Inhibited Non-small Cell Lung Cancer Cell Growth Research Articles

AC-93253 iodide, a novel Src inhibitor, suppresses NSCLC progression by modulating multiple Src-related signaling pathways

BackgroundThe tyrosine kinase Src is involved in the progression of many cancers. Moreover, inhibiting Src activity has been shown to obstruct several signaling pathways regulated by the EGFR. Thus, Src is a valuable target molecule in drug development. The purpose of this study was to identify compounds that directly or indirectly modulate Src to suppress lung cancer cell growth and motility and to investigate the molecular mechanisms underlying the effects of these compounds.MethodsHuman non-small cell lung cancer (NSCLC) cell lines (PC9, PC9/gef, A549, and H1975) with different EGFR statuses were tested by cytotoxicity and proliferation assays after AC-93253 iodide treatment. Src and Src-related protein expression in AC-93253 iodide-treated PC9, PC9/gef, and A549 cells were assessed by western blotting. The effects of AC-93253 iodide on cancer cell colony formation, invasion, and migration were assessed in PC9 and PC9/gef cells. The synergistic effects of gefitinib and AC-93253 iodide were evaluated by combination index (CI)-isobologram analysis in gefitinib-resistant cell lines. The efficacy of AC-93253 iodide in vivo was determined using nude mice treated with either the compound or the vehicle.ResultsAmong the compounds, AC-93253 iodide exhibited the most potent dose-independent inhibitory effects on the activity of Src as well as on that of the Src-related proteins EGFR, STAT3, and FAK. Furthermore, AC-93253 iodide significantly suppressed cancer cell proliferation, colony formation, invasion, and migration in vitro and tumor growth in vivo. AC-93253 iodide sensitized tumor cells to gefitinib treatment regardless of whether the cells were gefitinib-sensitive (PC9) or resistant (H1975 and PC9/gef), indicating that it may exert synergistic effects when used in combination with established therapeutic agents. Our findings also suggested that the inhibitory effects of AC-93253 iodide on lung cancer progression may be attributable to its ability to modulate multiple proteins, including Src, PI3K, JNK, Paxillin, p130cas, MEK, ERK, and EGFR.ConclusionsOur data suggest that AC-93253 iodide inhibits NSCLC cell growth and motility by regulating multiple Src-related pathways. Our findings may facilitate the development of therapeutic strategies and anti-tumor drugs that may be useful for treating lung cancer in the future.

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation.

The molecular pathogenesis of human lung cancer has not been completely clarified. Here, we reported that UBE2L3, a member of the ubiquitin-conjugating enzymes (E2s), were overexpressed in non-small-cell lung cancer (NSCLC) tissues compared with the non-tumor tissues. High expression of UBE2L3 was correlated with advanced tumor stage and adverse outcomes. Knockdown of UBE2L3 inhibited NSCLC cell growth while ectopic expression of UBE2L3 promoted NSCLC cell growth in a cell cycle dependent manner. The results of subcutaneous tumor xenograft studies revealed that knockdown of UBE2L3 attenuated the in vivo tumor growth. Mechanistically, we observed that UBE2L3 could interact with F-box protein Skp2, a member of the SCF (Skp2) ubiquitin ligase complex, and thus promoted the ubiquitination and proteasomal degradation of p27kip1. Furthermore, NSCLC cases with high level of UBE2L3 and low level of p27kip1 had worst prognosis, suggesting that combination of UBE2L3 and p27kip1 is a more powerful prognostic marker for NSCLC patients. Taken together, the current study presented a novel marker for predicting prognosis and a potential therapeutic target for NSCLC patients.

