimens revealed that increased CXCL12 and p-STAT3 expression correlates with enhanced lung cancer progression.ConclusionThese data suggest that CXCR4 contributes to CXCL12-mediated anti-apoptosis by activating JAK2/STAT3 pathway in NSCLC cells. Therefore, targeting CXCL12/CXCR4 signaling pathway reveals a potential therapeutic approach for NSCLC.

Long non-coding RNA NKILA inhibits migration and invasion of non-small cell lung cancer via NF-\u03baB/Snail pathway

BackgroundNumerous studies have shown that long non-coding RNAs (lncRNAs) play key roles during multiple cancer processes, such as cell proliferation, apoptosis, migration and invasion. The previous studies found that NKILA interacted with and suppressed the nuclear translocation of NF-KappaB, which influenced metastasis and prognosis in breast cancer. However the clinical significance and biological role of NKILA in non-small cell lung cancer (NSCLC) remains unknown.MethodsWe examined expression levels of NKILA in 106 pairs of NSCLC tissues and cell lines. The expression level of NKILA after TGF-β1 stimulation also was examined by qRT-PCR and validated by Chromatin immunoprecipitation (ChIP). Gain-of-function and loss-of-function assays were performed to examine the effect of NKILA on proliferation, migration and invasion of NSCLC cells. RNA immunoprecipitation (RIP), western blot and rescue experiments were carried out to reveal the interrelation between NKILA, NF-κB and EMT signal pathway.ResultsThe expression of NKILA was down-regulated in NSCLC cancer tissues compared with matched adjacent noncancerous tissues, and lower NKILA expression in tumor tissues were significantly correlated with lymph node metastasis and advanced TNM stage. We found that the expression of NKILA was mainly regulated by classical TGF-β signal pathway in NSCLC cells rather than NF-κB pathway reported in breast cancer. Gain and loss of function assays found that NKILA inhibited migration, invasion and viability of NSCLC cells. Mechanistic study showed that NKILA attenuated Snail expression via inhibiting the phosphorylation of IκBα and NF-κB activation, subsequently suppressed the expression of markers of epithelial-mesenchymal transition process.ConclusionsThe present study found that the expression of NKILA was downregulated in tumor tissues of NSCLC, which improved the metastasis of NSCLC patients. In vitro studies further clarified that the expression of NKILA was regulated through classical TGF-β signal pathway, which subsequently inhibited migration and invasion of NSCLC cells through interfering NF-κB/Snail signal pathway in NSCLC cells.

A novel pathway in NSCLC cells: miR-191, targeting NFIA, is induced by chronic hypoxia, and promotes cell proliferation and migration

MicroRNAs (miRs) have emerged as being important in cancer biology. miR‑191 is a conserved miRNA, which has been investigated in detail and is reported to be induced by hypoxia-inducible factor (HIF)‑1α and has an contributory action in the progression of breast, hepatic and pancreatic cancer. However, the effects of miR‑191 in the progression of lung cancer are a subject of debate. In the present study, it was found that the expression of miR-191 was significantly upregulated in non‑small cell lung cancer (NSCLC) cells in patients invivo. However, the levels of miR‑191 remained unchanged in SK‑MES‑1, A549 and NCI‑H460 NSCLC cell lines, compared with the level in the normal HBE lung cell line, however, the levels were markedly upregulated in these NSCLC cell lines under conditions of chronic hypoxia. Subsequently, an miR‑191 mimic was transfected into the NSCLC cell lines to examine its effect on the progression of the NSCLC cells invitro. The data obtained using MTT and Cell counting kit‑8 assays revealed that miR‑191 had no effect on the proliferation of the cells under normal condition, however, their proliferation was promoted under mild hypoxic conditions. In addition, the results of a Transwell migration assay showed that miR‑191 had a promoting effect on NSCLC cell migration under the conditions of chronic hypoxia. Furthermore, the TargetScan bioinformatics server and 3'-untranslated region luciferase reporter assay indicated that the transcription factor, nuclear factor 1α (NFIA) was a target of miR‑191. Subsequent western blot analysis showed that, in chronic‑hypoxia, the protein levels of NFIA and the tumor suppressor, CCAAT-enhancer-binding proteinα, were sharply reduced in A549 cells. In conclusion, miR‑191 was induced by chronic hypoxia and promoted the proliferation and migration of NSCLC cells under chronic hypoxic conditions. This promotion may be associated with its targeting of NFIA. The present findings may provide a potential molecular target for the therapeutic treatment of NSCLC.

