Christopher D. Coldren

Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non–small cell lung carcinoma

The modest response of patients with head and neck squamous cell carcinoma (HNSCC) and non–small cell lung carcinoma (NSCLC) to epithelial growth factor receptor tyrosine kinase inhibitors such as gefitinib and erlotinib indicates the need for the development of biomarkers to predict response. We determined gefitinib sensitivity in a panel of HNSCC cell lines by a 5-day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confirmed these responses with analysis of downstream signaling by immunoblotting and cell cycle arrest. Basal gene expression profiles were then determined by microarray analysis and correlated with gefitinib response. These data were combined with previously reported NSCLC microarray results to generate a broader predictive index. Common markers of resistance between the two tumor types included genes associated with the epithelial to mesenchymal transition. We confirmed that increased protein expression of vimentin combined with the loss of E-cadherin, claudin 4, and claudin 7 by immunoblotting was associated with gefitinib resistance in both HNSCC and NSCLC cell lines. In addition, the loss of the Ca2+-independent cell-cell adhesion molecules EpCAM and TROP2 in resistant lines was confirmed by immunofluorescence. Tumor xenografts derived from the gefitinib-sensitive UM-SCC-2 were growth-delayed by gefitinib, whereas the gefitinib-resistant 1483 xenografts were unaffected. These data support a role for epithelial to mesenchymal transition in establishing gefitinib resistance for both HNSCC and NSCLC, and indicate that clinical trials should address whether these biomarkers will be useful for patient selection. [Mol Cancer Ther 2007;6(6):1683–91]

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.

Baseline Gene Expression Predicts Sensitivity to Gefitinib in Non–Small Cell Lung Cancer Cell Lines

Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non–small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance. (Mol Cancer Res 2006;4(8):521–8)

Antitumor activity of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in non-small cell lung cancer cell lines correlates with gene copy number and EGFR mutations but not EGFR protein levels.

Purpose: Recognition that the epidermal growth factor receptor (EGFR) was a therapeutic target in non–small cell lung cancer (NSCLC) and other cancers led to development of the small-molecule receptor tyrosine kinase inhibitors gefitinib and erlotinib. Clinical trials established that EGFR tyrosine kinase inhibitors produced objective responses in a minority of NSCLC patients. We examined the sensitivity of 23 NSCLC lines with wild-type or mutated EGFR to gefitinib to determine genes/proteins related to sensitivity, including EGFR and HER2 cell surface expression, phosphorylated EGFR expression, EGFR gene copy number, and EGFR mutational status. Downstream cell cycle and signaling events were compared with growth-inhibitory effects. Experimental Design: We determined gefitinib sensitivity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, EGFR expression by fluorescence-activated cell sorting and immunohistochemistry, phosphorylated EGFR by Western blotting, EGFR gene copy number by fluorescence in situ hybridization, and EGFR mutation by sequencing. The cellular effects of gefitinib on cell cycle were determined by flow cytometry and the molecular effects of gefitinib EGFR inhibition on downstream signal proteins by Western blotting. Gefitinib in vivo effects were evaluated in athymic nude mice bearing sensitive and resistant NSCLC xenografts. Results: There was a significant correlation between EGFR gene copy number, EGFR gene mutations, and gefitinib sensitivity. EGFR protein was necessary but not sufficient for predicting sensitivity. Gefitinib-sensitive lines showed a G1 cell cycle arrest and inactivation of downstream signaling proteins; resistant cell lines had no changes. The in vivo effects mirrored the in vitro effects. Conclusions: This panel of NSCLC lines characterized for gefitinib response was used to identify predictive molecular markers of response to gefitinib. Several of these have subsequently been shown to identify NSCLC patients likely to benefit from gefitinib therapy.

ZEB1-responsive genes in non-small cell lung cancer.

The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.

Somatic Chromosome Abnormalities in the Lungs of Patients with Pulmonary Arterial Hypertension

RATIONALE Vascular remodeling in pulmonary arterial hypertension (PAH) involves proliferation and migration of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Previous studies have indicated that the endothelial cell proliferation is quasineoplastic, with evidence of monoclonality and instability of short DNA microsatellite sequences. OBJECTIVES To assess whether there is larger-scale genomic instability. METHODS We performed genome-wide microarray copy number analysis on pulmonary artery endothelial cells and smooth muscle cells isolated from the lungs of patients with PAH. MEASUREMENTS AND MAIN RESULTS Mosaic chromosomal abnormalities were detected in PAEC cultures from five of nine PAH lungs but not in normal (n = 8) or disease control subjects (n = 5). Fluorescent in situ hybridization analysis confirmed the presence of these abnormalities in vivo in two of three cases. One patient harbored a germline mutation of BMPR2, the primary genetic cause of PAH, and somatic loss of chromosome-13, which constitutes a second hit in the same pathway by deleting Smad-8. In two female subjects with mosaic loss of the X chromosome, methylation analysis showed that the active X was deleted. One subject also showed completely skewed X-inactivation in the nondeleted cells, suggesting the pulmonary artery endothelial cell population was clonal before the acquisition of the chromosome abnormality. CONCLUSIONS Our data indicate a high frequency of genetically abnormal subclones within PAH lung vessels and provide the first definitive evidence of a second genetic hit in a patient with a germline BMPR2 mutation. We propose that these chromosome abnormalities may confer a growth advantage and thus contribute to the progression of PAH.

Expression of cilium-associated genes defines novel molecular subtypes of idiopathic pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is an untreatable lung disease with a median survival of only 3–5 years that is diagnosed using a combination of clinical, radiographic and pathologic criteria. Histologically, IPF is characterised by usual interstitial pneumonia (UIP), a fibrosing interstitial pneumonia with a pattern of heterogeneous, subpleural regions of fibrotic and remodelled lung. We hypothesised that gene expression profiles of lung tissue may identify molecular subtypes of disease that could classify subtypes of IPF/UIP that have clinical implications. Methods and findings We collected transcriptional profiles on lung tissue from 119 patients with IPF/UIP and 50 non-diseased controls. Differential expression of individual transcripts was identified using an analysis of covariance (ANCOVA) model incorporating the clinical diagnosis of each patient as well as age, gender and smoking status. Validation was performed in an independent cohort of 111 IPF/UIP and 39 non-diseased controls. Our analysis identified two subtypes of IPF/UIP based on a strong molecular signature associated with expression of genes previously associated with fibrosis (matrix metalloproteinases, osteopontin, keratins), cilium genes and genes with unknown function. We demonstrate that elevated expression of cilium genes is associated with more extensive microscopic honeycombing and higher expression of both the airway mucin gene MUC5B and the metalloproteinase MMP7, a gene recently implicated in attenuating ciliated cell differentiation during wound repair. Conclusions Expression of cilium genes appears to identify two unique molecular phenotypes of IPF/UIP. The different molecular profiles may be relevant to therapeutic responsiveness in patients with IPF/UIP.

Development of an Integrated Genomic Classifier for a Novel Agent in Colorectal Cancer: Approach to Individualized Therapy in Early Development

Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.

Peripheral Blood Mononuclear Cell Gene Expression in Chronic Obstructive Pulmonary Disease

Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.