DNA Sequencing of Small Bowel Adenocarcinomas Identifies Targetable Recurrent Mutations in the ERBB2 Signaling Pathway.

PURPOSE: Little is known about the genetic alterations characteristic of small bowel adenocarcinoma (SBA). Our purpose was to identify targetable alterations and develop experimental models of this disease.Experimental Design: Whole-exome sequencing (WES) was completed on 17 SBA patient samples and targeted-exome sequencing (TES) on 27 samples to confirm relevant driver mutations. Two SBA models with ERBB2 kinase activating mutations were tested for sensitivity to anti-ERBB2 agents in vivo and in vitro. Biochemical changes were measured by reverse-phase protein arrays. RESULTS: WES identified somatic mutations in 4 canonical pathways (WNT, ERBB2, STAT3, and chromatin remodeling), which were validated in the TES cohort. Although APC mutations were present in only 23% of samples, additional WNT-related alterations were seen in 12%. ERBB2 mutations and amplifications were present in 23% of samples. Patients with alterations in the ERBB2 signaling cascade (64%) demonstrated worse clinical outcomes (median survival 70.3 months vs. 109 months; log-rank HR = 2.4, P = 0.03). Two ERBB2-mutated (V842I and Y803H) cell lines were generated from SBA patient samples. Both demonstrated high sensitivity to ERBB2 inhibitor dacomitinib (IC50 < 2.5 nmol/L). In xenografts derived from these samples, treatment with dacomitinib reduced tumor growth by 39% and 59%, respectively, whereas it had no effect in an SBA wild-type ERBB2 model. CONCLUSIONS: The in vitro and in vivo models of SBA developed here provide a valuable resource for understanding targetable mutations in this disease. Our findings support clinical efforts to target activating ERBB2 mutations in patients with SBA that harbor these alterations.

A miRNA signature associated with human metastatic medullary thyroid carcinoma.

MicroRNAs (miRNAs) represent a class of small, non-coding RNAs that control gene expression by targeting mRNA and triggering either translational repression or RNA degradation. The objective of our study was to evaluate the involvement of miRNAs in human medullary thyroid carcinoma (MTC) and to identify the markers of metastatic cells and aggressive tumour behaviour. Using matched primary and metastatic tumour samples, we identified a subset of miRNAs aberrantly regulated in metastatic MTC. Deregulated miRNAs were confirmed by quantitative real-time PCR and validated by in situ hybridisation on a large independent set of primary and metastatic MTC samples. Our results uncovered ten miRNAs that were significantly expressed and deregulated in metastatic tumours: miR-10a, miR-200b/-200c, miR-7 and miR-29c were down-regulated and miR-130a, miR-138, miR-193a-3p, miR-373 and miR-498 were up-regulated. Bioinformatic approaches revealed potential miRNA targets and signals involved in metastatic MTC pathways. Migration, proliferation and invasion assays were performed in cell lines treated with miR-200 antagomirs to ascertain a direct role for this miRNA in MTC tumourigenesis. We show that the members of miR-200 family regulate the expression of E-cadherin by directly targeting ZEB1 and ZEB2 mRNA and through the enhanced expression of tumour growth factor ß (TGFß)-2 and TGFß-1. Overall, the treated cells shifted to a mesenchymal phenotype, thereby acquiring an aggressive phenotype with increased motility and invasion. Our data identify a robust miRNA signature associated with metastatic MTC and distinct biological processes, e.g., TGFß signalling pathway, providing new potential insights into the mechanisms of MTC metastasis.

Plasma microRNA profiles for bladder cancer detection.