Abstract 4037: High throughput kinase inhibitor screen reveals novel inhibitor combinations acting in synergy with TUBB3/βIII-tubulin suppression in non-small cell lung cancer

Abstract Background: Non-Small Cell Lung Cancer (NSCLC) survival rates remain dismal and this malignancy is poorly responsive to current therapy. The mechanisms contributing to this resistance phenotype and aggressive behavior of this disease are not well defined. TUBB3/βIII-tubulin is aberrantly expressed in NSCLC and its expression correlates negatively with disease free survival of patients. Our group has previously shown that βIII-tubulin plays a functional role in chemotherapy resistance and tumorigenesis in NSCLC via modulation of the PTEN/AKT signaling pathway1, 2. This study aimed to identify kinase inhibitors that synergize with βIII-tubulin inhibition in NSCLC using a high-throughput cell-based screen. Methods: A high-throughput cell-based screen of a kinase inhibitor library was performed against our previously described1, 2 NSCLC H460 cells, expressing either βIII-tubulin shRNA or control non-targeting shRNA. The primary screen consisted of a total of 210 kinase inhibitors tested at a single, fixed concentration and cell viability determined using the Alamar blue assay. Secondary screens were performed using a five point dose response. Further validation of the kinase inhibitors was performed against two independent NSCLC, H460 and A549 expressing either βIII-tubulin shRNA or control non-targeting shRNA. Results: A number of kinase inhibitors that act on key signalling cascades were identified to act in synergy with TUBB3/βIII-tubulin knockdown to inhibit NSCLC cell growth. Interestingly, several different inhibitors which target mTOR were identified as top candidates for acting in combination with βIII-tubulin knockdown to inhibit NSCLC cell growth. Treatment of cells with clinically approved mTOR inhibitors, Temsirolimus and Rapamycin, resulted in a significant decrease in cell viability in the βIII-tubulin knockdown cells compared to control. Notably, mTOR acts downstream of AKT signalling and further highlights a potential connection with βIII-tubulin and the AKT signal transduction cascade. Other kinase inhibitors that act on key signaling cascades including Amuvatinib-Multi Kinase Inhibitor (Flt3, Met, Kit, c-Ret & PDGFR); Mubritinib-HER-2 inhibitor; and Neratinib HER2, EGFR, also acted in combination with βIII-tubulin knockdown to inhibit NSCLC cell growth. Conclusions: Our results suggest that clinically approved therapies designed to target mTOR combined with inhibition of TUBB3/βIII-tubulin may offer a novel therapeutic strategy for the treatment of NSCLC patients.

Inhibition of NOB1 by microRNA-330-5p overexpression represses cell growth of non-small cell lung cancer.

MicroRNAs (miRNAs) play critical roles in the development and progression of various cancers, including non-small-cell lung cancer (NSCLC). Studies have suggested that miR-330-5p is involved in the progression of several cancers. However, the role of miR-330-5p in NSCLC remains unclear. We investigated the effect on and mechanism of miR-330-5p in the progression of NSCLC. We found that miR-330-5p was significantly downregulated in NSCLC tissues and cell lines as detected by real-time quantitative polymerase chain reaction (RT-qPCR). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bromodeoxyuridine (BrdU), colony formation and cell cycle assays showed that overexpression of miR-330-5p markedly inhibited cell growth. AnnexinV-FITC/PI and caspase-3 activity assays showed that overexpression of miR-330-5p significantly promoted cell apoptosis of NSCLC cells. Bioinformatics analysis and dual-luciferase reporter assays confirmed NIN/RPN12 binding protein1 (NOB1) as a target gene of miR-330-5p. RT-qPCR and Western blot analysis showed that overexpression of miR-330-5p inhibited the expression of NOB1 as well as cyclinD1 and cyclin-dependent kinase4 in NSCLC cells. Moreover, overexpression of NOB1 markedly reversed the miR‑330-5p-mediated inhibitory effect on NSCLC cell growth. Correlation analysis showed that miR‑330-5p expression was inversely correlated with NOB1 mRNA expression in NSCLC tissues. Taken together, our results indicate that miR-330-5p inhibits NSCLC cell growth through downregulation of NOB1 expression. Our study suggests that miR-330-5p may serve as a potential therapeutic target for the treatment of NSCLC.

Dihydroartemisinin and gefitinib synergistically inhibit NSCLC cell growth and promote apoptosis via the Akt/mTOR/STAT3 pathway.

