Human Non-small Cell Lung Tumors Research Articles

Abstract 510: Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer

Abstract The contemporary landscape of cancer treatment has witnessed significant progress with FDA-approved targeted drugs. The FDA's Project Optimus has underscored the growing need for innovative pharmacodynamic endpoints to determine the optimal biological dose in clinical development. Flow cytometry, a potent analytical tool in clinical and preclinical labs, now plays a leading role in cancer research, enabling the evaluation of new drug effects on cell cycle regulation and consideration of rational drug combinations with approved drugs. At TD2, we strive to enhance the translatability of in vitro induced cell cycle arrest studies to in vivo biomarker proof of concept and efficacy. We employ a comprehensive approach, utilizing flow cytometry to assess cell cycle dynamics in cultured cells and in vivo tumor models after treatment with approved and novel drugs. This abstract focuses on the translational relevance of combined in vitro and in vivo testing of well-known drugs that disrupt the cell cycle, laying the foundation for optimized treatment strategies and potentially personalized cancer therapies. Human A549 non-small cell lung carcinoma cells were treated with either thymidine (2.0mM) or the microtubule-depolymerizing drug nocodazole (100ng/ml) for 18 hours, then examined by flow cytometry. Thymidine induced a greater than 85% S-phase arrest, while nocodazole induced a greater than 88% G2/M-phase arrest. A549 cells (5 × 106 cells/mouse) were subcutaneously implanted in SCID mice, followed by treatment with an FDA-approved cell cycle inhibitor, Palbociclib, or cisplatin chemotherapy. Palbociclib, a CDK4/6 inhibitor, and low-dose cisplatin treatment resulted in an increased accumulation of cells in the G0/G1 phase, greater than 80% for each. Both treatments exhibited promising anti-cancer activity, with tumor growth inhibition (TGI) of 69% and 87%, respectively. Our findings highlight flow cytometry's multifaceted utility in assessing cell cycle arrest, providing a comprehensive view of agent effects on cancer cells in culture and within the complex in vivo tumor microenvironment. Determining the impact of any new drug on specific cell cycle checkpoints could allow for a more strategic plan for combination therapy. Integrating flow cytometry could enable early biomarker monitoring of tumor cell cycle changes, offering insights into the therapeutic potential of new agents alone or in combination. This multidimensional evaluation contributes to understanding cancer biology and has the potential to guide the development of novel therapeutic strategies, optimize treatment regimens to identify optimal biological doses, and improve patient outcomes in the era of precision medicine. Citation Format: Erin E. Trachet, Emily Cluff, Ana Lemos de Matos, Damaris Diaz, Chelsea Holmquist, Paul Gonzales. Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 510.

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor β (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

NRF2 Activation Promotes Aggressive Lung Cancer and Associates with Poor Clinical Outcomes.

Stabilization of the transcription factor NRF2 through genomic alterations in KEAP1 and NFE2L2 occurs in a quarter of patients with lung adenocarcinoma and a third of patients with lung squamous cell carcinoma. In lung adenocarcinoma, KEAP1 loss often co-occurs with STK11 loss and KRAS-activating alterations. Despite its prevalence, the impact of NRF2 activation on tumor progression and patient outcomes is not fully defined. We model NRF2 activation, STK11 loss, and KRAS activation in vivo using novel genetically engineered mouse models. Furthermore, we derive a NRF2 activation signature from human non-small cell lung tumors that we use to dissect how these genomic events impact outcomes and immune contexture of participants in the OAK and IMpower131 immunotherapy trials. Our in vivo data reveal roles for NRF2 activation in (i) promoting rapid-onset, multifocal intrabronchiolar carcinomas, leading to lethal pulmonary dysfunction, and (ii) decreasing elevated redox stress in KRAS-mutant, STK11-null tumors. In patients with nonsquamous tumors, the NRF2 signature is negatively prognostic independently of STK11 loss. Patients with lung squamous cell carcinoma with low NRF2 signature survive longer when receiving anti-PD-L1 treatment. Our in vivo modeling establishes NRF2 activation as a critical oncogenic driver, cooperating with STK11 loss and KRAS activation to promote aggressive lung adenocarcinoma. In patients, oncogenic events alter the tumor immune contexture, possibly having an impact on treatment responses. Importantly, patients with NRF2-activated nonsquamous or squamous tumors have poor prognosis and show limited response to anti-PD-L1 treatment.