BACKGROUND: Bladder cancer (BC) is a burdensome disease with significant morbidity, mortality, and cost. The development of novel plasma-based biomarkers for BC diagnosis and surveillance could significantly improve clinical outcomes and decrease health expenditures. Plasma miRNAs are promising biomarkers that have yet to be rigorously investigated in BC. OBJECTIVE: To determine the feasibility and efficacy of detecting BC with plasma miRNA signatures. MATERIALS AND METHODS: Plasma miRNA was isolated from 20 patients with bladder cancer and 18 noncancerous controls. Samples were analyzed with a miRNA array containing duplicate probes for each miRNA in the Sanger database. Logistic regression modeling was used to optimize diagnostic miRNA signatures to distinguish between muscle invasive BC (MIBC), non-muscle-invasive BC (NMIBC) and noncancerous controls. RESULTS: Seventy-nine differentially expressed plasma miRNAs (local false discovery rate [FDR] <0.5) in patients with or without BC were identified. Some diagnostically relevant miRNAs, such as miR-200b, were up-regulated in MIBC patients, whereas others, such as miR-92 and miR-33, were inversely correlated with advanced clinical stage, supporting the notion that miRNAs released in the circulation have a variety of cellular origins. Logistic regression modeling was able to predict diagnosis with 89% accuracy for detecting the presence or absence of BC, 92% accuracy for distinguishing invasive BC from other cases, 100% accuracy for distinguishing MIBC from controls, and 79% accuracy for three-way classification between MIBC, NIMBC, and controls. CONCLUSIONS: This study provides preliminary data supporting the use of plasma miRNAs as a noninvasive means of BC detection. Future studies will be required to further specify the optimal plasma miRNA signature, and to apply these signatures to clinical scenarios, such as initial BC detection and BC surveillance.

p63 expression defines a lethal subset of muscle-invasive bladder cancers.

BACKGROUND: p63 is a member of the p53 family that has been implicated in maintenance of epithelial stem cell compartments. Previous studies demonstrated that p63 is downregulated in muscle-invasive bladder cancers, but the relationship between p63 expression and survival is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We used real-time PCR to characterize p63 expression and several genes implicated in epithelial-to-mesenchymal transition (EMT) in human bladder cancer cell lines (n = 15) and primary tumors (n = 101). We correlated tumor marker expression with stage, disease-specific (DSS), and overall survival (OS). Expression of E-cadherin and p63 correlated directly with one another and inversely with expression of the mesenchymal markers Zeb-1, Zeb-2, and vimentin. Non-muscle-invasive (Ta and T1) bladder cancers uniformly expressed high levels of E-cadherin and p63 and low levels of the mesenchymal markers. Interestingly, a subset of muscle-invasive (T2-T4) tumors maintained high levels of E-cadherin and p63 expression. As expected, there was a strongly significant correlation between EMT marker expression and muscle invasion (p<0.0001). However, OS was shorter in patients with muscle-invasive tumors that retained p63 (p = 0.007). CONCLUSIONS/SIGNIFICANCE: Our data confirm that molecular markers of EMT are elevated in muscle-invasive bladder cancers, but interestingly, retention of the "epithelial" marker p63 in muscle-invasive tumors is associated with a worse outcome.

Receptor heterodimerization: a new mechanism for platelet-derived growth factor induced resistance to anti-epidermal growth factor receptor therapy for bladder cancer.