Non‑small cell lung cancer (NSCLC) is among the leading causes of cancer‑associated mortality worldwide. In clinical practice, therapeutic strategies based on drug combinations are often used for the treatment of various types of cancer. The present study aimed to investigate the effects of the combination of dihydroartemisinin (DHA) and gefitinib on NSCLC. Cell Counting kit 8 assay was used to evaluate cell viability. Transwell assays were performed to investigate cellular migration and invasion, and cellular apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick‑end labeling assay. Flow cytometry was used to investigate cell cycle distribution and the expression levels of target proteins were determined using western blot analysis. The results of the present study demonstrated that DHA (5, 10, 20, 50 and 100µM) reduced cancer cell viability in a dose‑dependent manner in the NCI‑H1975 human NSCLC cell line and significantly enhanced gefitinib‑induced apoptosis. Furthermore, DHA and gefitinib co‑administration induced cell cycle arrest in G2/M phase, which was associated with a marked decline in the protein expression levels of G2/M regulatory proteins, including cyclin B1 and cyclin‑dependent kinase 1. The addition of DHA appeared to potentiate the inhibitory actions of gefitinib on the migratory and invasive capabilities of NCI‑H1975 cells. DHA and gefitinib co‑administration also downregulated the expression levels of phosphorylated (p)‑Akt, p‑mechanistic target of rapamycin, p‑signal transducer and activator of transcription 3 and B‑cell lymphoma 2 (Bcl‑2), and upregulated the expression of Bcl‑2‑associated X protein. In conclusion, the present results suggested that the combination of DHA and gefitinib may have potential as a novel and more effective therapeutic strategy for the treatment of patients with NSCLC.

Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells

BackgroundLitchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood.MethodsIn this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined.ResultsThe results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling.ConclusionThe results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.

MicroRNA-346 facilitates cell growth and metastasis, and suppresses cell apoptosis in human non-small cell lung cancer by regulation of XPC/ERK/Snail/E-cadherin pathway.

Determinants of growth and metastasis in cancer remain of great interest to define. MicroRNAs (miRNAs) have frequently emerged as tumor metastatic regulator by acting on multiple signaling pathways. Here we report the definition of miR-346 as a novel oncogenic microRNA that facilitates non-small cell lung cancer (NSCLC) cell growth and metastasis. XPC, an important DNA damage recognition factor in nucleotide excision repair was defined as a target for down-regulation by miR-346, functioning through direct interaction with the 3′-UTR of XPC mRNA. Blocking miR-346 by an antagomiR was sufficient to inhibit NSCLC cell growth and metastasis, an effect that could be phenol-copied by RNAi-mediated silencing of XPC. In vivo studies established that miR-346 overexpression was sufficient to promote tumor growth by A549 cells in xenografts mice, relative to control cells. Overall, our results defined miR-346 as an oncogenic miRNA in NSCLC, the levels of which contributed to tumor growth and invasive aggressiveness.

MicroRNA-3666-induced suppression of SIRT7 inhibits the growth of non-small cell lung cancer cells.

Sirtuin7 (SIRT7) plays an important role in many cancer types, but its function in non-small cell lung cancer (NSCLC) remains unclear. This study investigated the biological role and underlying mechanism of SIRT7 in NSCLC. Results showed that SIRT7 was highly expressed in NSCLC cell lines, as detected by real-time quantitative polymerase chain reaction and western blot analysis. SIRT7 knockdown by small interfering RNA (siRNA) significantly inhibited the growth of NSCLC cells and induced their apoptosis. Bioinformatics algorithms indicated that SIRT7 was a putative target of microRNA-3666 (miR-3666). Dual-luciferase reporter assay demonstrated that miR-3666 could target the 3'-untranslated region of SIRT7. Western blot analysis revealed that miR-3666 could regulate the protein expression of SIRT7. The miR-3666 overexpression significantly inhibited NSCLC cell growth. The restoration of SIRT7 protein expression significantly abrogated the effect of the miR-3666 overexpression. Moreover, SIRT7 depletion induced by siRNA or miR-3666 overexpression promoted the expression of pro-apoptotic genes. Taken together, our study suggests that SIRT7 functions as an oncogene in NSCLC, and miR-3666 can target SIRT7 to inhibit NSCLC cell growth by promoting the pro-apoptotic signaling pathway. Thus, this study provides novel insights into the development of new and potential treatments for NSCLC.

