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Purpose: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer.Experimental Design: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux.Results: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux.Conclusion: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity. Clin Cancer Res; 17(20); 6510–21. ©2011 AACR.

Supplementary Data from Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation

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Subgroup analyses from clinical studies have suggested that among metastatic non-small cell lung cancer (NSCLC) patients receiving chemotherapy, females may derive less benefit from the addition of the vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab than males. This has raised the question of whether estrogen may impact the response to anti-angiogenic therapy. To address this, we investigated the effects of estrogen on tumor growth, angiogenesis, and the response to bevacizumab in human xenograft models of NSCLC. We observed that estrogen induced marked resistance to bevacizumab, which was accompanied by a 2.3 fold increase in tumor vascular pericyte coverage (p=0.01) and an upregulation of pro-angiogenic factors VEGF and platelet derived growth factor-BB (PDGF-BB). We also investigated the role of infiltrating myeloid cells, a population which has been associated with resistance to anti-VEGF therapies. We observed that estrogen induced a greater than 2-fold increase (p = 0.001) in the recruitment of tumor infiltrating myeloid cells as well as concomitant increases in the myeloid recruitment factors granulocyte colony-stimulating factor (G-CSF) and chemokine ligand 1 (CXCL1). Blockade of the estrogen receptor pathway using fulvestrant re-sensitized tumors to VEGF targeting as evidenced by reduced tumor vasculature and an increase in overall survival in our NSCLC xenograft models. Collectively, these data provide evidence that estrogen may promote resistance to VEGF-targeted therapies, potentially by enhancing pericyte coverage and myeloid recruitment, and suggest that estrogen receptor blockade merits further investigation as an approach to enhance the effects of antiangiogenic therapy.

Introduction The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. Methods We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models in vitro and in vivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. Results NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. Conclusions In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor–naive and refractory settings.

Objective The aim of this study was to determine the effectiveness of the nurse-run annual wellness visit (AWV) in improving adherence to cancer screening recommendations for colonoscopies and/or mammograms. Background The Affordable Health Care Act provides Medicare beneficiaries access to AWVs. Nurse-run AWVs offer individualized education, reinforce health screening recommendations, and may enhance the patients' intent to complete the screenings. Methods A nonexperimental comparative study was conducted using data collected from chart audits comparing patients who only attended the AWV, patients who attended the AWV linked with a physician visit, and patients who have not attended an AWV. Results Patients who attended the AWV showed greater adherence to mammogram completion regardless of the link to the physician follow-up visit. Differences in adherence to colonoscopy recommendations were not significant, likely because of the low number of colonoscopies reported. Conclusions Nurse-run AWV clinics are associated with adherence to mammograms and show promise of increasing colonoscopy compliance.

Background: We investigated cytokines and angiogenic factors (CAFs) in patients with metastatic renal cell carcinoma (mRCC) treated in a randomized phase II clinical trial of sorafenib versus sorafenib+ interferon-a (IFN-a) that yielded no differences in progression-free survival (PFS). We aimed to link the CAF profile to PFS and select candidate predictive and prognostic markers for further study. Methods: The concentrations of 52 plasma CAFs were measured pretreatment (n = 69), day 28, and day 56 using multiplex bead arrays and enzyme-linked immunosorbent assay. We investigated the association between baseline levels of CAFs with PFS and posttreatment changes. Results: Unsupervised CAF clustering analysis revealed two distinct mRCC patient groups with elevated proangiogenic or proinflammatory mediators. A six-marker baseline CAF signature [osteopontin, vascular endothelial growth factor (VEGF), carbonic anhydrase 9, collagen IV, VEGF receptor-2, and tumor necrosis factor-related apoptosis-inducing ligand] correlated with PFS benefit (hazard ratio 0.20 versus 2.25, signature negative versus positive, respectively; P = 0.0002). While changes in angiogenic factors were frequently attenuated by the sorafenib+ IFN combination, most key immunomodulatory mediators increased. Conclusions: Using CAF profiling, we identified two mRCC patient groups, a candidate plasma signature for predicting PFS benefit, and distinct marker changes occurring with each treatment. This platform may provide valuable insights into renal cell carcinoma biology and the molecular consequences of targeted therapies.

