Lung Tumor Cell Growth Research Articles

Abstract 451: AICAR targets oncogenic signaling pathways of MUC1-CT

Abstract Lung cancer cells overexpress oncogenic mucin 1 (MUC1), whose C-terminal active subunit (MUC1-CT) is involved in lung tumor formation. Although a specific peptide against MUC1-CT has effectively blocked the MUC1 signaling pathway, intrinsic metabolites targeting MUC1 were not well studied. 5-aminoimidazole-4-carboxamide riboside (AICAR) is a purine biosynthesis intermediate and an analog of adenosine. Our study showed that high AICAR reduced lung tumor cell growth by increasing DNA damage and cell apoptosis. MUC1 was validated as one of the leading AICAR-binding proteins by virtual target screening and thermal stability assay. AICAR treatment dramatically degraded MUC1-CT at protein levels. Whole transcriptome analysis demonstrated that AICAR negatively regulated the Janus kinase (JAK) signaling pathway. Duolink proximity ligation assay revealed that JAK1 interacted with MUC1 directly, and AICAR treatment impaired this protein-protein interaction. Increased epidermal growth factor receptor (EGFR) activity was linked to upregulated expression of MUC1-CT in tissues from an EGFR transgenic mouse lung cancer model. AICAR treatment reduced phosphorylated EGFR, JAK1, and MUC1-CT in lung cancer cells. A mono-treatment with AICAR reduced tumor formation in a mouse lung xenograft model. Combination therapy with AICAR and small-molecule inhibitors against JAK1 or mutant EGFR reduced the growth of lung tissue-derived tumor organoids. Thus, our study has first disclosed a new ligand repressing the MUC1 activity in lung cancer that disrupts protein-protein interactions between MUC1-CT and other oncogenic proteins. Citation Format: Fareesa Aftab, Alice Rodriguez-Fuguet, Luis Silva, Ikei S. Kobayashi, Jiao Sun, Katerina Politi, Elena Levantini, Wei Zhang, Susumu S. Kobayashi, Wen Cai Zhang. AICAR targets oncogenic signaling pathways of MUC1-CT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 451.

Engineering oncogene-targeted anisamide-functionalized pBAE nanoparticles as efficient lung cancer antisense therapies.

Non-small cell lung cancer (NSCLC) is one of the leading causes of worldwide death, mainly due to the lack of efficient and safe therapies. Currently, NSCLC standard of care for consist on the use of traditional chemotherapeutics, non-selectively distributed through the whole body, thus causing severe side effects while not achieving high efficacy outcomes. Consequently, the need of novel therapies, targeted to modify specific subcellular routes aberrantly expressed only in tumor cells is still urgent. In this context, the delivery of siRNAs that can know-down overexpressed oncogenes, such as mTOR, could become the promised targeted therapy. However, siRNA effective delivery remains a challenge due to its compromised stability in biological fluids and its inability to cross biological and plasmatic membranes. Therefore, polymeric nanoparticles that efficiently encapsulate siRNAs and are selectively targeted to tumor cells could play a pivotal role. Accordingly, we demonstrate in this work that oligopeptide end-modified poly(beta aminoester) (OM-pBAE) polymers can efficiently complex siRNA in small nanometric particles using very low polymer amounts, protecting siRNA from nucleases attack. These nanoparticles are stable in the presence of serum, advantageous fact in terms of in vivo use. We also demonstrated that they efficiently transfect cells in vitro, in the presence of serum and are able to knock down target gene expression. Moreover, we demonstrated their antitumor efficacy by encapsulating mTOR siRNA, as a model antisense therapy, which showed specific lung tumor cell growth inhibition in vitro and in vivo. Finally, through the addition of anisamide functionalization to the surface of the nanoparticles, we proved that they become selective to lung tumor cells, while not affecting healthy cells. Therefore, our results are a first step in the discovery of a tumor cell-targeted efficient silencing nanotherapy for NSCLC patients survival improvement.

