Non-small Cell Lung Adenocarcinoma Research Articles

Use of a proliferation-based mRNA signature to predict outcome in early-stage non-small cell lung adenocarcinoma.

7023 Background: Adjuvant treatment of patients with early-stage lung adenocarcinoma is based on post-surgical pathological staging and patient performance status. Disparate outcomes within each staging group suggest that additional prognostic markers could improve our understanding of risk-benefit and potentially lead to better treatment decisions. A proliferation-based, mRNA expression profile was applied to public microarray data of surgically treated lung adenocarcinomas and a cohort of FFPE samples to test its potential prognostic utility. Methods: Public expression data (Director’s Consortium, DC) were derived from Affymetrix HG-U133A arrays. Clinical FFPE samples were assayed by quantitative PCR. A cell cycle progression (CCP) score was calculated from the expression average of 31 cell cycle genes normalized by 15 housekeeper genes. The prognostic value of the CCP score to predict stage I and II patient outcomes was evaluated by Cox proportional hazards analysis with disease-related death as the primary outcome measure. Results: In 256 DC cases, the CCP score was a significant predictor of death in univariate (p=0.0001) and multivariate analysis (p=0.001, HR 1.57, 95%CI 1.20-2.05) using age, stage, gender, smoking status and treatment as covariates. Similarly, in a second data set (GSE31210, n=204) the CCP score was highly associated with death (univariate, p=0.001; multivariate analysis, p=0.003, HR 1.81, 95% CI 1.24-2.66). Using quantitative PCR, the signature was applied to 381 FFPE samples with a median follow-up of 5 years collected at the MD Anderson Cancer Center and the European Institute for Oncology. In the presence of clinical covariates (as above and tumor size and pleural invasion), the CCP score remained the most significant predictor of death in univariate (p=0.0003) and multivariate analysis (p=0.007, HR 1.50, 95% CI 1.11-2.02). Conclusions: A 46 gene mRNA signature is a significant predictor of disease-related death in early-stage lung adenocarcinoma, providing independent prognostic value in the presence of clinical variables. This molecular predictor of cancer survival will be studied in additional cohorts for its ability to impact clinical treatment decisions.

Abstract 2516: Immunohistochemical characterization of a novel, highly sensitive monoclonal antibody to folate receptor α

Abstract Background: Folate receptor ≤ (FRα) has restricted expression in normal tissues but high-level expression in a subset of epithelial-derived tumors, including non-mucinous ovarian carcinomas and non-small cell lung (NSCL) adenocarcinomas. FRα has shown significant potential for targeted cancer therapy and several promising agents are in late-stage clinical development. As such, robust reagents are needed that can be used across diverse diagnostic platforms to identify patients who have FRα-expressing tumors. The immunohistochemical (IHC) characterization of a novel, high-affinity monoclonal antibody (MAb) against FRα is described. Methods: MAbs were generated to modified full-length FRα protein using standard methods. One MAb, 26B3.F2, displayed high affinity and the ability to bind FRα on both fresh/frozen and formalin-fixed, paraffin-embedded tissue sections. An optimized IHC protocol was developed and FRα expression was assessed on commercial tissue microarrays (US Biomax). Data were analyzed using an M-score, a newly introduced, weighted-intensity score that incorporates both the proportion of FRα-positive cells and the staining intensity. A board-certified pathologist scored the level of FRα expression. Results: Normal tissue staining for MAb 26B3.F2 demonstrated a limited distribution on focal epithelial surfaces of pancreas, lung, salivary gland, kidney, hypophysis, thyroid, and breast samples. Staining was mainly located in the lumen. The tissue distribution is consistent with the published literature. Strong staining was observed in 100% of serous ovarian samples and a significant proportion of ovarian endometrioid adenocarcinoma samples. FRα expression, as determined by MAb 26B3.F2 in NSCL cancer samples, demonstrated high discrimination between adenocarcinoma (positive) and squamous cell carcinomas (negative) (P < 0.001). Conclusion: MAb 26B3.F2 is a highly sensitive, robust agent suitable for identifying FRα expression by IHC and other diagnostic platforms. In addition to the expected distribution in normal tissue, MAb 26B3.F2 revealed high-level expression of FRα in ovarian cancer and NSCL adenocarcinoma samples, and the ability to discriminate differential expression between two NSCL cancer subtypes, squamous cell carcinoma and adenocarcinoma. Preliminary evidence therefore suggests that MAb 26B3.F2 IHC will be a useful tool to support further development of FRα-targeted therapeutics. 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 2516. doi:1538-7445.AM2012-2516