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.

Synthesis and biological evaluation of new [1,2,4]triazolo[4,3-a]pyridine derivatives as potential c-Met inhibitors

A series of [1,2,4]triazolo[4,3-a]pyrazine derivatives (4a–4i) were designed, synthesized and evaluated for their c-Met kinase inhibition and antitumor activity against SNU5 gastric cell line in vitro. Among these compounds, 4d was found to show the highest activity against c-Met and high selectivity against the tumor cells which are believed to be dependent on the c-Met oncogene amplification, because 4d selectively inhibited c-Met while had no effect on other 59 kinases. In vivo efficacy study on human gastric (MKN-45) and human non-small cell lung (NCI-H1993) tumor xenograft in nude mouse demonstrated that 4d·CH3SO3H had a better inhibiting activity than SGX-523 in a dose-dependent manner. When tested in mice, compound 4d·CH3SO3H was found to have biological half-lives and plasma exposure values higher than those of JNJ-38877605, and its long-term toxicity and acute toxicity turned out to be acceptable, all of which indicates that 4d·CH3SO3H is a desirable drug candidate.

CXCR1/2 antagonism with CXCL8/Interleukin-8 analogue CXCL8(3-72)K11R/G31P restricts lung cancer growth by inhibiting tumor cell proliferation and suppressing angiogenesis.

CXCR1 and CXCR2 together with cognate chemokines are significantly upregulated in a number of cancers, where they act as key regulators of tumor cell proliferation, metastasis, and angiogenesis. We have previously reported a mutant protein of CXCL8/Interleukin-8, CXCL8(3-72)K11R/G31P (G31P), which can act as a selective antagonist towards CXCR1/2 with therapeutic efficacy in both inflammatory diseases and malignancies. In this study, we investigated the effect of this ELR-CXC chemokine antagonist G31P on human non-small cell lung cancer cells and lung tumor progression in an orthotopic xenograft model. We report increased mRNA levels of CXCR1 and CXCR2 in human lung cancer tissues compared to normal counterparts. Expression levels of CXCR1/2 cognate ligands was determined by ELISA. CXCR1/2 receptor antagonism via G31P leads to decreased H460 and A549 cell proliferation and migration in a dose-dependent manner. G31P also enhanced apoptosis in lung cancer cells as determined by elevated levels of cleaved PARP, Caspase-8, and Bax, together with a reduced expression of the anti-apoptotic protein Bcl-2. In an in vivo orthotopic xenograft mouse model of human lung cancer, G31P treatment suppressed tumor growth, metastasis, and angiogenesis. At the molecular level, G31P treatment was correlated with decreased expression of VEGF and NFкB-p65, in addition to reduced phosphorylation of ERK1/2 and AKT. Our results suggest that G31P blockage of CXCR1 and CXCR2 can inhibit human lung cancer cell growth and metastasis, which offers potential therapeutic opportunities.

Design, synthesis and biological evaluation of tasiamide analogues as tumor inhibitors.

Eighteen analogues of the marine cytotoxic linear peptide tasiamide were designed, synthesized and screened for their inhibitory activities against the growth of human nasopharyngeal carcinoma (KB) and human non-small cell lung tumor (A549) cell lines. The results indicated that minor modifications of the C-terminuswith aromatic groups were tolerated, with the IC50 values between 1.29 and 12.88 μM against these two cancer cell lines. Truncation, minor modifications at the N-terminus or elimination of the N-methyl groups in N-Me-d-Gln and/or N-Me-d-Phe residues resulted in inactive analogues.