Abstract 2818: In vivo imaging of mitochondrial bioenergetics in lung cancer

Abstract Non-small cell lung cancer (NSCLC) is a histologically, genetically and metabolically heterogeneous disease. The mitochondria are essential regulators of cellular energy and metabolism and they play a critical role in sustaining growth and survival of lung tumor cells. However, our understanding of mitochondrial metabolism in cancer at an in vivo level has been limited thus leaving a large gap in our knowledge of how mitochondrial bioenergetics support tumor growth. To better study mitochondrial bioenergetics in lung tumors, we recently developed and validated a voltage sensitive, positron emission tomography (PET) tracer known as 4-[18F]fluorobenzyl triphenylphosphonium (18FBnTP) that we used to profile mitochondrial bioenergetics in autochthonous K-Ras driven mouse models of lung cancer (Momcilovic et al., (2019) Nature). The use of 18FBnTP PET imaging enabled us to functionally profile mitochondrial bioenergetics in live tumors and discover distinct functional mitochondrial heterogeneity conserved across different NSCLC tumor subtypes. In order to study mitochondria at the level of ultrastructure we coupled 18FBnTP PET with 3D serial block-face scanning electron microscopy (3D SBEM). By coupling these two techniques we are able to image and quantify mitochondria heterogeneity from whole tumors down to the ultrastructures of individual mitochondria within tumor cells. Our study reveals distinct organization of mitochondrial structure and function as lung tumors adapt during tumorigenesis. We anticipate that coupling 18FBnTP PET imaging with 3D SBEM will have dynamic applications beyond that of lung cancer and enrich our understanding how mitochondria impact human disease. Citation Format: Mingqi Han, Milica Momcilovic, Eric Bushong, Linsey Stiles, Orian Shirihai, Carla Koehler, Saman Sadeghi, Mark Ellisman, David B. Shackelford. In vivo imaging of mitochondrial bioenergetics in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2818.

Non-Thermal Intervention of Lung Tumor by Core-Shell Magnetic Nanoparticles in a Magnetic Field

K-Ras mutations result in normal cells dividing uncontrollably and becoming cancerous. The prognosis is currently poor for patients due to the lack of drugs that can effectively target these mutations. In this study, magnetic nanoparticles (MNPs) were prepared, characterized, and cooperated with a magnetic field to intervene in the growth of lung tumor cells. The rise in temperature of a stimulation coil was studied by numerical calculation. The non-thermal effects of MNPs under a magnetic force were analyzed. The cell experiments showed that the growth of A549 tumor cells slowed down. The result of a wound-healing assay also indicated that the migration of tumor cells was suppressed. Compared with magnetic stimulation without MNPs, MNPs enhanced the inhibitory effects of a magnetic field. This study suggests a new way to treat K-Ras driven lung tumors using non-thermal effects of MNPs without the side effects caused by thermal effects.

S-allylcysteine induces cytotoxic effects in two human lung cancer cell lines via induction of oxidative damage, downregulation of Nrf2 and NF-κB, and apoptosis.

In this study, we investigated the putative cytotoxic effect elicited by the garlic-derived compound S-allylcysteine (SAC) in two human cancer cell lines (HCC827 and NCI-H1975) in order to develop an experimental approach to the therapeutic potential of this molecule for lung cancer. Cells were incubated for 24, 48 and 72 h in the presence of SAC (10 or 20 mM), which resulted in a concentration- and time-dependent decrease in cell viability and culture confluence in both cell lines. These effects were contrasted with - and validated through - those observed in an immortalized but nontumorigenic epithelial cell line from human bronchial epithelium (BEAS-2B, negative control) and an adenocarcinoma human alveolar basal epithelial cell line (A549, positive control). SAC (20 mM at 72 h) also increased the oxidative damage to lipids, augmented apoptosis, and decreased the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the nuclear factor kappa B (NF-κB) proteins in HCC827 and NCI-H1975 cells. Our results establish the efficacy of SAC in reducing malignant growth and proliferation of lung tumor cells. This effect is mediated by the induction of oxidative damage associated with the downregulation of Nrf2 and NF-κB and their corresponding signaling pathways.

