Inhibits Lung Cancer Growth Research Articles

Inhibition of lung cancer growth and metastasis by DHA and its metabolite, RvD1, through miR-138-5p/FOXC1 pathway

BackgroundNon small cell lung cancer (NSCLC) is one of the most common cancers in the world. DHA is known to be capable of suppressing NSCLC cell proliferation and metastasis. However, the mechanisms by which DHA exhibits its antitumor effects are unknown. Here we aimed to identify the effects and mechanisms of DHA and its metabolites on lung cancer cell growth and invasion.MethodsAs measures of cell proliferation and invasion ability, the cell viability and transwell assays were used in vitro. Transgenic mfat-1 mice, which convert ω-6 PUFAs to ω-3 PUFAs, were used to detect the effect of endogenous DHA on tumor transplantation. An LC − MS/MS analysis identified the elevation of several eicosanoid metabolites of DHA. By using qPCR miRNA microarray, online prediction software, luciferase reporter assays and Western blot analysis, we further elucidated the mechanisms.ResultsAddition of exogenous DHA inhibited the growth and invasion in NSCLC cells in vitro. Endogenously produced DHA attenuated LLC-derived tumor growth and metastasis in the transgenic mfat-1 mice. Among the elevation of DHA metabolites, resolvin D1 (RvD1) significantly contributed to the inhibition in cell growth and invasion. MiRNA microarray revealed that the level of miR-138-5p was significantly increased after RvD1 treatment. MiR-138-5p mimics decreased cell viability and invasion; while miR-138-5p inhibitor abolished RvD1-mediated suppression of cell viability and invasion. The expression of FOXC1 was significantly reduced upon overexpression of miR-138-5p while luciferase reporter assay showed that FOXC1 was a direct target of miR-138-5p. In vivo, endogenous DHA by the mfat-1 transgene enhanced miR-138-5p expression and decreased FOXC1 expression. Furthermore, overexpression of FOXC1 reversed the inhibition in cell viability and invasion induced by RvD1 treatment.ConclusionsThese data identified the RvD1/miR-138-5p/FOXC1 pathway as a novel mechanism by DHA and its metabolite, RvD1, and the potential of targeting such pathway as a therapeutic strategy in treating NSCLC.

IL-24 Inhibits Lung Cancer Growth by Suppressing GLI1 and Inducing DNA Damage.

Aberrant expression of GLI1 is responsible for aggressive tumor behavior and survival due to its effects on the DNA damage response (DDR). We investigated whether interleukin (IL)-24, a tumor suppressor, inhibits GLI1 and the associated DDR pathway in human NSCLCs. IL-24 treatment reduces mRNA and protein expression of GLI1 in lung tumor cells, but not in normal cells. GLI1 reporter assay and mRNA studies demonstrated that IL-24 regulates GLI1 at the post-transcriptional level by favoring mRNA degradation. Associated with GLI1 inhibition was marked suppression of the ATM-mediated DDR pathway resulting in increased DNA damage, as evidenced by γ-H2AX foci and Comet assay. Furthermore, attenuation of GLI1-associated DDR by IL-24 increased caspase-3 and PARP activity, resulting in cancer cell apoptosis. GLI1 inhibition and overexpression confirmed that IL-24-mediated anti-tumor effects involved the GLI-dependent pathway. Finally, we observed that IL-24-mediated alteration in GLI1 is independent of the canonical hedgehog-signaling pathway. Our study provides evidence that IL-24 treatment induces DNA damage, and reduces GLI1 expression and offers an opportunity for testing IL-24-based therapy for inhibiting GLI1 in lung cancer.

Loss of thymidine kinase 1 inhibits lung cancer growth and metastatic attributes by reducing GDF15 expression.