Triterpenoid saponins from Eryngium yuccifolium ‘Kershaw Blue’

Phytochemical investigation on the root of Eryngium yuccifolium ‘Kershaw Blue’ resulted in the isolation and identification of two new polyhydroxyoleanene saponins, named eryngioside M and eryngioside N, together with 15 known triterpenoid saponins eryngiosides A-L, 21β-angeloyloxy-3β-[β-d-glucopyranosyl-(1→2)]-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyloxyolean-12-ene-15α,16α,22α,28-tetrol, saniculasaponin III, and saniculasaponin II. Their structures were established by extensive spectroscopic and chemical analyses. Eryngioside M and saniculasaponin II showed week cytotoxicity against human non-small cell lung tumor cells (A549) with GI50 values of 37.5±1.59μM and 35.5±1.11μM, respectively.

Cytotoxic triterpenoid saponins from husks of Aesculus californica (Spach) Nutt.

Fifteen polyhydroxyoleanene saponins, aesculiosides C1-C15 (1-15), were isolated from husks of Aesculus californica. Their structures were established by extensive spectroscopic and chemical analyses. The triterpenoid saponins from A. californica have greater structural diversity than those from any other investigated species thus far in the genus Aesculus. The chemotaxonomic characteristic of aesculiosides C1-C15 is that the unit attached to the C-3 of the aglycone is a glucopyranosyl moiety, instead of a glucuronopyranosyl group in the saponins that have been isolated from other Aesculus species. The saponins isolated from A. californica then provide important evolutionary and chemotaxonomic knowledge of the Aesculus genus, a well-known intercontinental disjunct genus in the Northern hemisphere. Aesculiosides C1-C15 (1-15) showed cytotoxicity to human non-small cell lung tumor (A549) with GI50 ranged from 3.76 to >25μM.

Abstract 2452: Tumor detection and measurement of responses to chemotherapy using human phosphatidylserine targeting antibody fragments

Abstract Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells and tumor cells in response to chemotherapy, irradiation and oxidative stresses in the tumor microenvironment. Binding of antibodies targeting PS on the tumor endothelial cells and tumors recruit immune cells and engage the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric anti-PS antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in Phase II trials. In the present study, we characterize the response to chemotherapy of human tumor xenografts in mice using near-infrared dye labeled PGN650, a human F(ab’)2 fragment that targets PS expression. NIR-PGN650 bound human PC-3luc prostate, BT474 breast and A549luc non-small cell lung tumors in SCID or nude mice in the absence of chemotherapy. Docetaxel treatment enhanced tumor targeting of NIR-PGN650 approximately 2-3-fold compared to imaging signals prior to chemotherapy. Peak tumor uptake of NIR-PGN650 was observed when docetaxel was delivered 24 hours prior to injection of NIR-PGN650. Using these parameters, NIR-PGN650 is able to detect responses to chemotherapy in both the primary tumor and metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2452. doi:1538-7445.AM2012-2452

Abstract A205: Targeting caspase-9/PP2A interaction as a new antitumor strategy.