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Aromatase inhibitors (AI) are rapidly becoming the first choice for hormonal treatment of estrogen receptor-α (ERα)–positive breast cancer in postmenopausal women. However, de novo and acquired resistance frequently occurs. We have previously identified a lysine to arginine transition at residue 303 (K303R) in ERα in premalignant breast lesions and invasive breast cancers, which confers estrogen hypersensitivity and resistance to tamoxifen treatment. Thus, we questioned whether resistance to AIs could arise in breast cancer cells expressing the ERα mutation. As preclinical models to directly test this possibility, we generated K303R-overexpressing MCF-7 cells stably transfected with an aromatase expression vector. Cells were stimulated with the aromatase substrate, androstenedione, with or without the AI anastrozole (Ana). We found that Ana decreased androstenedione-stimulated growth of wild-type cells, whereas K303R-expressing cells were resistant to the inhibitory effect of Ana on growth. We propose that a mechanism of resistance involves an increased binding between the mutant receptor and the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K), leading to increased PI3K activity and activation of protein kinase B/Akt survival pathways. Inhibition of the selective “addiction” to the PI3K/Akt pathway reversed AI resistance associated with expression of the mutant receptor. Our findings suggest that the K303R ERα mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and that targeting the PI3K/Akt pathway may be a useful strategy for treating patients with tumors resistant to hormone therapy. [Cancer Res 2009;69(11):4724–32]

Purpose: Estrogen receptor α (ERα) predicts the natural history of breast cancer without intervening therapy. Here, we have optimized the detection of a somatic mutation, an A908G transition of ERα, and examined its association with clinical and biological features of invasive breast cancer. Experimental Design: We compared two methods of sequencing to detect the A908G ERα mutation. We then used primer extension sequencing with genomic DNA isolated from invasive breast tumors to determine whether the mutation was associated with clinical outcome in 267 axillary node–negative and axillary node–positive breast tumors. The presence of the mutation and clinical variables were analyzed for association with recurrence-free survival and overall survival by Cox proportional hazards regression models. Results: We determined that dye-labeled terminator sequencing was not adequate for detection of the A908G ERα mutation. The mutation was detected at a high frequency (50%) in invasive breast tumors using primer extension sequencing, and was found to be associated with clinical measures of poor outcome, including larger tumor size and axillary lymph node positivity. Although the mutation was associated with recurrence-free survival in univariate analysis, it was not an independent predictor of outcomes in multivariate analysis. Conclusions: Consistent with our previous finding of this somatic ERα mutation in breast ductal hyperplasias, we now present evidence that the A908G mutation is present in invasive breast tumors using an optimized sequencing method. We find that the mutation is significantly associated with aggressive biological tumor features, and with an unfavorable prognosis, but was not an independent prognostic marker in untreated patients.

Alterations in migration and adhesion are critical to invasion and metastasis. To examine signaling pathways important for colon tumor metastasis, cells of increased migratory potential from the low migratory SW480 human colorectal carcinoma parental cell line were biologically selected by serial migration through modified Boyden chambers. Several sublines were obtained with statistically significantly increased migration relative to the parental cell line. One highly migratory population was single-cell cloned and characterized. The migratory clones exhibit a four- to five-fold increase in protein and mRNA expression of T-lymphoma invasion and metastasis gene 1 (Tiam1), a guanine nucleotide exchange factor. To determine directly the role of Tiam1 in the migration of these migratory sublines, the parental SW480 cell line was transfected with a plasmid encoding the Tiam1 protein, and single cell clones were established. Ectopic expression of Tiam1 in these clones led to morphologic changes identical to biologically selected clones and increased migration. Finally, the implantation of clones that overexpress Tiam1 into the cecum of athymic mice resulted in tumor growth in the spleen, liver, and lung, whereas parental cells do not form tumors by this route of injection. These results demonstrate that overexpression of Tiam1 contributes to the metastatic phenotype of colon cancer cells.

As early as the 1800s, the actions of estrogen have been implicated in the development and progression of breast cancer. The estrogen receptor (ER) was identified in the late 1950s and purified a few years later. However, it was not until the 1980s that the first ER was molecularly cloned, and in the mid 1990s, a second ER was cloned. These two related receptors are now called ERalpha and ERbeta, respectively. Since their discovery, much research has focused on identifying alterations within the coding sequence of these receptors in clinical samples. As a result, a large number of naturally occurring splice variants of both ERalpha and ERbeta have been identified in normal epithelium and diseased or cancerous tissues. In contrast, only a few point mutations have been identified in human patient samples from a variety of disease states, including breast cancer, endometrial cancer, and psychiatric diseases. To elucidate the mechanism of action for these variant isoforms or mutant receptors, experimental mutagenesis has been used to analyze the function of distinct amino acid residues in the ERs. This review will focus on ERalpha and ERbeta alterations in breast cancer.