Metabolic alterations that are critical for cancer cell growth and metastasis are one of the key hallmarks of cancer. Here, we show that thymidine kinase 1 (TK1) is significantly overexpressed in tumor samples from lung adenocarcinoma (LUAD) patients relative to normal controls, and this TK1 overexpression is associated with significantly reduced overall survival and cancer recurrence. Genetic knockdown of TK1 with short hairpin RNAs (shRNAs) inhibits both the growth and metastatic attributes of LUAD cells in culture and in mice. We further show that transcriptional overexpression of TK1 in LUAD cells is driven, in part, by MAP kinase pathway in a transcription factor MAZ dependent manner. Using targeted and gene expression profiling-based approaches, we then show that loss of TK1 in LUAD cells results in reduced Rho GTPase activity and reduced expression of growth and differentiation factor 15 (GDF15). Furthermore, ectopic expression of GDF15 can partially rescue TK1 knockdown-induced LUAD growth and metastasis inhibition, confirming its important role as a downstream mediator of TK1 function in LUAD. Collectively, our findings demonstrate that TK1 facilitates LUAD tumor and metastatic growth and represents a target for LUAD therapy.

MiR-1286 inhibits lung cancer growth through aerobic glycolysis by targeting PKM2.

This study aims to explore the effects of microRNA-1286 (miR-1286) on the development of non-small cell lung cancer (NSCLC) via the aerobic glycolysis pathway by targeting pyruvate kinase muscle isozyme M2 (PKM2). The mRNA levels of miR-1286 in NSCLC tissues and mouse tumor tissues were detected by q-PCR. MiR-1286 was knocked down and overexpressed separately in A549 cells. The effect of miR-1286 on cell proliferation was determined by CCK8 assay. Western blotting was used to measure the expression of PKM2 protein. Lactate production assay was used to detect the aerobic glycolysis in A549 cells. The effect of miR-1286 in vivo was determined by xenograft assay. The mRNA level of miR-1286 decreased in NSCLC tissues compared with paired, tumor adjacent normal tissues. In addition, miR-1286 inhibited A549 cell proliferation in vitro. Moreover, knockdown of miR-1286 increased PKM2 expression and lactate production. Thus, miR-1286 expression negatively correlated with PKM2 in A549 cells. At the same time, in vivo experiments also showed that miR-1286 suppressed the growth of A549 cells and PKM2 was the target gene of miR-1286. These data show that miR-1286 inhibits lung cancer proliferation via aerobic glycolysis by targeting PKM2, which suggests that the functions of miR-1286 in NSCLC may play a key role in tumor progression and that miR-1286 can be a promising predictive biomarker and potential therapeutic target for NSCLC.

Coated cationic lipid-nanoparticles entrapping miR-660 inhibit tumor growth in patient-derived xenografts lung cancer models

Lung cancer is the leading cause of cancer-related deaths. Late diagnosis and inadequate therapies contribute to poor outcomes. MicroRNAs (miRNAs) are small non-coding RNAs and are involved in lung cancer development. Because miRNAs simultaneously regulate several cancer-related genes, they represent an interesting therapeutic approach for cancer treatment. We have developed Coated Cationic Lipid-nanoparticles entrapping miR-660 (CCL660) and intraperitoneally administered (1.5 mg/Kg) twice a week for four weeks into SCID mice carrying subcutaneously lung cancer Patients Derived Xenografts (PDXs). Obtained data demonstrated that miR-660 is down-regulated in lung cancer patients and that its replacement inhibited lung cancer growth by inhibiting the MDM2-P53 axis. Furthermore, systemic delivery of CCL660 increased miRNA levels in tumors and significantly reduced tumor growth in two different P53 wild-type PDXs without off-target effects. MiR-660 administration reduced cancer cells proliferation by inhibiting MDM2 and restoring P53 function and its downstream effectors such as p21. Interestingly, anti-tumoral effects of CCL660 also in P53 mutant PDXs but with a functional p21 pathway were observed. Stable miR-660 expression inhibited the capacity of H460 metastatic lung cancer cells to form lung nodules when injected intravenously into SCID mice suggesting a potential role of miR-660 in metastatic dissemination.To investigate the potential toxic effects of both miRNAs and delivery agents, an in vitro approach revealed that miR-660 replacement did not induce any changes in both mouse and human normal cells. Interestingly, lipid-nanoparticle delivery of synthetic miR-660 had no immunological off-target or acute/chronic toxic effects on immunocompetent mice. Altogether, our results highlight the potential role of coated cationic lipid-nanoparticles entrapping miR-660 in lung cancer treatment without inducing immune-related toxic effects.