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvβ3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a KrasG12D-LSLp53fl/fl subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

Distinguishing Non-Small Cell Lung Adenocarcinoma Patients from Squamous Cell Carcinoma Patients and Control Individuals Using Serum Profiling

Goals of this study were to analyze the ability of mass spectrometry serum profiling to distinguish non-small cell lung adenocarcinoma from squamous cell carcinoma patients and healthy controls. Sera were obtained from 19 adenocarcinoma patients, 24 squamous cell carcinoma patients, and 21 controls. Identifications of significant mass-to-charge ratio (m/z) peak differences between these groups were performed using t-tests. A “leave one out” cross-validation procedure yielded discriminatory lung adenocarcinoma versus squamous cell carcinoma p and ROC curve values of <.0001 and 0.92, respectively. Test sensitivity and specificity were 84% and 79%, respectively. This approach could aid in lung cancer diagnosis and sub-typing.

Abstract B4: A “DNA replication stress” gene signature associated with a poor prognosis in early non-small cell lung cancer

Abstract The conventional clinical staging classification is not sufficient to predict the survival of patients who suffer from early lung cancer. Additional prognostic factors are then needed to better forecast their outcome. Since perturbation of the genome replication i.e. the so-called “replicative stress” is admitted to contribute to neoplasia from its early stages, we hypothesized that genes involved in such process may therefore represent a still under-explored source of such biomarkers. We specifically assessed in primary tumors and adjacent normal tissues from a series of 93 patients suffering from early- or mid-stage non-small cell lung adenocarcinoma the expression (RT-qPCR) of 77 “DNA replication” genes involved in either initiation/licensing at the 50,000 replication origins dispersed along the genome, elongation of DNA chains onto damaged or undamaged DNA, maintenance of stalled DNA replication forks or intra-S phase DNA damage processing and signaling. A 4-gene signature separated patients to high-risk and low-risk subgroups with significantly different survival. This prognostic effect was independent on age, sex, treatment, stage classification and expression of proliferation markers. We propose that a “cancer replisome” signature could be a predictor of the cancer survival and might also help understanding the molecular mechanisms underlying tumor progression in lung cancer patients.

Comparison of the clonogenic survival of A549 non-small cell lung adenocarcinoma cells after irradiation with low-dose-rate beta particles and high-dose-rate X-rays

Purpose: Lung cancer is the leading cause of cancer-related death. Among the new modalities to treat cancer, internal radiotherapy seems to be very promising. However, the achievable dose-rate is two orders of magnitude lower than the one used in conventional external radiotherapy, and data has to be collected to evaluate the cell response to highlight the potential effectiveness of low-dose-rate beta particles irradiation. This work investigates the phosphorus beta irradiation (32P) dose response on the clonogenicity of human A549 non-small cell lung adenocarcinoma cells and compares it to high-dose-rate X-irradiations results.Materials and methods: Cell survival was evaluated by a colony forming assay eight days after low-dose-rate 32P beta irradiations (0.8 Gy/h) and high-dose-rate X-ray irradiations (0.855 Gy/min).Results: Survival curves were obtained for both types of irradiations, and showed hyper-radiosensitivity at very low doses. Radiosensitivity parameters were obtained by using the linear-quadratic and induced-repair models.Conclusions: Comparison with high-dose-rate X-rays shows a similar surviving fraction, confirming the effectiveness of beta particles for tumor sterilization.

Serum Heparan Sulfate Concentration is Correlated with the Failure of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Treatment in Patients with Lung Adenocarcinoma

The epidermal growth factor receptor (EGFR) mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC. The pretreatment serum HS concentrations were determined using enzyme-linked immunosorbent assay in 83 patients with stage IV non-small cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) were analyzed. Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease than among those without progressive disease (p = 0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (p = 0.0022 and p = 0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (p = 0.0012 and p = 0.0003). We concluded that a high-serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising noninvasive and repeatable glycobiological biomarker in cancer treatment.

DNA Unwinding by ASCC3 Helicase Is Coupled to ALKBH3-Dependent DNA Alkylation Repair and Cancer Cell Proliferation

Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the activating signal cointegrator complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3'-5' DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain genomic integrity in a cell-type specific manner.

Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments

Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from whole cell lysate digests and compared it to the well-established SCX-TiO(2) workflow for phosphopeptide purification on a proteome-wide scale. We demonstrate the scaleabilty of our approach from 200 μg to 5 mg of total NCI-H23 non-small cell lung adenocarcinoma cell lysate digest and determine its quantitative reproducibility by label-free analysis of phosphopeptide peak areas from replicate purifications (median CV: 20% RSD). Finally, we combine this approach with immunoaffinity phosphotyrosine enrichment, enabling the identification of 3168 unique nonredundant phosphotyrosine peptides in two LC-MS/MS runs from 8 mg of HeLa peptides, each with 80% phosphotyrosine selectivity, at a peptide FDR of 0.2%. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in a variety of scenarios that is easy to implement in biomedical research and translational settings.

An analysis of serum heparan sulfate concentration and EGFR tyrosine kinase inhibitor treatment in patients with non-small cell lung adenocarcinoma.

10601 Background: The EGFR mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small-cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC. Methods: The pre-treatment serum HS concentrations were determined using ELISA in 83 patients with stage IV non-small-cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS) and overall survival (OS) were analyzed. Results: Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease (PD) than among those without PD (P=0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (P=0.0022 and P=0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (P=0.0012 and P=0.0003). Conclusions: We concluded that a high serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising non-invasive and repeatable glycobiological biomarker in cancer treatment.

Abstract 3617: Evaluation of folate receptor 1 (FOLR1) expression by calibrated immunohistochemistry identifies candidate tumor subtypes for targeting by IMGN853, an anti-FOLR1-maytansinoid conjugate

Abstract Background: The folate receptor α (FOLR1) is a GPI-linked glycoprotein reported to be highly expressed in cancers including ovarian, renal, brain, lung, and breast carcinomas, while having restricted distribution in normal tissues, including kidney and lung. This expression profile suggests that FOLR1 may be an attractive target for development of a therapeutic antibody-drug conjugate. The antibody maytansinoid conjugate (AMC), IMGN853, consists of a FOLR1-binding monoclonal antibody, M9346A, conjugated to the maytansinoid, DM4, attached via the cleavable sulfo-SPDB linker. IMGN853 exhibited potent anti-tumor activity in preclinical studies, supporting its development as a treatment for FOLR1-expressing cancers. Objective: The aim of the study was to identify potential patient populations for treatment with IMGN853 by determining the level, cellular distribution, and incidence of FOLR1 expression in carcinomas. Methods: A calibrated immunohistochemical (IHC) staining method was developed for assessing FOLR1 expression in formalin-fixed paraffin-embedded (FFPE) tissues. Staining conditions were established wherein the intensity of FOLR1 membrane staining observed in tumor tissues could be related to FFPE cell pellet controls, prepared from human tumor cell lines expressing known levels of FOLR1 (quantified by flow cytometry). This calibrated IHC staining method was applied to FOLR1-positive controls and a panel of tumor tissues including ovarian, renal, brain, lung, and breast carcinomas. The extent, pattern, and intensity of immunostaining were evaluated for each tissue, and the level of FOLR1 expression was estimated using comparison to the cell pellet controls. Results: Biopsy samples from patients with ovarian cancer and non-small cell lung adenocarcinoma or bronchiolalveolar carcinomas of the lung express significant levels of membrane-associated FOLR1. Expression in these indications was frequently observed exhibiting high, homogeneous, and cell membrane-localized patterns – properties considered favorable for AMC-targeting. In particular, a high percentage of the most common subtypes of ovarian cancer, serous (79%) and endometrioid (70%), expressed high (&amp;gt; 260,000 antigen sites per cell) and homogeneous levels of FOLR1. Likewise, a high percentage of the non-small cell lung adenocarcinoma (54%) and bronchioloalveolar carcinoma (100%) subtypes also expressed high (&amp;gt; 260,000 antigens per cell) levels. In contrast, FOLR1 expression in most non-small cell lung squamous carcinomas was undetectable. These findings support the development of IMGN853 for treatment of ovarian carcinomas and NSCL adenocarcinoma and bronchiolalveolar carcinomas, and provide a potential approach for assessing FOLR1 expression in patient tumors during clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2011-3617

Abstract 1041: Alterations of INPP4B, PIK3CA and pAkt of the PI3K pathway are associated with squamous cell carcinoma of the lung