Alternative splicing of S6K1 promotes non-small cell lung cancer survival

Ribosomal S6 kinase 1 (S6K1) that acts downstream of the mammalian target of rapamycin (mTOR) plays an important role in cell proliferation, protein translation, and cell survival. The gene RPS6KB1 encoding for S6K1 had been found to be alternatively spliced to form different isoforms. In this study, we identified that short isoforms of S6K1 splice variant were overproduced in non-small cell lung cancer (NSCLC). Moreover, suppression of S6K1 short isoforms inhibited NSCLC cell growth and induced apoptosis via upregulation of the BH3-only protein Bim in vitro and in vivo. Additionally, short isoforms of S6K1 activated mTORC1, leading to increased 4E-BP1 phosphorylation. Taken together, our findings suggested that S6K1 short isoforms were deregulated in NSCLC and promoted cell survival. Altogether, our study opens possibilities of new therapeutic approaches for NSCLC that selectively downregulate S6K1 shorter isoforms.

Abstract 1063: miR-195 represses the tumorigenesis of non-small cell lung cancer and synergizes with microtubule targeting agents

Abstract MicroRNAs (miRNAs) play important roles in nearly all cellular physiological and pathological pathways. Dysregulated miRNAs have been shown to contribute to tumorigenesis of many cancers, including lung cancer. The let-7 miRNA family, among the first miRNAs discovered, suppresses the growth of non-small cell lung cancer (NSCLC). Other miRNAs have been shown to regulate the response of cancer cells to anticancer agents, such as the microtubule-targeting agent (MTA) paclitaxel. These findings highlight the potential application of miRNAs as the next generation of therapeutic agents, either alone or in combination with chemotherapy. A high throughput screen was performed in NSCLC cells. miRNAs that both inhibited NSCLC cell growth and sensitized NSCLC cells to paclitaxel were validated by short- and long-term assays for cell viability. The targets of miRNAs were identified by expression profiling following miRNA transfection, validated by luciferase reporter assay, and confirmed by functional assays. We show that miR-195 causes G1 phase arrest of NSCLC cells by targeting CCND3, and leads to apoptosis and reduction of cell migration and invasion by targeting BIRC5. Conversely, the inhibition of cell growth by miR-195 is compromised by overexpressing CCND3 or BIRC5; the inhibition of cell migration and invasion by miR-195 is abrogated by overexpressing BIRC5. We also show that miR-195 synergizes with MTAs paclitaxel and eribulin to inhibit the growth of NSCLC cells by regulating CHEK1. Analysis of miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) shows that miR-195 expression is negatively correlated with BIRC5 and CHEK1 but not CCND3. Clinical data from TCGA show that miR-195 is significantly down-regulated in lung tumors compared to adjacent normal tissues and that its down-regulation in lung cancer patients is associated with worse survival. The TCGA data also show that BIRC5 and CHEK1 are significantly up-regulated in lung tumors and that their up-regulation is associated with worse survival. CCND3, however, is slightly downregulated in lung tumors and its expression is not correlated with patient survival. Intriguingly, we also show that miR-195 up-regulates the oncogene MYC, inhibition of which sensitizes NSCLC cells to miR-195. We show that Myc, reported to transcriptionally repress let-7c, also represses miR-195, and further identify a feedback loop between let-7c and miR-195 in which each inhibits the other. We identify miR-195 as a tumor suppressor and chemotherapy sensitizer in NSCLC mediated by its repression of CCND3, BIRC5 and CHEK1. We also uncover a feedback network between miR-195, Myc and let-7c. The ability of miR-195 and Myc to regulate each other indicates a mechanism by which NSCLC cells can become more sensitive to miR-195. The mutual inhibition between miR-195 and let-7c suggests an interesting regulatory loop between cancer-related miRNAs that deserves further study. Citation Format: Xiaojie Yu, Zhenze Zhao, Xiuye Ma, Liqin Du, Alexander Pertsemlidis. miR-195 represses the tumorigenesis of non-small cell lung cancer and synergizes with microtubule targeting agents. [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 1063.