Novel agent #2714 potently inhibits lung cancer growth by suppressing cell proliferation and by inducing apoptosis invitro and invivo.

The use of small molecule compounds to inhibit cell proliferation is one of the most promising approaches in cancer therapy. In the present study, a cell viability assay, flow cytometry analysis, western blotting and mouse xenograft models were used to investigate the anticancer activities of #2714 and its underlying mechanisms in lung cancer. The present in vitro results suggested that #2714 significantly inhibited the viability of the human non-small cell lung cancer line SPC-A1 in a concentration- and time-dependent manner, with a half-maximal inhibitory concentration value of 5.54 µM after 48 h of treatment. Additionally, #2714 inhibited SPC-A1 cell proliferation via the Wnt/β-catenin pathway and by impairing mitochondrial membrane potential. The protein expression levels of Wnt 3a, Wnt 5a/b, phosphorylated (p)-β-catenin, p-glycogen synthase kinase 3β, and p-mitogen-activated protein kinase 14 were downregulated following treatment with #2714. Furthermore, using a mouse xenograft model, #2714 was identified to significantly inhibit tumor growth and to decrease cancer cell proliferation in vivo. #2714 may represent a novel effective anticancer compound targeting lung cancer cells. Additionally, #2714 was able to induce apoptosis and decrease cell proliferation in SPC-A1 cells via the Wnt/β-catenin pathway.

Curcumin induces apoptosis in lung cancer cells via mitochondria-dependent signal pathways

Purpose: To investigate the effect of curcumin on the growth and viability of lung cancer cells.Methods: The viability of curcumin-treated A-427 and A-549 cells, and changes in their morphologies were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and phase-contrast microscopy, respectively. Flow cytometry and western blot assays were employed for the determination of apoptosis and changes in protein expressions, respectively.Results: Curcumin treatment altered the morphologies of A-427 and A-549 lung cancer cells. The viability of A-427 cells was reduced to 89, 71, 49, 26 and 25 %, respectively, on treatment with 5, 10, 15, 20 and 25 μM curcumin, while the corresponding decreases in viability of A-549 cells treated with the same doses of curcumin were 91, 74, 53, 31 and 29 %, respectively. Treatment with 20 % curcumin resulted in 62.87 % apoptosis in A-549 lung cancer cells. Curcumin increased the proportion of cells in G0/G1 phase in a concentration-dependent manner, and increased the expressions of p21 and p-p53 proteins in A-427 cells, while the expressions of cyclin E and Cdc25c were decreased. Curcumin treatment also enhanced the expressions of Bax, cleavage-caspase-8 and PARP in A-549 cells.Conclusion: Curcumin inhibits lung cancer growth by inducing apoptosis and cell cycle arrest. Thus, curcumin has a promising potential for the treatment of lung cancer.Keywords: Curcumin, Lung cancer, Apoptosis, Viability, Intrinsic, Reactive oxygen species

Anterior Gradient-2 monoclonal antibody inhibits lung cancer growth and metastasis by upregulating p53 pathway and without exerting any toxicological effects: A preclinical study

Increased drug resistance and acute side effects on normal organs are the major disadvantages of traditional cancer chemotherapy and radiotherapy. This has increased the focus on targeted therapeutic strategies such as monoclonal antibody-based cancer therapies. The major advantage of antibody-based therapies is the specific inhibition of cancer-related targets, with reduced off-target side effects. Anterior gradient-2 (AGR2) is a prometastatic and proangiogenic tumor marker that is overexpressed in multiple cancers. Therefore, anti-AGR2 antibodies may be potential therapeutic agents for treating different cancers. In the present study, we examined a novel anti-AGR2 monoclonal antibody mAb18A4 and found that this antibody inhibited lung cancer progression and metastasis without exerting any adverse side effects on the major organs and blood in mice. Moreover, we found that mAb18A4 activated p53 pathway and attenuated ERK1/2–MAPK pathway. Furthermore, mAb18A4-treated cancer cell lines showed attenuated proliferation and colony formation, enhanced apoptosis, increased p53 expression, and reduced phosphorylated ERK1/2 expression. Treatment with mAb18A4 significantly reduced tumor size and suppressed tumor metastasis in and increased the survival of different xenograft tumor models. In addition, mAb18A4 potently suppressed AGR2-induced angiogenesis. Results of pharmacokinetic and toxicological analyses confirmed the safety of mAb18A4 as an antitumor treatment.