Isoflurane Promotes Non-Small Cell Lung Cancer Malignancy by Activating the Akt-Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

BackgroundLung cancer is one of the leading causes of cancer mortalities worldwide, and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Surgery remains one of the front-line treatment options for NSCLC, but events within the perioperative period were found to affect cancer prognosis, such as anesthesia procedures. Isoflurane, a commonly used volatile anesthetic, enhances the malignant potential of renal, prostate, and ovarian cancer cells, but its effects on NSCLC development have not been previously reported.Material/MethodsCCK-8 and MTT cell proliferation assays were used to analyze NSCLC cell proliferation. Metastatic ability was examined by wound healing and transwell assays. We used Western blot analysis to study the mechanism of effect of Isoflurane in NSCLC development.ResultsWe demonstrated that isoflurane promotes proliferation, migration and invasiveness of NSCLC cells, as well as upregulation of the Akt-mTOR signaling pathway in NSCLC cells. Pharmacological inhibition of Akt-mTOR signaling abolished the ability of isoflurane to promote proliferation, migration, and invasion of NSCLC cells, indicating that isoflurane promotes NSCLC cell malignancy by activating the Akt-mTOR signaling pathway.ConclusionsIsoflurane promotes NSCLC proliferation, migration and invasion by activating the Akt-mTOR signaling pathway.

Knockdown of CUL4B Suppresses the Proliferation and Invasion in Non-Small Cell Lung Cancer Cells.

Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, was found to be overexpressed in many types of tumors. However, the expression pattern and role of CUL4B in non-small cell lung cancer (NSCLC) remain largely unknown. Therefore, in the present study, we investigated the role of CUL4B in NSCLC, and the underlying mechanism was also explored. Our results showed that CUL4B was highly expressed in NSCLC cell lines. Silencing CUL4B obviously inhibited proliferation and migration/invasion of NSCLC cells, and it also suppressed the epithelial–mesenchymal transition (EMT) progress in NSCLC cells. Furthermore, knockdown of CUL4B significantly inhibited the expression of β-catenin, cyclin D1, and c-Myc in NSCLC cells. Taken together, these results suggest that knockdown of CUL4B inhibited the proliferation and invasion through suppressing the Wnt/β-catenin signaling pathway in NSCLC cells. Therefore, CUL4B may represent a novel therapeutic target for the treatment of NSCLC.

PKIB promotes cell proliferation and the invasion-metastasis cascade through the PI3K/Akt pathway in NSCLC cells.

Lung cancer is one of the most common malignancies in the world, and non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Overgrowth of tumor cells usually results from the intensive proliferation of cancer cells, but the mechanisms by which the proliferation of cancer cells are promoted are currently unclear. Thus, it is necessary to determine the vital factors involved in regulating the growth of NSCLC. The MTT assay, BrdU assay, western blots, and migration and invasion assays were used in our study. Here, we found that PKIB (cAMP-dependent protein kinase inhibitor-β), a novel molecular target, was up-regulated in NSCLC tissues compared with the normal tissues adjacent to the tumors. Moreover, overexpression of PKIB promoted cell proliferation and potentiated the invasion and migration in A549 cells, whereas knocking down PKIB gene expression inhibited the proliferation and attenuated the invasive behavior and metastasis in H1299 cells. However, all of these effects of PKIB on cell proliferation and metastasis were reduced by inhibiting the PI3K/Akt pathway. Our results indicate that PKIB promotes cell proliferation and tumorigenesis by activating the PI3K/Akt pathway in NSCLC, implying that this is an important underlying mechanism that affects the progression of NSCLC.