MicroRNA-376c suppresses non-small-cell lung cancer cell growth and invasion by targeting LRH-1-mediated Wnt signaling pathway

MicroRNAs (miRNAs) that negatively regulate gene expression have emerged as novel therapeutic tools for cancer treatment. In this study, we investigated the potential role of Liver receptor homolog-1 (LRH-1), a novel oncogene, in non-small-cell lung cancer (NSCLC), and examined the regulation of LRH-1 by miRNAs. We found that LRH-1 was highly overexpressed in NSCLC cell lines. Knockdown of LRH-1 by small interfering RNA significantly inhibited NSCLC cell growth and invasion. miR-376c directly targeted the 3’-untranslated region (UTR) of LRH-1 and negatively regulated LRH-1 expression, as detected by dual-luciferase reporter assay, real-time quantitative polymerase chain reaction and Western blot analysis. Further data showed that miR-376c expression was inversely correlated with LRH-1 expression in clinical cancer samples. Overexpression of miR-376c could inhibit NSCLC cell growth and invasion as well as Wnt signaling. In contrast, depletion of miR-376c exhibited the opposite effects. Moreover, these effects of miR-376c overexpression were partially abrogated by overexpression of LRH-1. Taken together, these results indicate that LRH-1 is involved in regulating the growth and invasion of NSCLC cells and that miR-376c inhibits NSCLC cell growth and invasion by targeting LRH-1, providing a novel insight into the potential for development of anti-cancer drugs for NSCLC.

JMJD1A promotes tumorigenesis and forms a feedback loop with EZH2/let-7c in NSCLC cells.

Lung cancer is the most common cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) accounts for 80 to 85% of all lung cancer. Although the standard treatment regimen has been established, long-term survival for NSCLC patients is still generally poor. The histone demethylase Jumonji domain containing 1A (JMJD1A) has been proposed as an oncogene in several types of human cancer, but its clinical significance and functional roles in NSCLC remain largely unclear. In the present study, JMJD1A was frequently upregulated in NSCLC compared with para-carcinoma tissues. JMJD1A knockdown significantly inhibited NSCLC cell growth, migration, and invasion in vitro and tumorigenesis in vivo. Further experiments demonstrated that JMJD1A knockdown could decrease the expression of EZH2, which has been shown to play a crucial role in the carcinogenesis of NSCLC and, in turn, increase the expression of anti-tumor microRNA let-7c. Also, let-7c directly targeted the 3'-untranslated regions of JMJD1A and EZH2. Taken together, JMJD1A could promote NSCLC tumorigenesis. JMJD1A/EZH2/let-7c constituted a feedback loop and might represent a promising therapeutic target for NSCLC.

Chinese herbal medicine Xiaoji decoction inhibited growth of lung cancer cells through AMPKα-mediated inhibition of Sp1 and DNA methyltransferase 1

Ethnopharmacological relevanceXiaoji decoction (XJD), which was considered as a Chinese herbal prescription, has been used for cancer treatment, especially lung cancer, for decades to improve quality of life and prolong the patient survival. However, the molecular mechanisms underlying the therapeutic potential have not been well elucidated. Materials and methodsThe cell viability was examined by MTT assays. The phosphorylation and expression of AMP-activated protein kinase alpha (AMPKα), DNA methyltransferase 1 (DNMT1) and transcription factor Sp1 proteins were assessed by Western Blot. Exogenous expression of Sp1 and DNMT1 were performed by transient transfection methods. The effects of XJD on the growth of xenograft tumors were evaluated by in vivo bioluminescence imaging. ResultsWe showed that XJD inhibited growth of human non small cell lung cancer (NSCLC) cells in vitro. We also found that XJD increased phosphorylation of AMPKα and inhibited protein expression of DNTM1, the latter was not observed in the presence of the inhibitor of AMPK (compound C). Overexpression of DNTM1 reversed the effect of XJD on cell growth. In addition, XJD decreased Sp1 protein expression, which was eliminated by compound C. Conversely, exogenous expressed Sp1 abrogated XJD-inhibited DNTM1 protein expression. Interestingly, exogenous expression of DNMT1 feedback antagonized the XJD-induced phosphorylation of AMPKα. In in vivo studies, we found that XJD inhibited tumor growth in xenograft nude mice model, which was accompanied by induction of phosphorylation of AMPKα and suppression of DNMT1 protein from xenograft tumors. ConclusionOur results show that XJD inhibits NSCLC cell growth via AMPKα-mediated inhibition of transcription of Sp1, followed by the reduction of DNMT1 expression both in vitro and in vivo. The negative feedback regulation loop of AMPKα further demonstrates the critical role of DNMT1 in mediating the overall effects of XJD in this process. This study unveils novel molecular mechanism by which XJD controls NSCLC cell growth.