Abstract Background: Dysfunction in the regulation of apoptosis is associated with important pathologies, including cancers. While apoptosis is known to rely on the Bcl-2 family members and caspases, recent data suggest that the serine/threonine phosphatase PP2A is a key actor in regulating both Bcl-2 family members and caspases for cell life or cell death decision. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage, leading to their proposed use as vector for delivering therapeutic cargo. Focusing on both caspase-9 and PP2A, our goal was to develop cell penetrating peptides specifically designed against the caspase-9/PP2A interaction and evaluate their in vitro and in vivo therapeutic potential in various cancer cells and tumors. Materials and Methods: We have published the Drug Phosphatase Technology (DPT) based on the design of cell penetrating peptides obtained by association of a cell permeable shuttle, DPT-sh1 to a sequence of interest. We have also shown a direct interaction of human caspase-9/human PP2Ac, detected by co-immunoprecipitation and confirmed by far western. We decided to generate a peptide containing the previously published penetrating sequence (DPT-Sh1) associated to the site of interaction of human caspase-9 (DPT-C9h), in order to disturb the interaction caspase-9/PP2Ac. Using both mouse and human breast (BC) and non-small cell lung (NSCLC) cancer cells and tumors, we have then investigated the effect of DPT-C9 (specific to the sequence of the mouse caspase-9) and DPT-C9h (specific to the sequence of the human caspase-9) peptides. In in vivo experiments, DPT-C9 and DPT-C9h peptides were intraperitonealy administered at doses ranging from 1 to 50 mg/kg/day for up to 6 weeks. Results: We have demonstrated that both DPT-C9 and DPT-C9h peptides specifically blocked in vitro, in vivo, and ex vivo caspase-9/PP2A interaction, and induced caspase-9 activation and apoptosis in all tested cancer cell lines. In in vivo experiments, we have showed that DPT-C9 induced significant tumor growth inhibition (TGI) in mice bearing transgenic Polyoma Middle-T PymT mouse breast tumors and in mice bearing Kras mutated mouse NSCLC tumors. Similarly, DPT-C9h induced significant TGI in various primary human BC and NSCLC xenografts. Finally, no toxicity has been observed in treated mice, as well as no immunogenic response in immunodeficient mice. Conclusions: We have identified an interaction between caspase-9/PP2A that may be used as a new therapeutic target. Using the cell penetrating peptide DPT-C9h blocking this interaction, we have demonstrated that DPT-C9h had an impressive in vitro and in vivo therapeutic effect and therefore constitutes a new therapeutic approach of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A205.

Hypoxia-Dependent Inhibition of Tumor Cell Susceptibility to CTL-Mediated Lysis Involves NANOG Induction in Target Cells

Hypoxia is a major feature of the solid tumor microenvironment and is known to be associated with tumor progression and poor clinical outcome. Recently, we reported that hypoxia protects human non-small cell lung tumor cells from specific lysis by stabilizing hypoxia-inducible factor-1α and inducing STAT3 phosphorylation. In this study, we show that NANOG, a transcription factor associated with stem cell self renewal, is a new mediator of hypoxia-induced resistance to specific lysis. Our data indicate that under hypoxic conditions, NANOG is induced at both transcriptional and translational levels. Knockdown of the NANOG gene in hypoxic tumor cells is able to significantly attenuate hypoxia-induced tumor resistance to CTL-dependent killing. Such knockdown correlates with an increase of target cell death and an inhibition of hypoxia-induced delay of DNA replication in these cells. Interestingly, NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. To our knowledge, this study is the first to show that hypoxia-induced NANOG plays a critical role in tumor cell response to hypoxia and promotes tumor cell resistance to Ag-specific lysis.

Antitumor and antiangiogenic effects of GA-13315, a gibberellin derivative

This study showed that 13-chlorine-3,15-dioxy-gibberellic acid methyl ester (GA-13315), a gibberellin derivative, possessed high antitumor and antiangiogenic activity in vitro and in vivo. Cytotoxicity assays showed that GA-13315 was a potential and efficient antitumor compound, with inhibitory concentration 50 (IC(50)) values ranging from 0.13 to 30.28μg/ml in 12 human tumor cell lines, and it showed moderate toxicity to peripheral blood mononuclear cells with an IC(50) value of 14.2μg/ml. Administration of 0.5 or 2.5mg/kg GA-13315 for 23days significantly inhibited tumor growth of human non-small cell lung tumor (A549) xenografts, with relative growth rates ranging from 29.91% to 35.05%. Acute toxicity was determined in ICR mice, and the lethal dose 50 (LD(50)) was 4.19g/kg after intragastric administration. The high antitumor potency of GA-13315 occurred in parallel with its antiangiogenic activity. In vitro, GA-13315 inhibited recombinant human epithelial growth factor-induced chemotactic motility and capillary-like tube formation of primary cultured human endothelial cells. Furthermore, GA-13315 decreased the factor VIII(+) microvessel density and vascular endothelial growth factor expression in A549 tumors, indicating its antiangiogenic efficacy in vivo. These results indicate that the antiangiogenic activity of GA-13315 contributes to its anticancer properties. Further studies are needed to investigate the use of GA-13315 as an anticancer drug.