Abstract The aim of the study was to investigate how alterations in the PI3K pathway correlate with the non-small cell lung cancer subtypes squamous cell carcinoma (SSC) and adenocarcinoma (ADCA). We analyzed copy number variation (CNV) and protein expression of the newly identified phosphatase INPP4B, protein expression of pAkt, PDPK1 and PTEN as well as mutational status of PIK3CA and PTEN in 180 lung cancer cases. Nineteen % of all cases displayed loss of INPP4B copy whereas 47% lacked expression. Both CNV and expression analyses showed a correlation between loss of INPP4B and the squamous subtype (RR = 1.44, p = 0.06; RR = 1.86, p = 0.00003). Elevated level of pAkt expression was seen in 63% of all cases and also correlated specifically to SCC (RR = 1.71, p = 0.001). PDPK1 was expressed in 70% of all cases, more in male than female patients, but no difference between the tumor subtypes. Regarding PTEN, 50% of all cases displayed loss of expression, of which seven cases were identified with mutations in the phosphatase or C2 domains. We detected nine cases (5%) of PIK3CA mutations, all identified as the E545K hot spot mutation in the helical domain, all except one in SCC (RR = 1.69, p = 0.05). When analyzing all PI3K pathway components together, we show that patients with at least one alteration in the PI3K pathway are twice as likely to have SCC, than ADCA (RR = 2.01, p = 0.02). Interestingly, we also found a strong correlation between high pAkt expression and PTEN expression, which we speculate can be explained by a feedback-loop via EGR1 and ARF recently proposed by Yu et al, 2009. As comparison, we also analyzed MAPK pathway genes, where we identified fifteen KRAS mutations (8%) and one BRAF mutation (1%), significantly associated to ADCA. The intermediate insulin receptor substrate 1 (IRS-1) is involved in activation of both the PI3K and MAPK pathways and we were interested in studying the Gly972Arg polymorphism previously shown to be associated with prostate, colon and breast cancer. However, we could not detect any associations to any subgroup. In conclusion, we show here that several components of the PI3K pathway, alone and in combination, are correlated to development of squamous cell carcinoma of the lung. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2011-1041

Abstract 4357: Tumor initiating cells promoting angiogenesis as a model of tumor re-growth following chemotherapy

Abstract Tumor stem-like cells, also referred to as tumor initiating cells (TICs), are thought to display the only cells in the tumor with the proliferative capacity to seed tumor growth when transplanted into immune deficient mice, implying that they are critical in initiating and driving tumorigenesis. Previous studies linked TICs with angiogenesis, a process critical for tumor growth. We hypothesized that tumors enriched with TICs, in addition to their proliferative capacity, may be strong inducers of tumor angiogenesis in particular after chemotherapy. To study the pro-angiogenic capabilities of TICs, we isolated and characterized TIC populations from a series of human established cell lines; including U87 glioma, HT29 colon carcinoma, A549 non-small cell lung adenocarcinoma, MCF7 breast carcinoma, PANC1 pancreatic adenocarcinoma. We generated long-term cultures grown either as monolayers in serum-containing medium (‘TIC-low’) or as non-adherent tumor spheres in serum-free medium containing factors promoting stem cell growth (‘TIC-high’). Here we demonstrate that an increased expression of stem cell surface markers, a high activity of aldehyde dehydrogenase, a high expression of P21, a potent cyclin-dependent kinase inhibitor, and resistance to standard chemotherapy and radiotherapy were observed in TIC-high fractions of all cell lines in comparison to TIC-low fractions. TIC-high conditioned media of U87 and HT29 cells but not MCF7, A549, and PANC1, significantly induced endothelial cell migration, invasion, and aortic ring endothelial cell tube formation as opposed to TIC-low conditioned media. Matrigel plugs containing TIC-high conditioned media of U87 and HT29 were enriched with blood vessel capillaries and bone marrow derived proangiogenic cells (BMDCs) including hemangiocytes, and Gr-1+/CD11b+ myeloid derived suppressor cells, when compared to TIC-low conditioned media. No significant differences in BMDC colonization and endothelial cell number were observed in Matrigel plugs containing TIC-high conditioned media from MCF7, A549, and PANC1. Interestingly, the addition of an antiangiogenic drug to conditioned media of U87 and HT29 but not PANC1 inhibited angiogenesis using aortic ring assay. Results from experiments conducted on U87 and PANC1 tumor bearing mice to further investigate the angiogenesis properties of TICs following chemotherapy, will be presented in the meeting. Collectively, our results indicate that TICs of U87 and HT29 act as significant contributors to tumor angiogenesis and vasculogenesis by actively recruiting BMDCs and promoting endothelial cell function. Since TICs are resistant to chemotherapy they may contribute to tumor re-growth, at least in part, by inducing angiogenesis. These results indicate the necessity of new strategies for targeting TICs in combination with chemotherapy in order to improve clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2011-4357