Increased expression and/or activity of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, occurs commonly during colon tumor progression. To examine potential roles for c-Met in promoting metastasis, we compared the colon tumor cell line KM12C with low metastatic potential to the isogenic variants KM,12L4 and KM12SM with high metastatic potential. KM12C cells express c-Met with low levels of tyrosine phosphorylation in the absence of HGF. The high metastatic cells express a c-Met that is constitutively tyrosine phosphorylated, they have increased colony formation, and are minimally responsive to HGF relative to the parental cells. Tyrosine-phosphorylated beta-catenin was constitutively associated with c-Met in the more metastatic cells, but was inducible only after HGF addition in the less metastatic cells. Functions mediated by beta-catenin, including cell-cell adhesion and migration, and activation of the tcf (T-cell factor) family of transcription factors, were also elevated in the more metastatic KM12SM and L4 cells. Furthermore, analysis of the known tcf transcriptional target genes, cyclin D1, c-Myc, and uPAR, demonstrated increased expression in the high metastatic cells, correlating with the levels of tcf activity. Collectively, these results suggest that endogenous activation of c-Met in highly metastatic KM12SM CRC cells results in increased survival and growth under anchorage independent conditions, increased in vitro migration, and elevated levels of tcf target genes. Thus, beta-catenin association with activated c-Met may contribute to a more aggressive liver metastatic phenotype of these cells.

Purpose: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. Experimental Design: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. Results: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. Conclusion: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity. Clin Cancer Res; 17(20); 6510–21. ©2011 AACR.

Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1alpha pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1alpha. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1alpha and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.

Purpose: To determine the frequency of estrogen receptor α and β and progesterone receptor protein immunohistochemical expression in a large set of non–small cell lung carcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports. Experimental Design: Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors α and β and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients' clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status. Results: Estrogen receptors α and β were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for α and 42-56% for β; cytoplasm: Conclusions: Estrogen receptor α and β expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor α expression correlates with EGFR mutations. (Clin Cancer Res 2009;15(17):5359–68)

Although the androgen receptor (AR) is a known clinical target in prostate cancer, little is known about its possible role in breast cancer. We have investigated the role of AR expression in human breast cancer in response to treatment with the antiestrogen tamoxifen. Resistance to tamoxifen is a major problem in treating women with breast cancer. By gene expression profiling, we found elevated AR and reduced estrogen receptor (ER) alpha mRNA in tamoxifen-resistant tumors. Exogenous overexpression of AR rendered ERalpha-positive MCF-7 breast cancer cells resistant to the growth-inhibitory effects of tamoxifen in anchorage-independent growth assays and in xenograft studies in athymic nude mice. AR-overexpressing cells remained sensitive to growth stimulation with dihydrotestosterone. Treatment with the AR antagonist Casodex (bicalutamide) reversed this resistance, demonstrating the involvement of AR signaling in tamoxifen resistance. In AR-overexpressing cells, tamoxifen induced transcriptional activation by ERalpha that could be blocked by Casodex, suggesting that AR overexpression enhances tamoxifen's agonistic properties. Our data suggest a role for AR overexpression as a novel mechanism of hormone resistance, so that AR may offer a new clinical therapeutic target in human breast cancers.

Estrogen and its receptors alpha and beta (ERalpha and ERbeta) play a major role in tumor progression and approximately two-thirds of breast cancers express these functional receptors. Thus, the ER is a major target for current and developing therapies. Although most ER-positive tumors initially respond to hormonal therapies such as tamoxifen, many tumors will eventually become resistant to tamoxifen induced growth inhibition. This chapter will discuss molecular mechanisms that contribute to hormonal resistance of current therapies including ERalpha mutations, the roles of proliferation and apoptosis in tumor homeostasis and receptor coregulator proteins. Additionally, the role of nonclassical ERalpha signaling through growth factor receptors and the subsequent downstream-initiated signaling, and the role of the progesterone receptors will be discussed.