PURPOSE: Human bladder cancer cells resistant to anti-epidermal growth factor receptor therapy often co-express platelet-derived growth factor receptor-ß. We determined whether there is functional crosstalk between epidermal growth factor receptor and platelet-derived growth factor receptor-ß, and how this regulates biological functions in bladder cancer cases. MATERIALS AND METHODS: We determined heterodimerization and co-localization of epidermal growth factor receptor and platelet-derived growth factor receptor-ß by immunoprecipitation and confocal microscopy, respectively. We tested the antiproliferative effects of specific inhibitory monoclonal antibodies to each receptor by (3)H-thymidine uptake assay. We transfected the nonplatelet-derived growth factor receptor-ß expressing bladder cancer cell line UMUC5 with the platelet-derived growth factor receptor-ß gene. These cells were analyzed in vitro by (3)H-thymidine uptake and by Matrigel™ invasion assay, and in vivo for tumorigenicity, metastatic potential and orthotopic growth. In a treatment study nude mice were inoculated with orthotopic tumors and treated with the inhibitory antibodies alone and in combination. RESULTS: Immunoprecipitation revealed epidermal growth factor receptor/platelet-derived growth factor receptor-ß heterodimers in all platelet-derived growth factor receptor-ß expressing cell lines. Forced expression of platelet-derived growth factor receptor-ß in epidermal growth factor receptor sensitive UMUC5 cells (50% inhibitory concentration less than 10 nM) significantly decreased responsiveness to epidermal growth factor receptor inhibition (50% inhibitory concentration greater than 100 nM) and increased invasive potential 3-fold as well as tumorigenicity. Increased invasiveness was associated with epidermal growth factor triggered platelet-derived growth factor receptor-ß transactivation, increased mitogen activated protein kinase and glycogen synthase kinase-3ß phosphorylation, and decreased E-cadherin. Inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-ß receptors blocked cell invasion, decreased cell proliferation, reduced xenograft tumor growth and increased E-cadherin expression. CONCLUSIONS: In epidermal growth factor receptor expressing bladder cancer co-expression of platelet-derived growth factor receptor-ß has implications for tumor biology. Thus, it should be further evaluated as a strategy involving dual receptor targeting.

Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer.

Epithelial-to-mesenchymal transition (EMT) is a process that plays essential roles in development and wound healing that is characterized by loss of homotypic adhesion and cell polarity and increased invasion and migration. At the molecular level, EMT is characterized by loss of E-cadherin and increased expression of several transcriptional repressors of E-cadherin expression (Zeb-1, Zeb-2, Twist, Snail, and Slug). Early work established that loss of E-cadherin and increased expression of MMP-9 was associated with a poor clinical outcome in patients with urothelial tumors, suggesting that EMT might also be associated with bladder cancer progression and metastasis. More recently, we have used global gene expression profiling to characterize the molecular heterogeneity in human urothelial cancer cell lines (n = 20) and primary patient tumors, and unsupervised clustering analyses revealed that the cells naturally segregate into two discrete "epithelial" and "mesenchymal" subsets, the latter consisting entirely of muscle-invasive tumors. Importantly, sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) or type-3 fibroblast growth factor receptor (FGFR3) was confined to the "epithelial" subset, and sensitivity to EGFR inhibitors could be reestablished by micro-RNA-mediated molecular reversal of EMT. The results suggest that EMT coordinately regulates drug resistance and muscle invasion/metastasis in urothelial cancer and is a dominant feature of overall cancer biology.

The stabilization and targeting of surfactant-synthesized gold nanorods.

The strong cetyltrimethylammonium bromide (CTAB) surfactant responsible for the synthesis and stability of gold nanorod solutions complicates their biomedical applications. The critical parameter to maintain nanorod stability is the ratio of CTAB to nanorod concentration. The ratio is approximately 740,000 as determined by chloroform extraction of the CTAB from a nanorod solution. A comparison of nanorod stabilization by thiol-terminal PEG and by anionic polymers reveals that PEGylation results in higher yields and less aggregation upon removal of CTAB. A heterobifunctional PEG yields nanorods with exposed carboxyl groups for covalent conjugation to antibodies with the zero-length carbodiimide linker EDC. This conjugation strategy leads to approximately two functional antibodies per nanorod according to fluorimetry and ELISA assays. The nanorods specifically targeted cells in vitro and were visible with both two-photon and confocal reflectance microscopies. This covalent strategy should be generally applicable to other biomedical applications of gold nanorods as well as other gold nanoparticles synthesized with CTAB.

miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy.

PURPOSE: The epithelial-to-mesenchymal transition (EMT) is a cell development-regulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFR therapy. EXPERIMENTAL DESIGN: miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFR and ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3'-untranslated region/ERRFI-1/luciferse reporters. RESULTS: We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFR inhibitors-induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3'-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect. CONCLUSIONS: Members of the miR-200 family appear to control the EMT process and sensitivity to EGFR therapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.