Rac1 Targeting Suppresses Human Non-Small Cell Lung Adenocarcinoma Cancer Stem Cell Activity

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Use of novel radiosensitizers and radiation-specific miRNAs to modulate radiation response in non-small cell lung adenocarcinoma (NSCLC).

e17519 Background: Tumor response to radiotherapy in NSCLC remains markedly heterogeneous. Few successful radiosensitivity predictive assays exist that can be applied to individualize radiotherapeutic options. Methods: Using Bayesian regression analysis, a 52-gene expression signature that predicts sensitivity to external beam radiotherapy was developed and independently validated. On the basis of the signature, using Connectivity Map, novel candidate radiosensitizers were identified and tested in vitro by cell proliferation assays on predicted radioresistant NSCLC cell lines at three different radiation doses (3, 5 and 7 Gy). Cisplatin was used as a clinically relevant control. Furthermore, a systemic computational approach was developed to identify potential radiation-specific miRNAs, which were later transiently expressed and validated in vitro using the aforementioned NSCLC cell lines. Results: The 52-gene expression signature accurately predicted radiosensitivity in vitro and in individuals treated with radiation or chemoradiation independent of tissue. Tumor cell-specific novel agents, such as ethacrynic acid and docosahexaenoic acid have shown to radiosensitize NSCLC cell lines in a drug and radiation dose-indepependent manner. Finally, radiation-specific miRNAs (hsa-let-7b, hsa- let-7i and hsa-miR30a), which were shown to regulate RAS signaling pathway as evidenced from Western analyses, significantly radiosensitize (p<0.001 for all three miRNAs with respect to mock transfected controls) NSCLC cell lines. Conclusions: Comprehensive genomic analysis using gene expression signatures and miRNA data provides opportunities to better understand the mechanisms underlying radiation response and provides novel therapeutic opportunities in NSCLC. No significant financial relationships to disclose.

Pharmacokinetic properties and antitumor efficacy of the 5-fluorouracil loaded PEG-hydrogel

BackgroundWe have studied the in vitro and in vivo utility of polyethylene glycol (PEG)-hydrogels for the development of an anticancer drug 5-fluorouracil (5-FU) delivery system.MethodsA 5-FU-loaded PEG-hydrogel was implanted subcutaneously to evaluate the drug retention time and the anticancer effect. For the pharmacokinetic study, two groups of male rats were administered either an aqueous solution of 5-FU (control group)/or a 5-FU-loaded PEG-hydrogel (treated group) at a dose of 100 mg/kg. For the pharmacodynamic study, a human non-small-cell lung adenocarcinoma (NSCLC) cell line, A549 was inoculated to male nude mice with a cell density of 3 × 106. Once tumors start growing, the mice were injected with 5-FU/or 5-FU-loaded PEG-hydrogel once a week for 4 weeks. The growth of the tumors was monitored by measuring the tumor volume and calculating the tumor inhibition rate (IR) over the duration of the study.ResultsIn the pharmacokinetic study, the 5-FU-loaded PEG-hydrogel gave a mean residence time (MRT) of 8.0 h and the elimination half-life of 0.9 h; these values were 14- and 6-fold, respectively, longer than those for the free solution of 5-FU (p < 0.05). In the pharmacodynamic study, A549 tumor growth was significantly inhibited in the 5-FU-loaded PEG-hydrogel group in comparison to the untreated group beginning on Day 14 (p < 0.05-0.01). Moreover, the 5-FU-loaded PEG-hydrogel group had a significantly enhanced tumor IR (p < 0.05) compared to the free 5-FU drug treatment group.ConclusionWe suggest that 5-FU-loaded PEG-hydrogels could provide a useful tool for the development of an anticancer drug delivery system.