CircAKT3 inhibits glycolysis balance in lung cancer cells by regulating miR-516b-5p/STAT3 to inhibit cisplatin sensitivity.

Lung cancer was one of the most deadly cancers around the world. Circular RNA AKT3 (CircAKT3) was highly expressed in lung cancer and could inhibit cell proliferation, but there were few studies on the mechanism of specific regulation of drug resistance. Therefore, we aimed to provide new ideas and perspectives for the role of circAKT3 in the mechanism of tumor resistance. The levels of circAKT3, miR-516b-5p and STAT3 in lung cancer tissues and cells were examined using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assays. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to examine the sensitivity of cells treated under different conditions to cisplatin (DDP). A glucose assay kit and lactate assay kit were used to assess glycolysis and lactate production of cells treated with different plasmids and 2-deoxy-glucose (2-DG). Western blot analysis was used to detect the expression level of the hypoxia-inducible factor (HIF-1α) in A549 and H1299 cells. Starbase 3.0 predicted a targeted relationship between circAKT3 and miR-516b-5p, STAT3 and miR-516b-5p, and the relationship was proved by a dual-luciferase reporter assay. Knockdown of circAKT3 was used to study the effects of circAKT3 on tumor development in vivo. The levels of circAKT3 and STAT3 were upregulated, miR-516b-5p was downregulation in lung cancer tissues and cells. Functionally, circAKT3 knockdown improved cell sensitivity to DDP, and repressed glycolysis in lung cancer cells. Meanwhile, inhibition of HIF-1α-dependent glycolysis attenuated the circAKT3-induced increase of chemo-resistance in A549 cells. Mechanistically, miR-516b-5p was found to possess some binding sites with circAKT3. Noticeably, the inhibitory action of circAKT3 knockdown on DDP resistance and glycolysis was overturned through inhibitor of miR-516b-5p in lung cancer cells. Furthermore, besides, circAKT3 knockdown suppressed lung tumor cell growth by the miR-516b-5p/STAT3 axis in vivo. CircAKT3 inhibit cisplatin sensitivity of lung cancer cells at least partly through regulating miR-516b-5p/STAT3 axis-mediated glycolysis balance, providing a possible long noncoding RNA -targeted therapy for lung cancer.

Diplatin, a Novel and Low-Toxicity Anti-Lung Cancer Platinum Complex, Activation of Cell Death in Tumors via a ROS/JNK/p53-Dependent Pathway, and a Low Rate of Acquired Treatment Resistance.

Background: Platinum-based drugs prevail as the main treatment of lung cancer; this is caused by their relative effectiveness despite known side effects, such as neurotoxicity. The risk reward of the treatment and side effects is confronted when dosage is considered and when resistance to treatment develops. Development of new compounds that improve effectiveness and safety profiles addresses this ongoing need in clinical practice. Objectives: The novel water-soluble platinum complex, diplatin, was synthesized, and its antitumor potency and toxicology profile were evaluated in murine xenograft tumor models and in lung cancer cell lines. Methods: The effects of diplatin, cisplatin (DDP), and carboplatin (CBP) on the viability of nine lung tumor cell lines and one normal human lung epithelial cell line were evaluated using the MTT assay. Therapeutic index was calculated as LD50/ED50 to identify and compare the ideal therapeutic windows of the above compounds. Diplatin’s antitumor effects were assessed in lung xenograft tumors of nude mice; molecular mechanisms of therapeutic effects were identified. Results: Diplatin had desirable IC50 compared to CBP in a variety of cultured tumor cells, notably lung tumor cells. In the mouse xenograft lung tumor, diplatin led to a substantially improved therapeutic index when compared to the effects of DDP and CBP. Importantly, diplatin inhibited the growth of DDP-resistant lung tumor cells. Diplatin’s mode of action was characterized to be through cell cycle arrest in the G2/M phase and induction of lung tumor apoptosis via ROS/JNK/p53-mediated pathways. Conclusion: Diplatin was observed to have antitumor effects in mice with both greater potency and safety compared with DDP and CBP. These observations indicate that diplatin is promising as a potential treatment in future clinical applications.