ADAM10 as a therapeutic target for cancer and inflammation.

Both cancer and chronic inflammatory diseases are often marked by homeostatic signal transduction pathways run amok. Cleavage of membrane-bound substrates by extracellular metalloproteinases is frequently the rate limiting step in activating many of these pathways, resulting either in liberation of active ligands (shedding) or initiating further processing into bioactive cytoplasmic domains (regulated intramembrane proteolysis or RIP). ADAM10 is a member of the ADAM (A Disintegrin And Metalloproteinase) family of transmembrane metalloproteinases implicated in the RIPing and shedding of dozens of substrates that drive cancer progression and inflammatory disease, including Notch, E-cadherin, EGF, ErbB2 and inflammatory cytokines. ADAM10's emerging role as a significant contributor to these pathologies has led to intense interest in it as a potential drug target for disease treatment. Here we discuss some of the established functions of ADAM10 and the implications of its inhibition in disease progression.

Therapeutic IMC-C225 antibody inhibits breast cancer cell invasiveness via Vav2-dependent activation of RhoA GTPase.

PURPOSE: Abnormalities in the expression and signaling pathways downstream of epidermal growth factor receptor (EGFR) contribute to progression, invasion, and maintenance of the malignant phenotype in human cancers. Accordingly, biological agents, such as the EGFR-blocking antibody IMC-C225 have promising anticancer potential and are currently in various stages of clinical development. Because use of IMC-C225 is limited, at present, only for treatment of cancer with high EGFR expression, the goal of the present study was to determine the effect of IMC-C225 on the invasiveness of breast cancer cells with high and low levels of EGFR expression. EXPERIMENTAL DESIGN: The effect of IMC-C225 on invasion was studied using breast cancer cell lines with high and low levels of EGFR expression. RESULTS: The addition of EGF led to progressive stress fiber dissolution. In contrast, cells treated with IMC-C225 showed reduced invasiveness and increased stress-fiber formation. Interestingly, IMC-C225 pretreatment was accompanied by EGFR phosphorylation, as detected using an anti-phosphorylated tyrosine antibody (PY99), which correlated with phosphorylation of Vav2 guanine nucleotide exchange factor and activation of RhoA GTPase irrespective of EGFR level, and Vav2 interacted with EGFR only in IMC-C225-treated cells. The underlying mechanism involved an enhanced interaction between beta1 integrins and EGFR upon IMC-C225 treatment. CONCLUSION: Here, we defined a new mechanism for IMC-C225 that cross-links integrins with EGFR, leading to activation of RhoA and inhibition of breast cancer cell invasion irrespective of the level of EGFR in the cells, thus providing a rationale for using IMC-C225 in the metastatic setting independent of the levels of EGFR.

Is vascular endothelial growth factor modulation a predictor of the therapeutic efficacy of gefitinib for bladder cancer?