Astragaloside IV Enhances Cisplatin Chemosensitivity in Non-Small Cell Lung Cancer Cells Through Inhibition of B7-H3

Background: Chemoresistance is a major obstacle to successful chemotherapy for human non-small cell lung cancer (NSCLC). Astragaloside IV, the component of Astragalus membranaceus, has been reported to exhibit anti-inflammation, anti-cancer and immunoregulatory properties. In the present study, we investigated the role of astragaloside IV in the chemoresistance to cisplatin in NSCLC cells. Methods: We established astragaloside IV-suppressed NSCLC cell lines including A549, HCC827, and NCI-H1299 and evaluated their sensitivity to cisplatin in vitro. In addition, we examined the mRNA and protein levels of B7-H3 in response to cisplatin-based chemotherapy. Results: We showed that high doses of astragaloside IV (10, 20, 40 ng/ml) inhibited NSCLC cell growth, whereas low concentrations of astragaloside IV (1, 2.5, 5 ng/ml) had no obvious cytotoxicity on cell viability. Moreover, combined treatment with astragaloside IV significantly increased chemosensitivity to cisplatin in NSCLC cells. On the molecular level, astragaloside IV co-treatment significantly inhibited the mRNA and protein levels of B7-H3 in the presence of cisplatin. In addition, ectopic expression of B7-H3 diminished the sensitization role of astragaloside IV in cellular responses to cisplatin in NSCLC cells. Conclusion: These results demonstrate that astragaloside IV enhances chemosensitivity to cisplatin via inhibition of B7-H3 and that treatment with astragaloside IV and inhibition of B7-H3 serve as potential therapeutic approach for lung cancer patients.

Skp2 regulates non-small cell lung cancer cell growth by Meg3 and miR-3163.

Maternally expressed gene 3 (Meg3) encodes a long non-coding RNA that has been shown to play a role in tumorigenesis. Skp2 is a component of the E3 ubiquitin ligase SCF that specifically promotes the ubiquitination-associated degradation of CDK inhibitor p27, and has been shown to promote cancer cell growth in different types of cancers, including non-small cell lung cancer (NSCLC). Nevertheless, a regulatory relationship between Meg3 and Skp2 has not been acknowledged. Here, we showed that NSCLC specimens had significant higher levels of Skp2 and significantly lower levels of Meg3, compared to paired non-tumor lung tissue. The levels of Meg3 and Skp2 were inversely correlated in NSCLC specimens. Patients with low Meg3 levels had a poor survival. Overexpression of Meg3 decreased Skp2 protein and increased p27 protein, while depletion of Meg3 increased Skp2 protein and decreased p27 protein in NSCLC cells, without altering Skp2 mRNA. These data suggest that the Skp2 may be regulated by Meg3 at post-transcriptional level. Bioinformatics analyses showed that miR-3163 bound to 3'-UTR of Skp2 mRNA in NSCLC cells to inhibit its translation, which was supported by luciferase reporter assay. Meg3 augmented the effects of miR-3163 on Skp2 mRNA, possibly through binding-induced function enhancement, which was supported by the double fluorescent in situ hybridization showing co-localized intracellular Meg3 and miR-3163 signals in NSCLC cells. The miR-3163 levels in NSCLC were not different from in NT, suggesting that the regulation of Skp2 in NSCLC by miR-3163 may require coordination of Meg3. Thus, our data suggest that Meg3 and miR-3163 may coordinate suppression of translation of Skp2 mRNA in NSCLC cells to inhibit NSCLC cell growth.