Abstract 4629: FOXA2 controls tumor-associated inflammation inKRAS-mutant lung cancer

Abstract Background: Although the role of the transcription factor FOXA2 in lung cancer has been investigated using mouse and human lung cancer cell lines, to our knowledge, the role of FOXA2 in autochthonous lung tumors in GEMM (Genetically Engineered Mouse Model) has not been determined. Methods: In order to determine the role of FOXA2 in autochthonous lung tumor cells, we deleted FOXA2 in lung epithelia of Kras-mutant lung cancer model mice (Scgb1a1-Cre;[tetO]-Kras4bG12D;Foxa2flox/flox, a GEMM) and assessed survival and lung histology of the GEMM. Results: Deletion of FOXA2 in autochthonous Kras-mutant lung tumor cells significantly extended the survival of Kras-mutant lung cancer model mice (median survival from 8 weeks to 23 weeks; n>14 for each group). Histological analysis indicated that FOXA2 was required for the growth of lung tumor cells and the recruitment of tumor-associated macrophages. Conclusion: FOXA2 promotes primary KRAS-mutant lung tumors in part by controlling tumor-associated inflammation. Citation Format: Koichi Tomoshige, Minzhe Guo, Iris Fink-Baldauf, William Stuart, Yutaka Maeda. FOXA2 controls tumor-associated inflammation inKRAS-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4629.

Abstract 2048: Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells

Abstract Lung cancer is a very aggressive malignant form of cancer, and is one of the biggest public health challenges facing the United States and many other countries. Although incidence rates have been stabilized, an estimated 154,050 Americans are expected to die from lung & bronchus cancer in 2018, accounting for approximately 25.3 percent of all cancer deaths. Lung cancer is a difficult disease to detect in its early stages, with greater than 50% of patients diagnosed with lung cancer presenting with metastatic disease. Early detection of lung cancer is an important opportunity for decreasing mortality while it is still treatable and curable. The overall 5-year survival rate is ~15 percent. Thus, it is essential to better understand the mechanisms that initiate lung carcinogenesis and find easy-use biomarkers for more accurate lung cancer detection. Due to heterogeneity of lung cancers, a panel of biomarkers should be used for more accurate lung cancer detection and classification. Signal transducer and activator of transcription 3 (Stat3) is well known for its lung cancer-promoting activity. To assess the consequences of STAT3 persistent activation in the lung, a doxycycline-controlled CCSP-rtTA/(tetO)7-Stat3C bitransgenic mouse model was generated that over-expresses STAT3C (a constitutively active form of STAT3) in alveolar type II (AT II) epithelial cells. In sequential steps, Stat3C over-expression up-regulated pro-inflammatory molecules, increased inflammatory cell infiltration and caused adenocarcinomas in the lung. Using this lung tumor model, thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, Stat3 downstream inducible secretory protein biomarkers potentially can be used as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood. Citation Format: Xinchun Ding. Stat3 downstream transthyretin stimulates tumor growth through regulation of tumor, immune and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2048.

Abstract 2731: A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis

Abstract We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Bing Zhu, Veronica Ramirez-Alcantara, Antonio Ward, Kristy Berry, Adam B. Keeton, Michael R. Boyd, Yulia Maxuitenko, Xi Chen, Gary A. Piazza. A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2731.

Transthyretin Stimulates Tumor Growth through Regulation of Tumor, Immune, and Endothelial Cells.