PURPOSE: The epidermal growth factor receptor inhibitor gefitinib (Iressa) is currently being studied in patients with bladder cancer and it has significant anti-angiogenic activity. We investigated the relationship between the modulation of vascular endothelial growth factor (Santa Cruz Biotechnology, Santa Cruz, California) expression and the biological efficacy of gefitinib for bladder cancer. MATERIALS AND METHODS: In vitro the 4 bladder cancer cell lines 253JB-V, UMUC-3, KU-7 and UMUC-13 were treated with gefitinib and vascular endothelial growth factor secretion was measured. The effects of gefitinib on vascular endothelial growth factor promoter, proliferation, cell cycle and downstream signals were evaluated. In vivo 253JB-V and UMUC-13 were injected into nude mice and tumors were treated with 2 mg gefitinib per day. Tumor kinetics were determined and the levels of phospho-epidermal growth factor receptor (Biosource), vascular endothelial growth factor, phospho-vascular endothelial growth factor (Cell Signaling Technology), angiogenesis and apoptosis were measured. RESULTS: Epidermal growth factor receptor (Neomarkers, Fremont, California) phosphorylation was blocked efficiently in all cell lines at concentrations of 0.5 microM or greater. Gefitinib (1 microM) induced an accumulation of cells in G0/G1 without apoptosis in 253J B-V cells, whereas it had no effect in other cell lines. Gefitinib inhibited vascular endothelial growth factor secretion in 253JB-V and UMUC-13 (concentration inhibiting a 50% response 0.5 and 0.1 microM, respectively) but not in UMUC-3 or KU-7. Gefitinib decreased vascular endothelial growth factor promoter activity in 253JB-V and UMUC-13 by 40% to 60%. In vivo the growth of 253JB-V tumors was significantly inhibited by gefitinib, whereas no effect was demonstrated in UMUC-13 tumors. Vascular endothelial growth factor expression and vascular endothelial growth factor receptor activation were significantly decreased in 253JB-V tumors and to a greater extent in resistant UMUC-13 tumors. Gefitinib inhibited angiogenesis and induced apoptosis in sensitive 253JB-V tumors only. CONCLUSIONS: Epidermal growth factor receptor blockade exerts an anti-angiogenic effect on bladder cancer cells, in part by modulating vascular endothelial growth factor expression. However, down-regulation of vascular endothelial growth factor expression is not sufficient to inhibit bladder cancer growth and it should not be used as a predictor of the therapeutic efficacy of gefitinib.

Sensitivity to epidermal growth factor receptor inhibitor requires E-cadherin expression in urothelial carcinoma cells.

PURPOSE: Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of urothelial carcinoma, but a clinical response can be expected in only a small proportion of patients. The aim of this study was to define molecular markers of response to cetuximab therapy in a panel of urothelial carcinoma cell lines. EXPERIMENTAL DESIGN: Eleven cell lines were investigated for antiproliferative response to cetuximab based on [(3)H]thymidine incorporation. A variety of markers, including EGFR expression, phosphorylation, and gene amplification, as well as the expression of other growth factor receptors, their ligands, and markers of epithelial-to-mesenchymal transition were investigated. Cohen's kappa statistic was used to estimate the agreement between response and expression of these markers. E-cadherin was silenced by small interfering RNA in two sensitive cell lines, and the effect on the response to cetuximab was measured. RESULTS: We were able to identify a panel of relevant markers pertaining especially to alternate growth factor receptor expression and epithelial-to-mesenchymal transition that predicted response to cetuximab. The data suggested that expression of intact HER-4 (kappa, 1.00; P = 0.008), E-cadherin (kappa, 0.81; P = 0.015), and beta-catenin (kappa, 0.81; P = 0.015) and loss of expression of platelet-derived growth factor receptor beta (kappa, 0.57; P = 0.167) were associated with response to cetuximab therapy. Silencing E-cadherin in two sensitive cell lines reduced responsiveness to cetuximab in both (P < 0.001). CONCLUSIONS: A panel of predictive markers for cetuximab response has been established in vitro and is currently being evaluated in a prospective clinical trial of neoadjuvant EGFR-targeted therapy. Most importantly, E-cadherin seems to play a central role in modulation of EGFR response in urothelial carcinoma.

Distinctive expression pattern of ErbB family receptors signifies an aggressive variant of bladder cancer.