BMP7 signaling via BMPR1A, BMPR1B inhibits the proliferation of lung large carcinoma NCI-H460 cell

背景与目的已有的研究发现骨形成蛋白7（bone morphogenetic protein 7, BMP7）具有抑制和促进多种肿瘤发生发展的双重作用，但其对肺癌细胞增殖的影响及其具体机制尚不明确。本实验首先检测了外源性BMP7对肺癌细胞增殖的影响，然后通过在肺癌细胞系中阻断不同的Ⅰ型受体，观察其对BMP7生物学作用的影响，以探讨不同的Ⅰ型受体在BMP7信号传导过程中的作用。方法应用RT-PCR及MTT方法分别检测4种非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞系和人支气管上皮细胞系（HBE）中BMP7 Ⅰ型受体的表达情况及外源性BMP7对肺癌细胞增殖能力的影响，并联合运用抗体阻断的方法阻断NCI-H460细胞中内源性Ⅰ型抗体，采用MTT法检测BMP7对NCI-H460细胞增殖的影响，分析不同的Ⅰ型受体在BMP7信号传导过程中的作用。结果NCI-H460细胞系中三种Ⅰ型受体均有表达。外源性BMP7抑制了肺大细胞癌NCI-H460细胞的增殖（P=0.002）。运用特异性抗体阻断NCI-H460细胞内源性BMPR1A、BMPR1B、BMPR1A+BMPR1B后BMP7对NCI-H460增殖的抑制作用明显减弱（P=0.003, P=0.014, P < 0.001），而阻断ACVR1A后BMP7对NCI-H460增殖的抑制作用无明显变化（P=0.074）。结论BMP7通过激活BMPR1A、BMPR1B两种Ⅰ型受体抑制NCI-H460细胞的增殖。

Antitumor activity of noscapine in human non-small cell lung cancer xenograft model

An antitussive plant alkaloid, Noscapine HCl (Nos) displays anticancer activity and has a safe pharmacological profile in humans. The current study was aimed to investigate the in vitro and in vivo anti tumor activity of Nos to determine possible mechanisms of anti tumor activity for treatment of non-small cell lung cancer (NSCLC). In vitro cytotoxicity of Nos was studied in H460 cells treated with different doses of Nos (10-160 microM) for 72 h and cell viability was determined using crystal violet assay. Apoptosis in H460 cells was evaluated by TUNEL assay after treatment of cells for 72 h with 30 and 40 microM doses of Nos. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and on day 4 onwards Nos was administered orally at dose of 300, 450 and 550 mg/kg/day for 24 days. As a control, xenografted tumors were separately treated with Docetaxel (10 mg/kg i.v. bolus on day 5, 11, 17, 23). The tumor volumes were measured every five days. Expression of PARP, Bcl(2, )Bax, and caspase-3 families of proteins was measured by Western Blotting (WB), while TUNEL and Immunohistochemical methods were utilized to determine DNA fragmentation and cleaved caspase-3 levels respectively. Nos inhibited growth of H460 cells with the IC50 values of 34.7 +/- 2.5 microM. Nos at 30 and 40 microM doses caused apoptosis as evidenced by nuclear condensation in treated H460 cells. Nos caused 49, 65 and 86% reduction in the xenografted tumor volumes at a dose of 300 (P < 0.05), 450 (P < 0.01), 550 mg/kg/day (P < 0.01), respectively, when compared to controls. Nos-dependent suppression of xenografted tumor growth involved up regulation of PARP, Bax, caspase-3 and repression of Bcl(2) expression. An increase in Bax/Bcl(2) ratio suggests involvement of a mitochondrial mediated apoptotic processes. Our studies revealed a non significant (P > 0.05) increase in Bax/Bcl(2) ratio with Nos at a dose of 300 mg/kg/day, while a significant (P < 0.001) increase in Bax/Bcl(2) ratio was observed with Nos doses of 450 and 550 mg/kg/day. Further, Nos caused elevated apoptosis in tumor xenografts as evidenced by enhanced expression of caspase-3 and positive TUNEL staining in regressed tumor tissues, thus suggesting induction of apoptosis by mitochondrial pathway. Our studies suggest that potent antitumor activity of Nos against NSCLC cells. Oral administration of Nos showed significant reduction in tumor volume in human non-small cell lung tumor xenograft in nude mice in a dose dependant manner. Thus, Nos is a promising novel chemotherapeutic agent for the treatment of human lung cancer.