Early detection of lung cancer offers an important opportunity to decrease mortality while it is still treatable and curable. Thirteen secretory proteins that are Stat3 downstream gene products were identified as a panel of biomarkers for lung cancer detection in human sera. This panel of biomarkers potentially differentiates different types of lung cancer for classification. Among them, the transthyretin (TTR) concentration was highly increased in human serum of lung cancer patients. TTR concentration was also induced in the serum, bronchoalveolar lavage fluid, alveolar type II epithelial cells, and alveolar myeloid cells of the CCSP-rtTA/(tetO)7-Stat3C lung tumor mouse model. Recombinant TTR stimulated lung tumor cell proliferation and growth, which were mediated by activation of mitogenic and oncogenic molecules. TTR possesses cytokine functions to stimulate myeloid cell differentiation, which are known to play roles in tumor environment. Further analyses showed that TTR treatment enhanced the reactive oxygen species production in myeloid cells and enabled them to become functional myeloid-derived suppressive cells. TTR demonstrated a great influence on a wide spectrum of endothelial cell functions to control tumor and immune cell migration and infiltration. TTR-treated endothelial cells suppressed T cell proliferation. Taken together, these 13 Stat3 downstream inducible secretory protein biomarkers potentially can be used for lung cancer diagnosis, classification, and as clinical targets for lung cancer personalized treatment if their expression levels are increased in a given lung cancer patient in the blood.

Long non-coding RNA LOC285194 functions as a tumor suppressor by targeting p53 in non-small cell lung cancer.

Recently, LOC285194 has shown a potential tumor-suppressor function in several types of human cancers, but its function in non-small cell lung cancer (NSCLC) remains unknown. This study intended to investigate the biological role of LOC285194 and its clinical significance in NSCLC. LOC285194 was detected by qRT-PCR, and its correlation with clinicopathological features of NSCLC was analyzed. The expression of LOC285194 was knocked down or ectopically expressed in lung cancer cells (A549 and H1299) and tumor cell growth, migration and invasion in vitro were investigated. In addition, the interaction of LOC285194 and target proteins was assessed by RNA pulldown and RNA immunoprecipitation in vitro. The results revealed that the expression of LOC285194 was significantly lower in tumor tissues when compared with the corresponding non-tumor tissues (P<0.001). Its expression was correlated with the tumor size (P=0.027). Kaplan-Meier analysis revealed that patients with lower LOC285194 expression had worse disease-free survival and overall survival rates (P<0.05). RNA protein interaction analysis revealed that p53 was the direct binding target of LOC285194 in NSCLC. Bioinformatics analyses suggested that depletion of LOC285149 could affect its antitumor function through the KRAS/BRAF/SMEK pathway. Our findings indicated that LOC285194 was a novel non-coding prognostic indicator and contributed to tumor suppression by targeting p53 in NSCLC, suggesting that it may be a non-coding target for NSCLC gene therapy.

NFATc1 Promotes Antitumoral Effector Functions and Memory CD8+ T-cell Differentiation during Non-Small Cell Lung Cancer Development.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release-activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non-small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619-33. ©2018 AACR.

Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors selectively suppress growth by blocking β-catenin and MAPK signaling.

Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of β-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic β-catenin and MAPK signaling.

Abstract 4972: Novel Ras inhibitor DC070-547 potently and selectively blocks Ras-RBD binding, EGFR binding to Ras signaling complex, EGFR activation of Ras signaling, and growth of Ras-driven lung tumor cells