PURPOSE: Expression of various members of the ErbB family (epidermal growth factor receptor/ErbB-1, ErbB-2, ErbB-3 and ErbB-4) is associated with disease stage and survival in patients with urothelial carcinoma. We examined the correlation of ErbB family receptor expression with the progression of urothelial carcinoma and survival. MATERIALS AND METHODS: A urothelial carcinoma tissue array was constructed from 248 archival paraffin blocks and quality control studies were ascertained. The tissue microarray was stained for epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4, and analyzed using an automated reader. Patient data included grade, stage, growth pattern, recurrence and survival. RESULTS: Kaplan-Meier estimates of 5-year overall and recurrence-free survival were 58% and 27%, respectively. Patients with high grade, invasive or nonpapillary disease had a worse prognosis than patients with low grade, superficial or papillary disease (p <0.0001). High epidermal growth factor receptor or low ErbB-4 expression was associated with nonpapillary, high grade and invasive tumors as well as with significantly shorter recurrence-free and overall survival (p <0.002, 0.028 and 0.047, respectively). Levels of ErbB-2 and ErbB-3 expression were not associated with overall or recurrence-free survival. CONCLUSIONS: The expression profiles of ErbB-4 and epidermal growth factor receptor are prognostic in urothelial carcinoma. They may help in selecting patients at high risk with bladder cancer for more aggressive therapy.

Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

PURPOSE: Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. MATERIALS AND METHODS: We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. RESULTS: In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. CONCLUSIONS: Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment. This inhibition may prove beneficial for treating superficial bladder cancer with adenovirus mediated interferon-alpha and hopefully contribute to a decreased recurrence rate of this neoplasm.

Gefitinib reverses TRAIL resistance in human bladder cancer cell lines via inhibition of AKT-mediated X-linked inhibitor of apoptosis protein expression.

In a previous study, we found that the small-molecule epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839, Iressa) blocked cell proliferation at biologically relevant concentrations in approximately one third (6 of 17) of human bladder cancer cell lines examined. Here, we studied the effects of gefitinib on apoptosis in a representative subset of the same panel of cells. The drug had modest effects on DNA fragmentation as a single agent at concentrations that produced strong growth inhibition (< or =1 micromol/L) and also failed to promote apoptosis induced by conventional chemotherapeutic agents (gemcitabine and paclitaxel). However, gefitinib did interact with recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce high levels of apoptosis in gefitinib-responsive but not gefitinib-unresponsive lines. The molecular mechanisms involved down-regulation of active AKT and X-linked inhibitor of apoptosis protein (XIAP) expression and were mimicked by chemical inhibitors of the phosphatidylinositol 3-kinase/AKT pathway but not of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway. Furthermore, direct small interfering RNA-mediated knockdown of AKT resulted in down-regulation of XIAP and TRAIL sensitization, and knockdown of XIAP itself was sufficient to reverse TRAIL resistance. Together, our results show that EGFR pathway activation limits TRAIL-induced apoptosis via an AKT- and XIAP-dependent mechanism in EGFR-dependent human bladder cancer cells, providing the conceptual framework for a further evaluation of the combination in relevant preclinical in vivo models.

Molecular correlates of gefitinib responsiveness in human bladder cancer cells.

We characterized the effects of the small molecule epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839, Iressa) on cell proliferation in a panel of 17 human bladder cancer cell lines. Gefitinib inhibited DNA synthesis in a concentration-dependent fashion in 6 of 17 lines. Growth inhibition was associated with p27(Kip1) accumulation and decreased cyclin-dependent kinase 2 activity. Gefitinib also inhibited baseline EGFR, AKT, and extracellular signal-regulated kinase (ERK) phosphorylation in the EGFR-dependent cells maintained in serum-free medium, whereas it had no effect on baseline EGFR or ERK phosphorylation in the EGFR-independent cells. Analyses of candidate markers of EGFR dependency revealed that the gefitinib-sensitive cells expressed higher surface EGFR levels than the gefitinib-resistant lines. Gefitinib-sensitive cells generally expressed higher levels of E-cadherin and lower levels of vimentin than the gefitinib-resistant cells, but these correlations were not perfect, suggesting that these markers of epithelial-mesenchymal transition cannot be used by themselves to prospectively predict EGFR-dependent growth. Together, our results show that bladder cancer cells are markedly heterogeneous with respect to their sensitivity to EGFR antagonists. Although surface EGFR levels and epithelial-mesenchymal transition status seem to roughly correlate with responsiveness, they cannot be used by themselves to identify bladder tumors that will be sensitive to EGFR-directed therapy. However, comparing levels of p27(Kip1) or DNA synthesis before and after gefitinib exposure does identify the drug-sensitive cells.