Small-cell lung cancer growth inhibition: synergism between NMDA receptor blockade and chemotherapy.

BackgroundSmall-cell lung cancer (SCLC) has a poor prognosis since there is currently no effective therapy for commonly recurring disease. In our previous study, both primary and recurrent human tumors have been shown to express functional N-methyl-D-aspartate (NMDA) receptors, and blockade of these receptors with GluN1 and GluN2B antagonists decreased tumor cell viability in vitro, and growth of tumor xenografts in nu/nu mice.Materials and methodsIn this study, we examine the influence of the GluN2B antagonist ifenprodil and the channel-blocker antagonist memantine, on cell viability and growth of tumor xenografts of recurrent SCLC (rSCLC) in mice.ResultsBoth antagonists significantly reduced cell viability and levels of components of the ERK1/2 pathway, increased apoptosis, and at very safe levels significantly reduced the growth of tumors in mice. Each antagonist and topotecan had additive effects to reduce cell viability with significant synergy demonstrated for the case of memantine. More significantly, combination treatments of xenografts in mice with ifenprodil and the chemotherapeutic agent topotecan produced clear additive effects that completely stopped tumor growth. Moreover, the ifenprodil and topotecan combination showed excellent supra-addition or synergy of inhibition for tumors ≤300 mm in size (P=4.7E−4). Combination treatment of memantine with topotecan also showed clear addition but, unlike ifenprodil, no synergy for the doses chosen.ConclusionSince topotecan is a drug of choice for treatment of rSCLC, our findings suggest that combining this agent with NMDA receptor blockade using the GluN2B antagonist, ifenprodil, will significantly improve patient outcomes.

Anticancer effects of curcumin on nude mice bearing lung cancer A549 cell subsets SP and NSP cells.

Curcumin is a key polyphenolic curcuminoid extracted from the root of turmeric rhizome Curcuma longa Linn, which is a frequently used Chinese herb for the treatment of cancer. The aim of the present study was to investigate the mechanism of the inhibitory effects of curcumin on nude mice with lung cancer A549 cell subsets side population (SP) and non-SP (NSP) cells. BALB/c mice were subcutaneously injected with the tumor cells of A549 SP or NSP subsets consisting of 1×109 cells/l (0.2 ml in total). After 16 days of inoculation with A549, the mice were intraperitoneally injected with curcumin (100 mg/kg, 0.2 ml) once every other day, eight times in total. A series of assays were performed to detect the effects of curcumin on: i) Tumor weight and size; ii) Notch and hypoxia inducible factor 1 (HIF-1) mRNA expression by quantitative polymerase chain reaction; and iii) vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) by immunohistochemistry. It was determined that curcumin decreased the tumor weight and size, downregulated the expression of Notch and HIF-1 mRNA and suppressed the VEGF and NF-κB expression. These results indicated that curcumin inhibited lung cancer growth through the regulation of angiogenesis mediated by VEGF signaling.

Bioreducible Poly(Amino Ethers) Based mTOR siRNA Delivery for Lung Cancer.