Abstract A novel series of compounds that potently and selectively inhibits the growth of tumor cells harboring constitutively activated Ras relative to cells lacking activated Ras were identified by screening a proprietary library of indene derivatives in a phenotypic, cell-based assay. Lead-optimization produced a drug development candidate, DC070-547, which showed strong antitumor activity at doses not causing any discernible toxicity in preclinical mouse models. Here we characterize the underlying mechanism of growth inhibition in lung tumor cells. A panel of non-small cell lung cancer lines with constitutively activated Ras were highly sensitive to DC070-547 with IC50 values as low as 2 nM, while normal airway epithelial cells were essentially insensitive. Transfection of wild-type ras H322 bronchioalveolar tumor cells with mutant ras (G12V) confirmed that activated Ras is required for the selective growth inhibitory activity of DC070-547. Ras-RBD binding assays showed that DC070-547 disrupts Ras-RBD binding at low nanomolar concentrations that parallel those required to inhibit the growth of lung tumor cells with activated Ras. Similar concentrations of DC070-547 were found to inhibit the binding of phosphorylated EGFR (Y1068) to Ras immunoprecipitates in mutant ras transfected H322 cells, but not in control H322 cells. DC070-547 also inhibited the binding of SOS, Grb2, Gab1, S338 phosphorylated c-Raf (pc-Raf), S473 phosphorylated Akt-1 (pAkt-1) and T202/Y204 phosphorylated Erk1/2 (pERK1/2) to Ras or EGFR immunoprecipitates. To determine if DC070-547 can inhibit EGF-stimulated Ras signaling, serum-starved mutant ras transfected H322 cells were treated with EGF and probed for effects on Ras signaling components. DC070-547 caused a concentration-dependent inhibition of EGF-induced Y1068-EGFR as measured in Ras immunoprecipitates, and also reduced pc-Raf, pAkt-1, pErk1/2 and pGab1 (Y627) levels in Ras or EGFR immunoprecipitates. In addition, DC070-547 caused a concentration-dependent decrease in Erk1/2 and Akt-1-mediated phosphorylation of Bad proteins (S112, S136 and S155) to induce apoptosis. These results show that DC070-547 prevents Ras-RBD binding to block EGF-induced Raf/MAPK and Akt signaling to potently and selectively inhibit the growth of lung tumor cells harboring constitutively activated Ras. Support provided by NCI grants 1R01CA197147 and 1R21CA182941. Citation Format: Bing Zhu, Xi Chen, Jacob Valiyaveettil, Joshua Canzoneri, Kevin Lee, Kate Saville, Kristy Berry, Luciana Barnes, Tyler Maddox, Ashley Lindsey, Antonio Ward, Veronica Ramirez-Alcantara, Adam Keeton, Michael Boyd, Gary Piazza. Novel Ras inhibitor DC070-547 potently and selectively blocks Ras-RBD binding, EGFR binding to Ras signaling complex, EGFR activation of Ras signaling, and growth of Ras-driven lung tumor cells [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 4972. doi:10.1158/1538-7445.AM2017-4972