Mutations within the kinase domain and truncations of the epidermal growth factor receptor are rare events in bladder cancer: implications for therapy.

PURPOSE: It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma. EXPERIMENTAL DESIGN: The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls. RESULTS: Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas. CONCLUSIONS: When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

Schedule dependent efficacy of gefitinib and docetaxel for bladder cancer.

PURPOSE: We determined the sequence specific efficacy of gefitinib and docetaxel treatment for bladder cancer. This combination was selected because it is currently under study in a phase II clinical trial. MATERIALS AND METHODS: In vitro antiproliferative effects of gefitinib, docetaxel and a combination were determined in the 4 bladder cancer cell lines 253J B-V, UM-UC-3, KU-7 and UM-UC-13 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle analysis was analyzed using flow cytometry and propidium iodide labeling. Epidermal growth factor receptor downstream signaling was assessed by Western blot analysis. In vivo nude mice were injected subcutaneously with 253J B-V cells and treated with placebo, gefitinib, docetaxel, docetaxel followed by gefitinib or gefitinib followed by docetaxel. Tumor kinetics were established. RESULTS: Gefitinib demonstrated antiproliferative effect against 253J B-V cells (50% inhibitory concentration less than 0.5 muM) but no apoptotic effect in vitro, whereas docetaxel demonstrated antiproliferative and apoptotic effects. When gefitinib and docetaxel were combined, gefitinib enhanced the apoptotic and antiproliferative effects of docetaxel only when gefitinib was administered following docetaxel pretreatment. Apoptosis increased from 45% to 66%. In vivo there were significant differences in tumor weight in mice treated with combination therapy vs gefitinib or docetaxel alone. Importantly improved efficacy was observed when docetaxel was followed by gefitinib administration compared with gefitinib followed by docetaxel (mean tumor weight 42 vs 93 mg, p = 0.022). Sequence specific efficacy was not observed in UM-UC-3, UM-UC-13 and KU-7 cells, which are resistant to gefitinib. CONCLUSIONS: Docetaxel followed by gefitinib demonstrated sequence specific efficacy against gefitinib sensitive bladder cancer compared with gefitinib followed by docetaxel or either drug alone. Accordingly gefitinib administration concurrently or after chemotherapy might be the sequence of choice and it should be considered for future clinical trials.

Uncoupling between epidermal growth factor receptor and downstream signals defines resistance to the antiproliferative effect of Gefitinib in bladder cancer cells.

Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3beta (GSK-3beta). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3beta activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-beta (PDGFRbeta) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3beta in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3beta and of nuclear cyclin D1.

Clinical applications for targeted therapy in bladder cancer.

The tremendous amount of data accumulated through genomics, proteomics, and metabolomic technologies has not led to a definitive understanding of the mechanisms underlying cancer. The challenge remains as to how to integrate all of the relevant knowledge and data in a systematic manner so that researchers can gain the knowledge needed to devise the best therapeutic and diagnostic strategies. Human transitional cell carcinoma of the bladder is genetically heterogeneous, and it is surrounded by a complex tissue microenvironment involving vasculature, stromal cells, and connective tissue. One of the most challenging problems facing cancer researchers is the lack of correlation between in vitro cell lines and animal tumor models and human in vivo tumors. A few promising approaches are being devised that will help address this issue in the coming years. One such approach is the measurements of molecular levels of receptors, ligands, pathways components, and so on, directly in human tumors through in vivo imaging, or through proteomic profiling, as it has been proposed as standard protocol for cancer diagnostics and therapeutics.