Suppression of non-small cell lung tumor development by the let-7 microRNA family

Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that let-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-Ras(G12D)-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, let-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-Ras(G12D) largely rescued let-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden.

Response to Comment on “Characterization of Human Lung Tumor-Associated Fibroblasts and Their Ability to Modulate the Activation of Tumor-Associated T Cells”

We have two independently derived sets of new data that establish that B7-H1 is expressed by fibroblasts in the microenvironment of human non-small cell lung tumors. First, our flow cytometry data identify fibroblasts within disrupted lung tumor biopsy tissue through staining with a mAb specific for

IL-12/TGF-β1 Regulation of TCR Signal Transduction in Human Memory T Cells Associated with Malignant and Non-malignant Chronic Inflammatory Microenvironments

RATIONALE: T cells persisting in the microenvironments of primary human non-small cell lung tumors, synovial fluid of rheumatic joints and nasal polyps demonstrate similar phenotypes identifying effector memory T cells yet these cells remain unresponsive to the increasing tissue pathology.METHODS: Effector memory T cells were isolated from the primary, human, non-small cell lung tumors, nasal polyps and synovial fluid of rheumatic joints and evaluated for their response to TCR stimulation via CD3+CD28 cross-linking by nuclear translocation of NF-κB and NFAT under confocal microscopy. Response to pulsation with cytokines including TNF-α and IL-12, and low pH elution of surface proteins was examined to delineate the nature of the TCR anergy.RESULTS: Effector memory T cells from these chronic inflammatory microenvironments were found to persist in a TGF-β1 -mediated anergic state. Using IL-12, effector memory T cell anergy can be reversed both in vitro and in vivo resulting in the activation, proliferation and T cell mediated eradication of tumor cells from the xenografts.CONCLUSIONS: Effector memory T cells persist in chronic inflammatory microenvironments of malignant and non-malignant tissues by a reversible, regulatory mechanism mediated by TGF-β1. RATIONALE: T cells persisting in the microenvironments of primary human non-small cell lung tumors, synovial fluid of rheumatic joints and nasal polyps demonstrate similar phenotypes identifying effector memory T cells yet these cells remain unresponsive to the increasing tissue pathology. METHODS: Effector memory T cells were isolated from the primary, human, non-small cell lung tumors, nasal polyps and synovial fluid of rheumatic joints and evaluated for their response to TCR stimulation via CD3+CD28 cross-linking by nuclear translocation of NF-κB and NFAT under confocal microscopy. Response to pulsation with cytokines including TNF-α and IL-12, and low pH elution of surface proteins was examined to delineate the nature of the TCR anergy. RESULTS: Effector memory T cells from these chronic inflammatory microenvironments were found to persist in a TGF-β1 -mediated anergic state. Using IL-12, effector memory T cell anergy can be reversed both in vitro and in vivo resulting in the activation, proliferation and T cell mediated eradication of tumor cells from the xenografts. CONCLUSIONS: Effector memory T cells persist in chronic inflammatory microenvironments of malignant and non-malignant tissues by a reversible, regulatory mechanism mediated by TGF-β1.