Abstract 1140: Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer

Abstract BACKGROUND: Screening a focused library of indene derivatives for PDE10 inhibitory activity identified novel leads with potent and selective tumor cell growth inhibitory activity. ADT-030 emerged from lead optimization chemistry with excellent drug-like properties and oral bioavailability. Here we characterize the anti-tumor activity of ADT-030 in human lung tumor cells and an orthotopic mouse model of lung cancer. METHODS: Growth inhibitory activity of ADT-030 was measured in a panel of human lung tumor cell lines by ATP quantification following 72 h of treatment. The effect of ADT-030 on intracellular cGMP/cAMP was measured in whole cell lysates using a competitive ELISA assay. PDE inhibitory activity of ADT-030 was evaluated in lysates of human lung tumor cells and by recombinant PDE isozymes using the IMAP fluorescence polarization PDE assay. Activation of PKG signaling and suppression of β-catenin levels in response to ADT-030 treatment was evaluated by Western blot using whole cell lysates of human lung tumor cells. ADT-030 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity of ADT-030 was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1x106 A549 lung tumor cells and treating once daily by oral administration at dosages ranging from 25 - 125 mg/kg. Tumor growth was monitored by in situ bioluminescence using IVIS as well as necropsy and pathological grading after 4 weeks of treatment. RESULTS: ADT-030 inhibited the growth of human lung tumor cell lines with IC50 values in the low micromolar range by inducing apoptosis, while appreciably higher concentrations were required to affect the growth of normal human airway epithelial cells. ADT-030 treatment of human lung tumor cells increased both intracellular cGMP and cAMP levels, activated PKG and suppressed β-catenin within the same concentration range as required for tumor cell growth inhibition. Pharmacokinetic studies in mice demonstrated a half-life suitable for once a day dosing. Tissue distribution studies revealed appreciably higher concentrations of ADT-030 in lungs relative to plasma and other tissues, with the highest accumulation measured in the parenchyma. ADT-030 was well tolerated in mice implanted with A549 tumor cells and displayed strong anti-tumor activity as evident by reduced luminescence, tumor grading, and double-blinded pathological evaluation. CONCLUSIONS: ADT-030 represents a prospective drug development candidate with favorable drug-like properties that concentrates in lung after oral administration exhibiting a strong anti-tumor activity in a pre-clinical mouse model. The mechanism of lung tumor cell growth inhibition involves PDE10 inhibition, elevation of cGMP, activation of PKG, and attenuation of β-catenin. Citation Format: Veronica Ramirez-Alcantara, Bing Zhu, Xi Chen, Rajkumar Savai, Prema Subbarayal, Michele A. Schuler, Kevin J. Lee, Ashley S. Lindsey, Kristy L. Berry, Dennis Otali, Joshua Canzoneri, Jacob Valiyaveettil, Adam Keeton, Lori Coward, Gregory Gorman, William Grizzle, Michael Boyd, Gary A. Piazza. Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer [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 1140. doi:10.1158/1538-7445.AM2017-1140

Molecular Mechanisms Underlying the Inhibitory Effects of Qingzaojiufei Decoction on Tumor Growth in Lewis Lung Carcinoma.

Objective: Qingzaojiufei decoction (QD) is an empirical herbal formula from traditional Chinese medicine that is used for the treatment of lung-related diseases. However, the effect of QD on the growth of lung tumor cells has not been investigated. The aim of this study was to examine the antitumor activity of QD in Lewis lung carcinomas (LLC) in vivo and in vitro, and to elucidate the underlying mechanisms. Methods: The LLC cells were used to assess the antitumor activity of QD by Cell Counting Kit-8 assay in vitro. In vivo, mice were randomly assigned to 5 groups (n = 10/group): the model control (MC) group was intragastrically administered physiological saline (0.9% NaCl) twice daily from day 2 after tumor implantation for 2 weeks. The QD groups were intragastrically administered QD twice daily from 2 weeks before to 2 weeks after tumor implantation for 4 weeks. The mRNA levels were detected by quantitative polymerase chain reaction, the proteins expression was determined by immunohistochemistry or western blotting. Results: Compared with the model group, QD showed inhibition of proliferation of LLC cells and reductions in tumor weight and proliferating cell nuclear antigen protein expression. Furthermore, QD up-regulated p53 mRNA expression, and downregulated c-myc and Bcl-2 mRNA expression, while MMP-9, VEGF, and VEGFR protein expression was suppressed. Phosphorylated ERK1/2 levels were also reduced by QD in a dose-dependent manner. Conclusion: Our findings suggest that QD inhibited lung tumor growth and proliferation, by activation of tumor suppressor genes, inactivation of oncogenes, suppressing the potential for invasion and metastasis, and attenuating angiogenesis. The ERK/VEGF/MMPs signaling pathways may play an important role in QD-induced inhibition of malignant tumor cell proliferation.