Abstract 4372: Phosphodiesterase 10A inhibition suppresses lung tumor cell growth by activating PKG to inhibit ras and Wnt signaling

Abstract We recently reported that phosphodiesterase 10A (PDE10) is overexpressed in colon tumors and essential for colon tumor cell growth (Li et al., Oncogene 2014), but a role in lung cancer has not been well studied. Human non-small cell lung cancer (NSCLC) cell lines were found to express appreciably higher PDE10 levels compared with normal airway epithelial cells, while silencing of PDE10 expression with siRNA or inhibition of PDE10 activity with small molecule inhibitors selectively inhibits NSCLC cell growth. Here we study the mechanism by which PDE10 inhibitors suppress NSCLC cell growth. At concentrations effective for inhibiting NSCLC cell growth, the PDE10 inhibitor, PQ10 rapidly increases intracellular cGMP levels as determined by a luminescence biosensor assay. Within the same concentration range and time period, PQ10 activates PKA and PKG, demonstrated by an increase in phosphorylation of the substrate, VASP at Ser157 and Ser239, respectively. Under the same conditions, PQ10 suppresses the phosphorylation of Erk1/2 (Thr202/Tyr204), MEK1/2 (Ser217/Ser221) and down-stream p90RSK (Ser380). Similarly, PQ10 increases the phosphorylation of β-catenin at Ser552, but showed no effect on phospho-Ser675 levels. After longer durations of treatment, PQ10 reduces β-catenin levels, as well as proteins that are regulated by β-catenin-dependent TCF/LEF-transcriptional activity, such as survivin and cyclin D1. These effects were mimicked by a novel structurally unrelated PDE10 inhibitor, ADT-020 that selectively inhibits the growth of NSCLC cells and increases cGMP levels in a manner similar to PQ10. ADT-020 also activates PKG in NSCLC cells demonstrated by higher phospho-Ser239 VASP levels and lower β-catenin and survivin levels. These observations suggest that PDE10 inhibition and activation of PKG can simultaneously suppress ras and Wnt signaling that are essential for lung tumor cell growth. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Bing Zhu, Kevin Lee, Joshua Canzoneri, Veronica Ramirez-Alcantara, Sara Sigler, Bernard Gary, Ethan Butler, Adam Keeton, Xi Chen, Michael Boyd, Gary Piazza. Phosphodiesterase 10A inhibition suppresses lung tumor cell growth by activating PKG to inhibit ras and Wnt signaling. [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 4372. doi:10.1158/1538-7445.AM2015-4372

MiR-143 inhibits NSCLC cell growth and metastasis by targeting Limk1.

MicroRNAs (miRNAs) have essential roles in carcinogenesis and tumor progression. Here, we investigated the roles and mechanisms of miR-143 in non-small cell lung cancer (NSCLC). miR-143 was significantly decreased in NSCLC tissues and cell lines. Overexpression of miR-143 suppressed NSCLC cell proliferation, induced apoptosis, and inhibited migration and invasion in vitro. Integrated analysis identified LIM domain kinase 1 (Limk1) as a direct and functional target of miR-143. Overexpression of Limk1 attenuated the tumor suppressive effects of miR-143 in NSCLC cells. Moreover, miR-143 was inversely correlated with Limk1 expression in NSCLC tissues. Together, our results highlight the significance of miR-143 and Limk1 in the development and progression of NSCLC.

5‐Demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis

Tangeretin (TAN) and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human nonsmall cell lung cancer (NSCLC) cells. Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50 s of 5DT were 79-fold, 57-fold, and 56-fold lower than those of TAN in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while TAN at tenfold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21(Cip1/Waf1), and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7-4.9 fold higher than those of TAN after the cells were treated with 5DT or TAN at the same concentration. Our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by TAN, which is partially due to the higher intracellular uptake of 5DT than TAN.

Berberine Targets AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth.

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

A Novel Muscarinic Antagonist R2HBJJ Inhibits Non-Small Cell Lung Cancer Cell Growth and Arrests the Cell Cycle in G0/G1

Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.