Lung cancer is one of the leading causes of deaths in the United States, but currently available therapies for lung cancer are associated with reduced efficacy and adverse side effects. Small interfering RNA (siRNA) can knock down the expression of specific genes and result in therapeutic efficacy in lung cancer. Recently, mTOR siRNA has been shown to induce apoptosis in NSCLC cell lines but its use is limited due to poor stability in biological conditions. In this study, we modified an aminoglyocisde-derived cationic poly (amino-ether) by introducing a thiol group using Traut's reagent to generate a bio-reducible modified-poly (amino-ether) (mPAE). The mPAE polymer was used to encapsulate mTOR siRNA by nanoprecipitation method, resulting in the formation of stable and bio-reducible nanoparticles (NPs) which possessed an average diameter of 114nm and a surface charge of approximately +27mV. The mTOR siRNA showed increased release from the mTS-mPAE NPs in the presence of 10mM glutathione (GSH). The polymeric mTS-mPAE-NPs were also capable of efficient gene knockdown (60 and 64%) in A549 and H460 lung cancer cells, respectively without significant cytotoxicity at 30μg/ml concentrations. The NPs also showed time-dependent cellular uptake for up to 24h as determined using flow cytometry. Delivery of the siRNA using these NPs also resulted in significant inhibition of A549 and H460 cell proliferation in vitro, respectively. The results demonstrate that the mPAE polymer based NPs show strong potential for siRNA delivery to lung cancer cells. It is anticipated that future modification can help improve the efficacy of nucleic acid delivery, leading to higher inhibition of lung cancer growth in vitro and in vivo.

Abstract 2664: Identification of two small molecules with small cell lung cancer growth inhibition response profiles different from etoposide/topotecan

Abstract Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic cancer with an overall 5-year survival rate of <5% whose treatment has not changed in the past 20 years and has been designated by the US Congress and the National Cancer Institute as a “recalcitrant cancer.” SCLCs are characterized by high initial response rates to chemotherapy (platin + etoposide) and radiation therapy but relapse is almost universal within 1 year. A NCI screen of 526 clinical or clinical investigational agents in 63 SCLC lines found large response phenotype differences (Polley, JNCI (2016) 108(10): djw122) but, in general, SCLC lines were either “sensitive” or “resistant” to all drugs tested and there was no correlation with tumor molecular biomarkers, or prior chemotherapy treatment. Thus, new drugs are needed (preferably with tumor associated molecular biomarkers) that have different profiles than available agents. As part of an NCI Cancer Treatment Discovery and Development Network (CTD2N) consortium we have screened large chemical libraries and identified chemical compounds that kill subsets of non-small cell lung cancers (NSCLCs) but not normal lung epithelial cells thus displaying a “therapeutic window” and tumor specificity. We first tested 116 of these compounds against 4 SCLC lines and found 30 that displayed dose dependent SCLC toxicity. We have so far tested 2 (SW034510, SW140154) compounds against a panel of 22 SCLC lines, which were also in the NCI screen, along with etoposide and topotecan as comparators, using 8-point dose response curves, for effect on SCLC growth inhibition. The SCLC lines (all with p53 and Rb mutations) were also phenotyped for expression of Myc family members (c-, N-, and L-), lineage transcription factors ASCL1 and NeuroD1 and neuroendocrine mRNAs. As found by Polley et al, etoposide and topotecan response phenotypes were correlated with each other (r2 = 0.7). By contrast, our two compounds, SW034510 and SW140154, response phenotypes did not correlate with etoposide/topotecan (r2 values of <0.08, and 0.1 respectively) and did not correlate with each other. Both agents cause cell death as assessed by the CellTox Green Assay suggesting efficacy across the ASCL1/NeuroD1, Myc family expression profiles. Cleaved caspase-3 western blot shows SW034510 causes caspase induced apoptosis. Cell cycle analysis suggests SW140154 causes a G1 cell cycle arrest. SARM1 gene expression anti-correlates with SW140154 IC50s. In conclusion, starting with chemical compounds that have selectivity for NSCLC, we have identified two new chemical compounds with specificity for subsets of NSCLC over normal lung epithelial cells, and which also inhibit the growth/kill subsets of SCLC in patterns different from the established therapy. Thus, these two new compounds provide potentially new therapeutic opportunities for SCLC patients resistant to current chemotherapies. Citation Format: Allison A. Mootz, Michael Peyton, Paul Yenerall, Kimberley Avila, Kenneth Huffman, Tomohiro Haruki, Mahboubeh Papari-Zareei, Victor Stastny, Luc Girard, Elizabeth McMillan, Michael Roth, John MacMillan, Bruce Posner, Michael White, Gazdar Adi, John Minna. Identification of two small molecules with small cell lung cancer growth inhibition response profiles different from etoposide/topotecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2664.