Abstract 4028: Oncogenic KRAS mutations induce PD-L1 overexpression through MAPK pathway activation in non-small cell lung cancer cells

Abstract The proto-oncogene KRAS is a frequently mutated oncogenic drivers in non-small cell lung cancer (NSCLC) and druggable molecular targets for KRAS-mutated NSCLC have been under investigation. Recent studies have reported promising results of nivolumab, a programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, for previously treated NSCLC patients and suggested that the programmed-death ligand 1 (PD-L1) is a candidate surrogate marker for the anti-PD-1 therapy. By microarray analysis, we found that short hairpin RNA (shRNA)-mediated stable knockdown of mutant KRAS downregulated the expression of the CD274 gene (encoding PD-L1) in the NSCLC cell lines, H358 (-2.68 fold-change) and H441 (-1.98 fold-change). Additionally, we confirmed that small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, reduced CD274 mRNA expression in these cell lines. As expected, the CD274 expression levels were higher in these cell lines when examining the expression in a panel of NSCLC cell lines by quantitative RT-PCR analysis. Furthermore, CD274 mRNA expression was downregulated by MEK and ERK inhibitors (U0126, FR180204, selumetinib) in H358 and H441 cells. On the other hand, the expression analysis also revealed that CD274 mRNA levels vary among KRAS-mutated cell lines. These results suggest that oncogenic KRAS mutations induce PD-L1 overexpression through activation of the MAPK pathway in NSCLC cells, whereas other unknown mechanisms that modulate PD-L1 expression underlie in different KRAS-mutated NSCLCs. Citation Format: Yosuke Miura, Noriaki Sunaga, Kyoichi Kaira, Yusuke Tsukagoshi, Takashi Osaki, Reiko Sakurai, Takeshi Hisada, Luc Girard, John D. Minna, Masanobu Yamada. Oncogenic KRAS mutations induce PD-L1 overexpression through MAPK pathway activation in non-small cell lung cancer cells. [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 4028.

Abstract B08: Keap1–Nrf2 interaction suppresses cell motility in lung adenocarcinomas by targeting the S100P protein

Abstract Purpose: Kelch-like ECH-associated protein 1 (Keap1) is an E3 ligase participated in the cellular defense response against oxidative stress through nuclear factor erythroid-2–related factor 2 (Nrf2). However, the role of Keap1 in regulating cancer motility is still controversial. We investigated the contribution of the Keap1–Nrf2 axis in the progression of non-small cell lung cancer (NSCLC). Experimental Design: The expression of Keap1 and Nrf2 was examined via immunohistochemistry, real-time PCR, and Western blot analysis in a cohort of NSCLC tissues and cells. A series of in vivo and in vitro assays was performed to elucidate the contribution of the Keap1–Nrf2 axis in lung cancer mobility and progression. Results: Keap1 expression was decreased in specimens from NSCLC patients with lymph node metastasis compared with patients without metastasis. Higher Keap1 expression levels were correlated with the survival ofNSCLC patients. Moreover,manipulation of Keap1 expression affected cell migration/invasion abilities. Depletion of Nrf2 relieved the migration promotion imposed by Keap1 suppression. Mechanistic investigations found that S100Pwas downregulated in both Keap1-overexpressing and Nrf2-knockdown NSCLC cells. Overexpression of Keap1 and knockdown of Nrf2 both suppressed S100P expression in NSCLC cells. Knockdown of S100P inhibited cell migration in highly invasive NSCLC cells and also relieved the migration promotion imposed by Keap1 suppression in weakly invasive NSCLC cells. Conclusions: Our findings suggest that Keap1 functions as a suppressor of tumor metastasis by targeting the Nrf2/S100P pathway in NSCLC cells. In addition, overexpression of Keap1 may be a novel NSCLC treatment strategy and/or useful biomarker for predicting NSCLC progression. Citation Format: Kuo-Tai Hua, Min-Liang Kuo. Keap1–Nrf2 interaction suppresses cell motility in lung adenocarcinomas by targeting the S100P protein. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B08.

Stathmin1 increases radioresistance by enhancing autophagy in non-small-cell lung cancer cells.

