Daniel T. Merrick

Fibroblast Growth Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-Small-Cell Lung Cancer Cells

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(±)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

Insulin-like Growth Factor Receptor 1 (IGF1R) Gene Copy Number Is Associated With Survival in Operable Non–Small-Cell Lung Cancer: A Comparison Between IGF1R Fluorescent In Situ Hybridization, Protein Expression, and mRNA Expression

PURPOSE The purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non-small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis. PATIENTS AND METHODS One hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181). RESULTS IGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis. CONCLUSION IGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway—involving AKT, CREB, Bcl-xL, survivin, and Bcl-2—downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

Oral Iloprost Improves Endobronchial Dysplasia in Former Smokers

There are no established chemopreventive agents for lung cancer, the leading cause of cancer death in the United States. Prostacyclin levels are low in lung cancer and supplementation prevents lung cancer in preclinical models. We carried out a multicenter double-blind, randomized, phase II placebo-controlled trial of oral iloprost in current or former smokers with sputum cytologic atypia or endobronchial dysplasia. Bronchoscopy was performed at study entry and after completion of six months of therapy. Within each subject, the results were calculated by using the average score of all biopsies (Avg), the worst biopsy score (Max), and the dysplasia index (DI). Change in Avg was the primary end point, evaluated in all subjects, as well as in current and former smokers. The accrual goal of 152 subjects was reached and 125 completed both bronchoscopies (60/75 iloprost, 65/77 placebo). Treatment groups were well matched for age, tobacco exposure, and baseline histology. Baseline histology was significantly worse for current smokers (Avg 3.0) than former smokers (Avg 2.1). When compared with placebo, former smokers receiving oral iloprost exhibited a significantly greater improvement in Avg (0.41 units better, P = 0.010), in Max (1.10 units better, P = 0.002), and in DI (12.45%, P = 0.006). No histologic improvement occurred in current smokers. Oral iloprost significantly improves endobronchial histology in former smokers and deserves further study to determine if it can prevent the development of lung cancer. Cancer Prev Res; 4(6); 793–802. ©2011 AACR.

Analysis of c-ErbB1/Epidermal Growth Factor Receptor and c-ErbB2/HER-2 Expression in Bronchial Dysplasia: Evaluation of Potential Targets for Chemoprevention of Lung Cancer

Purpose: Lung cancer is preceded by a premalignant phase during which intervention could decrease associated morbidity and mortality. Molecular characterization of factors involved in controlling progression of bronchial dysplasias will provide markers of premalignant change and identify targets for chemoprevention. Experimental Design: Immunohistochemical analysis of epidermal growth factor receptor (EGFR; c-ErbB1/EGFR), HER-2/neu (c-ErbB2/HER-2), Ki-67, and minichromosome maintenance protein 2 (MCM2) expression in bronchial dysplasia was undertaken to characterize molecular alterations associated with the progression of these lesions in 268 bronchoscopically obtained biopsies from 134 subjects. Results: Analysis of biopsies with the most severe diagnosis from each subject showed a linear relationship between increasing marker expression and severity of dysplastic change for EGFR (P < 0.001), Ki-67 (P < 0.001), and MCM2 (P = 0.001) but not HER-2 (P = 0.102). Increased expression of either EGFR or HER-2 was associated with increased levels of Ki-67 and MCM2 expression, and combined overexpression of these receptors was associated with the highest levels of proliferation, suggesting a synergistic effect. Finally, the lack of an associated trend toward increased EGFR expression when comparing the worst and best biopsies within each subject indicated a potential field effect in the induction of EGFR expression. Conclusions: The results suggest a prominent role for EGFR overexpression in the development and progression of bronchial dysplasia and provide rationale for exploring inhibition of EGFR signaling in lung cancer chemoprevention.

Depletion of Tumor-Associated Macrophages Slows the Growth of Chemically Induced Mouse Lung Adenocarcinomas

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4–6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Loss of Transforming Growth Factor Beta Type II Receptor Increases Aggressive Tumor Behavior and Reduces Survival in Lung Adenocarcinoma and Squamous Cell Carcinoma

Purpose: Lung adenocarcinoma and lung squamous cell carcinoma (SCC) are the most common non–small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of TGFβ type II receptor (TGFβRII) promotes lung adenocarcinoma and SCC carcinogenesis. Experimental Design: We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinicopathologic parameters. To determine whether sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic KrasG12D to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. Results: Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of KrasG12D-initiated adenocarcinoma and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion. Conclusions: Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII shows haploid insufficiency suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis. Clin Cancer Res; 18(8); 2173–83. ©2012 AACR.