Methionine gamma lyase from Clostridium sporogenes increases the anticancer effect of doxorubicin in A549 cells and human cancer xenografts.

The anti-cancer efficacy of methionine γ-lyase (MGL) from Clostridium sporogenes (C. sporogenes) is described. MGL was active against cancer models in vitro and in vivo. The calculated EC50 values for MGL were 4.4U/ml for A549, 7.5U/ml for SK-BR3, 2.4U/ml for SKOV3, and 0.4U/ml for MCF7 cells. The combination of doxorubicin (DOX) and MGL was more effective for A549 human lung cancer growth inhibition than either agent alone in vitro and in vivo. MGL reduced the EC50 of doxorubicin from 35.9μg/mL to 0.01-0.265μg/mL. The growth inhibitory effect of DOX + MGL on A549 xenografts in vivo was reflective of the results obtained in vitro. The inhibition rate of tumor growth in the combined arm was 57%, significantly higher than that in the doxorubicin (p = 0.033)-alone arm.

Liver kinase B1 suppresses growth of lung cancer cells through sonic hedgehog signaling pathway.

Lung cancer is one of life-threatening cancers in the worldwide. Liver kinase B1 (LKB1) has been reported to be closely related to cancers; however, the underlying mechanism of LKB1 in lung cancer remains unclear. In our study, a LKB1 specific shRNA was employed to down-regulate LKB1 levels and a LKB1 over-expression plasmid was constructed to up-regulate LKB1 levels. Thereafter, growth of lung cancer cells was assessed by MTT assay and flow cytometry. Effects of LKB1 on the activation of sonic hedgehog (Shh) signaling pathway were detected by Western blot. Effects of LKB1 on lung cancer growth and Shh signaling pathway activation were also assessed in vivo. Our results showed that LKB1 inhibited proliferation of lung cancer cells and induced their apoptosis. Moreover, LKB1 inhibited Shh signaling pathway activation. Our in vivo study also showed that LKB1 inhibited lung cancer growth in vivo and modulated Shh signaling pathway. Treatment with cyclopamine, a Shh signaling pathway inhibitor, reversed the effects of LKB1 silencing and enhanced the effects of LKB1 over-expression. Results of our study demonstrate that LKB1 inhibits lung cancer growth in vitro and in vivo through Shh signaling pathway.

2-anilino-4-amino-5-aroylthiazole-type compound AS7128 inhibits lung cancer growth through decreased iASPP and p53 interaction.

Lung cancer is the leading cause of cancer‐related death worldwide. Thus, developing novel therapeutic agents has become critical for lung cancer treatment. In this study, compound AS7128 was selected from a 2‐million entry chemical library screening and identified as a candidate drug against non‐small cell lung cancer in vitro and in vivo. Further investigation indicated that AS7128 could induce cell apoptosis and cell cycle arrest, especially in the mitosis stage. In addition, we also found that iASPP, an oncogenic protein that functionally inhibits p53, might be associated with AS7128 through mass identification. Further exploration indicated that AS7128 treatment could restore the transactivation ability of p53 and, thus, increase the expressions of its downstream target genes, which are related to cell cycle arrest and apoptosis. This occurs through disruption of the interactions between p53 and iASPP in cells. Taken together, AS7128 could bind to iASPP, disrupt the interaction between iASPP and p53, and result in cell cycle arrest and apoptosis. These findings may provide new insight for using iASPP as a therapeutic target for non‐small cell lung cancer treatment.

