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.

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1.

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.

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.

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.

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.

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.

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.

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.

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.

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.

The epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa) suppresses c-Src and Pak1 pathways and invasiveness of human cancer cells.

PURPOSE: Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including those of the head and neck and breast. Accordingly, agents such as the EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa) are promising, biologically based treatments that are in various stages of preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival; this study explored the effect of ZD1839 on the stimulation of c-Src and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival of cancer cells. EXPERIMENTAL DESIGN: We examined the effect of ZD1839 on biochemical and functional assays indicative of directional motility and cell survival, using human head and neck squamous cancer cells and breast cancer cells. RESULTS: ZD1839 effectively inhibited c-Src activation and Pak1 activity in exponentially growing cancer cells. In addition, ZD1839 suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Grb2-binding Y1068 sites, c-Src phosphorylation on Y215, and Pak1 activity. ZD1839 also blocked EGF-induced cytoskeleton remodeling, redistribution of activated EGFR, and in vitro invasiveness of cancer cells. CONCLUSIONS: These studies suggest that the EGFR-TKI ZD1839 may cause potent inhibition of the Pak1 and c-Src pathways and, therefore, have potential to affect the invasiveness of human cancer cells deregulated in these growth factor receptor pathways.

Linkage of rapid estrogen action to MAPK activation by ERalpha-Shc association and Shc pathway activation.

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells

Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215.

To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.

The role of mitogen-activated protein (MAP) kinase in breast cancer.

Mitogen-activated protein kinase (MAP kinase) cascades transmit and amplify signals involved in cell proliferation as well as cell death. These signal transduction pathways serve as an indicators of the intensity of trafficking induced by various growth factor, steroid hormone, and G protein receptor mediated ligands. Three major MAP kinase pathways exist in human tissues, but the one involving ERK-1 and -2 is most relevant to breast cancer. Peptide growth factors acting through tyrosine kinase containing receptors are the major regulators of ERK-1 and -2. Estradiol, progesterone, and testosterone can act non-genomically via membrane associated receptors to activate MAP kinase as can various other ligands acting through heterotrimeric G protein receptors. Recent studies demonstrate that breast cancers frequently contain an increased proportion of cells with the activated form of MAP kinase. In estrogen receptor positive breast tumors, MAP kinase pathways can exert "cross talk" effects at the level of ER induced transcription as well as at the level of the cell cycle. Estradiol stimulates cell proliferation by mechanisms which involve activation of MAP kinase, either through rapid, non-transcription effects or by increasing growth factor production and consequently MAP kinase. Progesterone and androgens also stimulate MAP kinase through both of these two mechanisms. Strategies used to treat hormone dependent breast cancer appear to result in upregulation of MAP kinase activation. Direct experimental data demonstrate that the pressure of estradiol deprivation results in the upregulation of MAP kinase in breast cancer cells growing in tissue culture and as xenografts. A number of investigators have now studied the expression of activated MAP kinase in human breast cancer tissues by enzymatic assay and by immunohistochemical techniques. Approximately half of breast tumors express more activated MAP kinase than does the surrounding benign tissue. Studies show a trend toward higher MAP kinase activity in primary tumors of node positive than in node negative patients. However, larger numbers of patients must be studied for these results to achieve statistical significance. The up-regulation of MAP kinase activity does not represent mutations of Ras, but appears to result from enhancement of growth factor pathway activation. No data are yet available on the relationship between MAP kinase activation and apoptosis. Additional studies are now needed to determine the precise relationship between MAP kinase activation and tumor proliferation, apoptosis, and degree of invasiveness as well as on disease free and overall survival.

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.