Radioresistance has been demonstrated to be involved in the poor prognosis of patients with non-small-cell lung cancer (NSCLC). However, the underlying mechanism remains largely unclear. Investigation on special therapeutic targets associated with radioresistance shows promises for the enhancement of clinical radiotherapy effect toward NSCLC. This study aimed to reveal the role of Stathmin1 (STMN1) in radioresistance in NSCLC as well as the underlying mechanism. Our data showed that the protein levels of STMN1 were significantly upregulated in NSCLC cells subjected to radiation, accompanied with the activation of autophagy. Knockdown of STMN1 expression enhanced the sensitivity of NSCLC cells to X-ray, and the radiation-induced autophagy was also inhibited. Molecular mechanism investigation showed that knockdown of STMN1 expression upregulated the activity of phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway in NSCLC cells. Moreover, the activation of PI3K/mTOR signaling showed an inhibitory effect on the autophagy and radioresistance induced by STMN1 in NSCLC cells. In addition, luciferase reporter assay data indicated that STMN1 was a direct target gene of miR-101, which had been reported to be an inhibitor of autophagy. Based on these data, we suggest that as a target gene of miR-101, STMN1 promotes the radioresistance by induction of autophagy through PI3K/mTOR signaling pathway in NSCLC. Therefore, STMN1 may become a potential therapeutic target for NSCLC radiotherapy.

Cinobufagin inhibits tumor growth by inducing intrinsic apoptosis through AKT signaling pathway in human nonsmall cell lung cancer cells.

The cinobufagin (CB) has a broad spectrum of cytotoxicity to inhibit cell proliferation of various human cancer cell lines, but the molecular mechanisms still remain elusive. Here we observed that CB inhibited the cell proliferation and tumor growth, but induced cell cycle arrest and apoptosis in a dose-dependent manner in non-small cell lung cancer (NSCLC) cells. Treatment with CB significantly increased the reactive oxygen species but decreased the mitochondrial membrane potential in NSCLC cells. These effects were markedly blocked when the cells were pretreated with N-acetylcysteine, a specific reactive oxygen species inhibitor. Furthermore, treatment with CB induced the expression of BAX but reduced that of BCL-2, BCL-XL and MCL-1, leading to an activation of caspase-3, chromatin condensation and DNA degradation in order to induce programmed cell death in NSCLC cells. In addition, treatment with CB reduced the expressions of p-AKTT308 and p-AKTS473 and inhibited the AKT/mTOR signaling pathway in NSCLC cells in a time-dependent manner. Our results suggest that CB inhibits tumor growth by inducing intrinsic apoptosis through the AKT signaling pathway in NSCLC cells.

MicroRNA-26b suppresses the metastasis of non-small cell lung cancer by targeting MIEN1 via NF-κB/MMP-9/VEGF pathways

MicroRNAs (miRNAs) are involved in tumor initiation and progression. MiR-26b was reported to be significantly downregulated in non-small cell lung cancer (NSCLC). However, the underlying mechanisms of miR-26b involvement in the development and progression of NSCLC remains poorly understood. In the present study, we report that miR-26b suppresses cell metastasis in NSCLC through targeting migration and invasion enhancer 1 (MIEN1). We found that miR-26b was significantly downregulated and MIEN1 was significantly upregulated in both NSCLC tissues and cells lines. The expression levels of miR-26b were negatively related to those of MIEN1 mRNA in clinical NSCLC tissues. Furthermore, MIEN1 was confirmed to be a direct target of miR-26b by dual-luciferase reporter assay and MIEN1 expression was downregulated by miR-26b in NSCLC cells. In terms of function, transwell and wound healing assays demonstrated that the miR-26b remarkably inhibited invasion and migration of NSCLC cells, which was simulated by siRNA knockdown of MIEN1 and reversed by pcDNA/MIEN1 overexpression of MIEN1. Finally, we found that miR-26b could regulate NF-κB/MMP-9/VEGF pathway in NSCLC cells. In conclusion, this study revealed that miR-26b suppresses NSCLC metastasis by targeting MIEN1 via NF-κB/MMP-9/VEGF pathways, implicating a potential prognostic biomarker and therapeutic target for NSCLC treatment.