Expression of histone deacetylase 1 gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor

Objective To investigate the expression of histone deacetylase 1 (HDAC1) gene in human lung cancer cell line A549 and the effect on the radiation sensitivity of nude mouse transplantation tumor. Methods HDAC1 small interfering RNA (siRNA) and recombinant expression vector pcDNA3.1-HDAC1 were transfected with A549 lung cancer cells, respectively. Stable transfected cell lines were screened by G418 selection. reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression levels of HDAC1 mRNA and HDAC1 protein in the cell; cell proliferation was measured by cell counting kit-8 (CCK-8). Clone formation experiment was used to observe the effect of up-regulation and down-regulation of HDAC1 expression on the radiation sensitivity of A549 cells, cell apoptosis was detected by transferase-mediated dUTP nick-end labelling (TUNEL) method. Nude mice experiment of transplantable tumor was applied for in vivo detection of the influence of up-regulated and down-regulation of HDAC1 expression combined with X-ray irradiation in the inhibition of lung cancer growth. Results The expression of HDAC1 gene and protein were significantly lower to 1.86±0.09 and 2.41±0.10 (P=0.015, 0.006) in HDAC1 cell line A549, and the HDAC1 up-regulation group were significantly higher to 3.65±0.14 and 5.41±0.12 (P=0.012, 0.008); The radiation sensitivity of lung cancer A549 cells was increased in the down-regulation group of HDAC1 (P=0.025), and the radiation sensitivity of lung cancer A549 cells was decreased (P=0.009)in the HDAC1 up-regulation group. Down-regulation of HDAC1 inhibited the proliferation of A549 lung cancer, increased the rate of apoptosis to (42.17±3.19)% (P=0.008), proliferation of A549 cells was significantly enhanced in HDAC1 up-regulated group, associated with reduced percentage of apoptotic cells (9.11±1.46)% (P=0.006); Average volume in the nude mice of HDAC1 down-regulated group (547.37±89.68) mm3 were significantly smaller than the control group (1 238.44±212.36) mm3 (P=0.008); furthermore, mean volume of tumor was greater in the HDAC1 up-regulated group than in the control group (1 681.27±195.37) mm3 (P=0.006). After 20 Gy γ irradiation, the average volume of HDAC1 down-regulated group were significantly decreased (214.15±54.26) mm3 (P=0.011). Compared with that before irradiation, the average volume of the tumor in the nude mice was not significantly changed after irradiation (P=0.264). Conclusion Down-regulated expression of HDAC1 can inhibit the growth of human lung cancer A549 cells in nude mice, enhance the radiation sensitivity of tumor cells, whereas up-regulated expression of HDAC1 can produce tumor radiation resistance. Key words: Lung cancer; A549 cell; Histone deacetylase 1; Transplantable tumor; Nude mice

Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5′UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5′UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

Targeted blockade of TGF-\u03b2 and IL-6/JAK2/STAT3 pathways inhibits lung cancer growth promoted by bone marrow-derived myofibroblasts

To investigate the role of TGF-β and IL-6 in myofibroblasts (MFs) — lung cancer cell interactions, lung cancer cells (Lewis and CTM-167 cell lines) were stimulated by IL-6, MF-conditioned medium (MF-CM) or MFs, with or without TGF-β signaling inhibitor — SB431542 and/or JAK2/STAT3 inhibitor — JSI-124. MFs were stimulated by TGF-β, cancer cell-CM or cancer cells, with or without SB431542 and JSI-124. Cell proliferation, the levels of cytokines, expression of mRNA and protein were determined. Mice bearing xenograft tumors were intraperitoneally treated with SB431542 or JSI-124 and monitored for up to 45 days. In co-culture systems, MFs secreted high levels of IL-6, while cancer cells produced high levels of TGF-β. Recombinant IL-6 and MF-CM activated STAT3 and upregulated TGF-β in cancer cells. In contrast, cancer cell-CM or TGF-β stimulated MFs to produce IL-6. Blockade of JAK2/STAT3 and TGF-β signaling by specific inhibitors significantly inhibited cell proliferation in vitro and tumor growth in vivo of lung cancer cells. Our study demontrated that the TGF-β and IL-6/JAK2/STAT3 signaling pathways form a positive feedback signaling loop that mediated the interactions between MFs and lung cancer cells. Targeted inhibiton of this signaling loop could be a new approach for lung cancer prevention and therapy.