Inhibition of human non-small cell lung tumors by a c-Met antisense/U6 expression plasmid strategy.

c-Met is a receptor tyrosine kinase whose activation by hepatocyte growth factor (HGF) can lead to transformation and tumorigenicity in a variety of tumors. We investigated the effects of suppressing c-Met protein expression in human non-small cell lung tumors. Expression plasmids containing either sense or antisense sequences of the human c-met gene were constructed under control of the U6 snRNA promoter. A U6 control plasmid was also constructed that did not contain any c-met sequence. These constructs have been examined both in vitro and in an in vivo tumor xenograft model. The c-Met protein was downregulated by 50-60% in two lung cancer cell lines that were transiently transfected with the c-Met antisense versus U6 control. Tumor cells treated with the c-Met antisense construct also show decreased phosphorylation of c-Met and MAP kinase when exposed to exogenous HGF. Lung cancer cells were grown as xenografts in mice and treated by intratumoral liposome-mediated transfer of the c-Met sense, antisense or U6 control plasmids. The treatment of lung tumors with c-Met antisense versus U6 control plasmid resulted in the downregulation of the c-Met protein expression, a 50% decrease in tumor growth over a 5-week treatment period and an increased rate of apoptosis. These results suggest that targeting the HGF/c-Met pathway may be an effective novel strategy to treat lung cancer patients.

Targeted radiotherapy with [(90)Y]-SMT 487 in mice bearing human nonsmall cell lung tumor xenografts induced to express human somatostatin receptor subtype 2 with an adenoviral vector.

Novel approaches to increasing the therapeutic efficacy of targeted radiotherapy of cancer are required. One strategy to achieve this goal is to induce high-level expression of a receptor on the surface of tumor cells that can be targeted with a radiolabeled peptide. The objectives of this study were to 1) induce somatostatin receptor (SSTr2) expression in tumor cells using an adenovirus encoding the SSTr2 gene (AdSSTr2), 2) demonstrate tumor localization of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors, and 3) show therapeutic efficacy with [(90)Y]-DOTA-D-Phe(1)-Tyr(3)-octreotide ([(90)Y]-SMT 487). SSTr2 expression was validated in vitro by the binding and subsequent internalization of [(111)In]-DTPA-D-Phe(1)-octreotide (21.3% per mg of total protein) in A-427 cells infected with AdSSTr2. In vivo imaging confirmed 5- to 10-fold greater uptake 5.5 hours after intravenous administration of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors relative to control tumors. For therapy studies, mice bearing established subcutaneous A-427 tumors were given two intratumoral injections of AdSSTr2 1 week apart, followed by an intravenous injection of 400 microCi or 500 microCi [(90)Y]-SMT 487 at 2 and 4 days after each adenoviral administration. Control animals either were not treated or were administered 500 microCi [(90)Y]-SMT 487 with no AdSSTr2 injection. These studies showed that untreated animals and animals treated with no virus and 500 microCi [(90)Y]-SMT 487 had median tumor quadrupling times of 16 and 25 days, respectively. Mice administered AdSSTr2 and either 400 microCi or 500 microCi of [(90)Y]-SMT 487 demonstrated significantly longer median tumor quadrupling times of 40 and 44 days, respectively (P < 0.02). These studies are the first to demonstrate in vivo therapeutic efficacy using a radiolabeled peptide targeted to a receptor expressed on the surface of tumor cells following gene transfer. Future studies will focus on the optimization of this approach.