Abstract 2273: Angiotensin-(1-7) reduces angiogenesis and fibrosis in orthotopic breast cancer

Abstract Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven-amino acid peptide hormone of the renin-angiotensin system with anti-proliferative properties. We previously showed that Ang-(1-7) significantly inhibited the growth of non-small cell lung adenocarcinoma in a xenograft mouse model in association with a reduction in angiogenesis. In a recently completed Phase I clinical trial conducted in the Wake Forest University Comprehensive Cancer Center, we reported that Ang-(1-7) significantly reduced the pro-angiogenic factor placental growth factor (PlGF) in four patients who had stable disease for greater than 3 months, suggesting that Ang-(1-7) is a novel anti-angiogenic drug for the treatment of cancer. In this study, BT-474 human epidermal growth factor receptor 2 (HER2) over-expressed, estrogen receptor (ER) positive breast cancer cells or ZR-75-1 ER positive breast cancer cells were injected into mammary fat pads of athymic mice and treated with Ang-(1-7), to determine the effect of the drug on breast cancer. After the tumors grew to 100-200 mm3, mice were treated with either saline or 24 µg/kg/h Ang-(1-7) for 18 days. Treatment with Ang-(1-7) reduced tumor volume (68.2% in BT-474 tumors and 77.0% in ZR-75-1 tumors) and tumor weight (38.9% in BT-474 tumors and 50% in ZR-75-1 tumors), indicating that the heptapeptide inhibits breast tumor growth. Vessel density was also decreased 50% by the heptapeptide, demonstrating that Ang-(1-7) reduced angiogenesis in breast tumors. Tumoral fibrosis, measured by picrosirius red collagen staining, was attenuated in Ang-(1-7)-treated breast tumors. The heptapeptide reduced interstitial fibrosis from 4.91 ± 0.96%/field to 1.2 ± 0.2 in BT474 tumors and from 23.3 ± 2.4%/field to 8.3 ± 0.8 in ZR-75-1 tumors, in association with a 66% reduction in collagen I deposition. Treatment with Ang-(1-7) also reduced perivascular fibrosis in BT-474 tumors, from 49.3 ± 3.2% fibrosis/vessel to 13.4 ± 2.2% fibrosis/vessel. Ang-(1-7) markedly attenuated the in vitro growth of fibroblasts isolated from orthotopic breast tumors. Treatment of tumoral fibroblasts with Ang-(1-7) reduced fibronectin protein by 40%, in association with a 50% decrease in transforming growth factor-β (TGF-β). Ang-(1-7) also caused a 2-fold increase in the mitogen-activated protein (MAP) kinase phosphatase DUSP1, with an associated 50% decrease in MAP kinase ERK1 and ERK2 activities, suggesting that the heptapeptide increases DUSP1 to reduce MAP kinase signaling. This is the first report that Ang-(1-7) targets the tumor microenvironment, by inhibiting the growth of tumor-associated fibroblasts to reduce fibrosis. Collectively, these results suggest that Ang-(1-7) may serve as a first-in-class chemotherapeutic agent for breast cancer, targeting the tumor microenvironment through a reduction in angiogenesis and a decrease in tumor fibrosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2273.

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Delta) complemented the Rin1 depletion effects, and overexpression of Rin1Delta had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.

The Cell Line Secretome, a Suitable Tool for Investigating Proteins Released in Vivo by Tumors: Application to the Study of p53-Modulated Proteins Secreted in Lung Cancer Cells

Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. p53, a major tumor suppressor protein very frequently mutated and/or inactivated in cancer cells, is known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we characterized the p53-dependent secretome of a lung tumor model in vitro (H358 human nonsmall cell lung adenocarcinoma cell line with a homozygous deletion of p53) and demonstrate that the modulation of exported proteins can also be detected in vivo in the plasma of tumor-bearing mice. We used a clone of H358, stably transfected with a tetracycline-inducible wild-type p53-expressing vector. With the use of iTRAQ labeling and LC-MALDI-MS/MS analysis, we identified 909 proteins released in vitro by the cells, among which 91 are p53-modulated. Three proteins (GDF-15, FGF-19, and VEGF) were also investigated in H358/TetOn/p53 xenograft mice. The ELISA dosage on total tumor protein extracts confirmed the influence of p53 on the release of these proteins in vivo. Moreover, the GDF-15 concentration was measured in the plasma and its p53-dependent modulation was confirmed. To our knowledge, this is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.