Recent developments in biomarkers for the early detection of lung cancer: perspectives based on publications 2003 to present.

Purpose of review Given the poor prognosis associated with lung cancer, the ability to diagnose lung cancer in its early stages is considered crucial to achieving decreased lung cancer mortality. Herein, we discuss recent advances in biomarker discovery and evaluation of their potential application in the clinical setting. Recent findings Novel potential biomarkers have been identified via new techniques including cDNA microarray analysis, comparative genomic hybridization, and proteomics. These factors have been evaluated in various validation studies including tissue microarrays, RT-PCR, and assays of bioactivity. The characterization of these potential biomarkers through analysis of pathways that have been associated with neo-plastic transformation may help to identify the precursor lesions that are associated with subsequent progression to invasive carcinoma. In addition, the past year has also produced intriguing results regarding the detection of biomarkers in easily accessible screening specimens such as sputa, serum, and exhaled breath. Recent advances in these aspects of biomarker identification for the early detection of lung cancer are reviewed. Summary Identification of new candidate biomarkers and improved applications of previously detected biomarkers show great promise for the ultimate establishment of practical lung cancer screening. While recent studies engender optimism for the creation of clinically applicable screening tests, the biomarkers that have been identified need larger, follow-up validational studies and further characterization as to their biologic importance.

Increased Insulin-Like Growth Factor 1 Receptor Protein Expression and Gene Copy Number in Small Cell Lung Cancer

Purpose: Identification of new therapies in small cell lung cancer (SCLC) is urgently needed. Insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. Knowledge about IGF1R protein expression, gene copy number, and the prognostic relevance of these features in SCLC is limited. Methods: We analyzed IGF1R protein expression and gene copy number in primary tumors from 90 patients with SCLC (67 men and 23 women) who underwent pulmonary resection. IGF1R expression assessed by immunohistochemistry with H scores from 0 to 400 was evaluable in 84 patients and IGF1R gene copy number assessed by silver in situ hybridization technique in 81 patients. Results: Median H score for IGF1R protein expression was 88 (range, 0–400), and the proportion of positive immunostaining using cutoff H score of 10 was 74%. Increased IGF1R gene copy number (an average of four or more copies per cell) was found in 15 cases (18.5%), five of whom (6.2%) showed gene amplification. There was a significant correlation between protein expression and gene copy number (r = 0.49, p < 0.005). IGF1R expression and gene copy number did not associate with clinicopathological factors such as patient age, tumor size, lymph node involvement, stage, and survival. Conclusions: SCLC is characterized by frequent high-IGF1R protein expression, increased gene copy number, and occasional occurrence of true gene amplification. These features may have important implications for future anti-IGF1R therapeutic approaches.

Sputum Cytologic Atypia Predicts Incident Lung Cancer: Defining Latency and Histologic Specificity

Background: There is a need for early detection methods for lung cancer. Radiologic imaging may be more sensitive for peripheral cancers than for cancers arising in the central airways, from which bronchial epithelial cells are exfoliated into the sputum. Methods: Sputum samples were collected at baseline and periodically thereafter in a cohort of smokers and former smokers with chronic obstructive lung disease. The association between cytologic atypia and incident lung cancer was assessed by hazard ratios (HR; 95% confidence intervals) using Cox regression and by odds ratios (95% confidence intervals) using logistic regression, adjusting for potential confounding factors. Results: We observed 174 incident lung cancers in a cohort of 2,521 people over 9,869 person-years of observation. Risk for incident lung cancer was increased among those with cytologic atypia graded as moderate or worse (adjusted HR, 2.37; 1.68-3.34). The association between sputum atypia and lung cancer incidence was greatest for those sputum samples collected 5 months or less before the diagnosis of lung cancer (odds ratio, 10.32; 5.34-19.97). The association was substantially stronger for squamous cell lung cancers (HR, 5.13; 2.89-9.10) than for adenocarcinomas (HR, 1.85; 0.94-3.65). Conclusion: Cytologic atypia is a marker for increased lung cancer risk. These cytologic changes seem to arise from late events that are most apparent for cancers arising in the central respiratory airways. Whether cytologic atypia might complement radiologic imaging in a combined approach to lung cancer, early detection requires additional evaluation of those two methods used together. (Cancer Epidemiol Biomarkers Prev 2008;17(1):158–63)