Ski regulates Smads and TAZ signaling to suppress lung cancer progression.

Ski, the transforming protein of the avian Sloan-Kettering retrovirus, displays both pro- and anti-oncogenic activities in human cancer. The mechanisms underlying these conflicting observations have not been fully understood. Herein, we investigated the mechanism underlying the tumor suppressor activity of Ski. To investigate the effect of Ski re-activation on TGF-β and Hippo/TAZ pathway, we measured its effect on the endogenous Smad target genes (PAI-1 and P15INK4B ) and TAZ target gene CTGF. The results revealed that Ski exerted its inhibitory activity in TGF-β1/Smad signaling pathway. Ski inhibited TAZ by increasing their phosphorylation by Lats2 and did not alter the localization of TAZ. Ski inhibited lung cancer growth and invasion. Ski methylation correlated with decreased mRNA expression in human lung cancer cell lines. Thus, Ski inhibited the function of TGF-β and TAZ through multiple mechanisms in human lung cancer.

Abstract 4565: Forced expression of GITRL in cancer cells enhances adenovirus-mediated in situ vaccination

Abstract Our approach involves a platform of killing cancer using more potent oncolytic viruses-based immunotherapy strategies. These replication competent adenoviruses are targeted to the Rb pathway to generate tumor-selectivity. The second generation of these therapeutic agents, Delta-24-RGD, was successfully translated to the clinical setting and is currently been tested in Phase I studies in several institutions in the USA and in Europe for the treatment of patients suffering from recurrent gliomas. Preliminary data from these clinical trials showed that 10 to 15% of Delta-24-RGD-treated tumors undergo complete regression. Agonistic treatments targeting co-stimulatory tumor necrosis factor receptor superfamily (TNFRSF), such as GITR (CD357), have been shown to enhance the proliferation and activation of T cells. Moreover, in preclinical tumor efficacy studies, these agonistic signals have shown potent tumoricidal activity. Different from antibodies, co-stimulatory ligands can be easily incorporated into replication competent oncolytic adenoviruses. Infection of cancer cells with these armed viruses will lead to the expression in their cell membranes of the co-stimulatory molecule that will directly interact with the tumor infiltrating lymphocytes to amplify and enhance the anti-tumor T-cell activity. In this study, we have developed an armed Delta-24-RGD carrying the cDNA of the mouse GITRL, Delta-24-GREAT. Treatment of glioma-bearing mice with intracranial injection of Delta-24-GREAT increased mice survival (P < 0.0001, long-rank test) and inhibited lung cancer growth in subcutaneous models. Infection of the tumor elicited an inflammatory response increasing the populations of CD4+ and CD8+ T cells versus treated controls. Importantly, 2 weeks after the adenovirus treatment a subset of brain hemispheric cells were positive for GITRL. In addition, co-culture experiments with tumor cells infected with viruses and splenocytes isolated from treated glioma-bearing mice demonstrated a response against the cancer cells as assessed by ELISA analyses of IFN-γ. Demonstrating the generation of anti-tumor memory, the surviving animals did not show evidence of tumor growth after re-challenging with GL261 glioma cell implantation in the contralateral hemisphere. However, survivors of GL261 tumors did not survive after the re-challenge was performed by intracranial implantation of B16/F10 melanoma cells, strongly indicating that the immune response was specific for GL261 glioma antigens. This is the first study with an oncolytic adenovirus expressing GITRL and our results strongly indicate that oncolytic adenoviruses armed with molecules of the TNFRSF may be of future clinical interest for the treatment of patients with cancer. Citation Format: Yisel A. Rivera-Molina, Francisco Puerta Martinez, Teresa Nguyen, Hong Jiang, Xuejun Fan, Rehnuma Shifat, Mohammad Belayat Hossain, Verlene K. Henry, Caroline C. Carrillo, Candelaria Gomez-Manzano, Juan Fueyo. Forced expression of GITRL in cancer cells enhances adenovirus-mediated in situ vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2017-4565