SPOP promotes SIRT2 degradation and suppresses non-small cell lung cancer cell growth

SIRT2 is a NAD-dependent deacetylase and inhibition of SIRT2 has a broad anticancer activity. Here we report that SPOP binds to SIRT2 and mediates its degradation by the 26S proteasome, which can be blocked by MG132 treatment. We also found that the levels of SPOP significantly decreased, while the levels of SIRT2 significantly increased in non-small cell lung cancer (NSCLC) cell lines, compared to normal bronchial epithelial cell line and NSCLC specimens, compared to the paired non-tumor lung tissue. Furthermore, SPOP can suppress NSCLC cell growth. Notably, mutations in NSCLC inhibit the abilities of SPOP to degrade SIRT2 and suppress NSCLC cell growth. These results reveal a novel regulation of SIRT2 by SPOP mediated degradation, which is important for the growth of lung tumor cells.

Abstract 1238: A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding

Abstract Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Screening using a phenotypic assay designed to select for Ras inhibitors and iterative chemical synthesis, identified a preclinical drug development candidate with attractive drug-like properties designated as DC070-547. Here we evaluated the sensitivity of non-small cell lung cancer (NSCLC) cell lines to DC070-547. The compound potently suppresses the growth of human A549, H460, HOP62, H1299, H1975 lung tumor cells having high levels of activated Ras with low nanomolar IC50 values. By contrast, normal human airway epithelial cells (NHAECs) and H322 lung tumor cells with low levels of activated Ras displayed IC50 values in the mid micromolar range. The Ras selectivity of DC070-547 was confirmed by ectopic expression of mutant H-Ras-G12V (H-Rasm) in H322 cells using a retroviral construct, in which the IC50 value of DC070-547 for growth inhibition was reduced approximately 2,500 fold compared with vector control cells. To study the mechanism of action, Ras activation status was measured by Ras-Raf pull-down assays using GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose and followed by western blotting with Ras antibodies. Direct binding to Ras was apparent by the ability of DC070-547 to inhibit Ras-Raf binding at concentrations that inhibit Ras-dependent tumor cell growth. Pull-down assays also showed a dose-dependent inhibition of Ras-GTP levels in intact H-Rasm-H322 cells treated with DC070-547 at the same concentration range. Western blot analysis of Ras-immunoprecipitated proteins revealed that DC070-547 also attenuated high levels of EGFR phosphorylation at Y1068 in H-Rasm-H322 cells but not in control cells. These results demonstrate novel Ras inhibitory activities of DC070-547 in NSCLC cell lines. DC070-547 and its pipeline analogs are being evaluated for antitumor efficacy in preclinical mouse models of lung cancers. Citation Format: Bing Zhu, Xi Chen, Veronica Ramirez-Alcantara, Kevin Lee, Jacob Valiyaveettil, Joshua Canzoneri, Sara Sigler, Kristy Berry, Ashley Lindsey, Adam Keeton, Michael R. Boyd, Gary A. Piazza. A novel Ras inhibitor potently and selectively suppresses lung tumor cell growth by blocking Ras-Raf binding. [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 1238.

Seven New Tetrahydroanthraquinones from the Root of Prismatomeris connata and Their Cytotoxicity against Lung Tumor Cell Growth.

The root of Prismatomeris connata has been used in China for centuries as the medicinal herb “Huang Gen” (HG), but its phytochemicals or active ingredients are not well understood. In this study, we performed chemical analysis of the ethyl acetate fraction of a HG ethanol extract. We thus isolated seven new tetrahydroanthraquinones, prisconnatanones C–I (compounds 1–7) from the root of P. connata and identified their structures using spectroscopic analyses. Their absolute configurations were established by both modified Mosher’s and Mo2OAc4 methods, and ORD techniques. Their cytotoxicity was tested in a panel of human lung tumor cells (H1229, HTB179, A549 and H520 cell lines). Prisconnatanone I (7) showed the highest activity, with an IC50 value ranging from 2.7 µM to 3.9 µM in the suppression of tumor cell growth, and the others with chelated phenolic hydroxyls exhibited relatively lower activity (IC50: 8–20 µM). In conclusion, these data suggest that some of the natural tetrahydroanthraquinones in HG are bioactive, and hydroxylation at C-1 significantly increases the cytotoxicity of these compounds against lung tumor cell growth.