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.

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.

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines.

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.

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.

Bortezomib abolishes tumor necrosis factor-related apoptosis-inducing ligand resistance via a p21-dependent mechanism in human bladder and prostate cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines that induces apoptosis in some tumor cells but not in normal cells. Unfortunately, many human cancer cell lines are refractory to TRAIL-induced cell death, and the molecular mechanisms underlying resistance are unclear. Here we report that TRAIL resistance was reversed in human bladder and prostate cancer cell lines by the proteasome inhibitor bortezomib (PS-341, Velcade). Synergistic induction of apoptosis occurred within 4 to 6 hours in cells treated with TRAIL plus bortezomib and was associated with accumulation of p21(WAF-1/Cip-1) (p21) and inhibition of cyclin-dependent kinase (cdk) activity. Roscovitine, a specific cdk1/2 inhibitor, also sensitized cells to TRAIL. Silencing p21 expression reduced levels of DNA fragmentation by 50% in cells treated with bortezomib and TRAIL, confirming that p21 was required for the response. Analysis of the TRAIL pathway revealed that caspase-8 processing was enhanced in a p21-dependent fashion in cells exposed to TRAIL and bortezomib as compared with cells treated with TRAIL alone. Thus, all downstream components of the pathway (Bid cleavage, cytochrome c release, and caspase-3 activation) were amplified. These data strongly suggest that p21-mediated cdk inhibition promotes TRAIL sensitivity via caspase-8 activation and that TRAIL and bortezomib should be combined in appropriate in vivo models as a possible approach to solid tumor therapy.

Smac mimetic reverses resistance to TRAIL and chemotherapy in human urothelial cancer cells.

PURPOSE: inhibitors of apoptosis proteins (IAPs) have been shown to contribute to resistance of neoplastic cells to chemotherapy and to biologic antineoplastic agents. Consequently, new agents are being developed targeting this family of proteins. In a panel of bladder cancer cell lines, we evaluated a Smac mimetic that antagonizes several IAPs for its suitability for bladder cancer therapy. Experimental design: A panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-A alone, TRAIL alone, chemotherapy alone, compound-A plus TRAIL, and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally, siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. RESULTS: single-agent compound-A had little effect, but compound-A augmented TRAIL- and chemotherapy-induced apoptosis. Immunoblotting showed that combination treatment with compound-A and TRAIL resulted in cleavage of procaspase-3 and procaspase-7, activation of which irreversibly commits cells to apoptosis. Immunoprecipitation of XIAP showed displacement of active caspase-3 fragments from XIAP, supporting the proposed mechanism of action. Furthermore, siRNA-mediated silencing of XIAP similarly sensitized these cells to apoptosis. EXPERIMENTAL DESIGN: a panel of seven bladder cancer cell lines were evaluated for sensitivity to the Smac mimetic compound-Alone, TRAIL alone, Chemotherapy alone, compound-A plus TRAIL and compound-A plus chemotherapy by DNA fragmentation analysis. IAP levels and caspase activation were examined by western blotting. Release of caspase-3 from X-linked inhibitor of apoptosis protein (XIAP), the most effective IAP, was assessed by immunoprecipitation and western blotting. Finally siRNA knockdown of XIAP was correlated with the sensitivity of cells to apoptosis induced by compound-A plus TRAIL by DNA fragmentation and western blotting. CONCLUSION: our results suggest that targeting of XIAP with the Smac mimetic compound-A has the potential to augment the effects of a variety of chemotherapeutic and biologic therapies in bladder cancer.

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth, metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice.

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.