Overexpression and/or activation of c-Met, the protein tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF), and the protein tyrosine kinase, Src, have been implicated in the progression and metastasis of human colorectal carcinoma (CRC). Previously, through ribozyme-mediated c-Met downregulation, we demonstrated a causal role for c-Met in CRC tumorigenesis and the production of liver metastases from the highly metastatic CRC cell line, KM20. Analysis of signaling intermediates downstream of c-Met demonstrated a specific reduction of Src activity with minimal effect on Erk1/2 and Akt following c-Met downregulation. As Src has been shown to upregulate Vascular Endothelial Growth Factor (VEGF), we sought to determine if the reduced tumor size and incidence could be due to reduced vessel formation in the c-Met downregulated clones. Here we show that tumors produced from c-Met downregulated cells have significantly reduced vessel density in tumors, correlating with a reduction in VEGF production. Additionally, the Src selective inhibitor, PP2, significantly reduced basal VEGF production in KM20 parental cells, and this effect could be rescued by HGF treatment. PP2 reduced basal proliferation, migration, and anchorage-independent growth. Furthermore, PP2 treatment demonstrated a dose-dependent decrease in HGF induced migration. In contrast, PP2 treatment only had a minor effect on HGF induced anchorage-independent growth. Collectively, these data demonstrate a significant role for c-Met-mediated Src activation in processes involved in CRC tumorigenesis and liver metastasis.

Estrogen and its receptors α and β (ERα and ERβ) play a major role in tumor progression and approximately two-thirds of breast cancers express these functional receptors. Thus, the ER is a major target for current and developing therapies. Although most ER-positive tumors initially respond to hormonal therapies such as tamoxifen, many tumors will eventually become resistant to tamoxifen induced growth inhibition. This chapter will discuss molecular mechanisms that contribute to hormonal resistance of current therapies including ERα mutations, the roles of proliferation and apoptosis in tumor homeostasis and receptor coregulator proteins. Additionally, the role of nonclassical ERα signaling through growth factor receptors and the subsequent downstream-initiated signaling, and the role of the progesterone receptors will be discussed.

It has long been appreciated that estrogenic signaling contributes to breast cancer progression. c-Src is also required for a number of processes involved in tumor progression and metastasis. We have previously identified the K303R mutant estrogen receptor α (ERα) that confers hypersensitivity to low levels of estrogen. Because ERα and c-Src have been shown to interact in a number of different systems, we wanted to evaluate the role of c-Src kinase in estrogen-stimulated growth and survival of ERα-positive breast cancer cells. MCF-7 cells stably expressing the mutant receptor showed increased c-Src kinase activity and c-Src tyrosine phosphorylation when compared with wild-type ERα-expressing cells. A c-Src inhibitor, AZD0530, was used to analyze the biological effects of pharmacologically inhibiting c-Src kinase activity. MCF-7 cells showed an anchorage-dependent growth IC50 of 0.47 μmol/L, which was increased 4-fold in the presence of estrogen. In contrast, cells stably expressing the mutant ERα had an elevated IC50 that was only increased 1.4-fold by estrogen stimulation. The c-Src inhibitor effectively inhibited the anchorage-independent growth of both of these cells, and estrogen was able to reverse these effects. When cells were treated with suboptimal concentrations of c-Src inhibitor and tamoxifen, synergistic inhibition was observed, suggesting a cooperative interaction between c-Src and ERα. These data clearly show an important role for ERα and estrogen signaling in c-Src–mediated breast cancer cell growth and survival. Here, we show that c-Src inhibition is blocked by estrogen signaling; thus, the therapeutic use of c-Src inhibitors may require inhibition of ERα in estrogen-dependent breast cancer. [Mol Cancer Ther 2006;5(12):3023–31]

The expression of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, frequently increases during prostate tumor progression. However, whether reduced c-Met expression inhibits tumor growth and metastasis has not been ascertained.c-Met expression was reduced by infection of an adenovirus expressing a c-Met ribozyme into the highly metastatic human prostate cancer cell line PC3-LN4. In vitro, effects on c-Met, Akt, and extracellular signal-regulated kinase 1/2 expression and phosphorylation, Src expression and activity, and vascular endothelial growth factor expression were determined, as were effects on cell migration and invasion. Prostate tumor formation and metastasis to regional lymph nodes in nude mice were examined after both ex vivo and in vivo infection of cells.Infection of PC3-LN4 cells with the Ad-c-Met-expressing ribozyme decreased steady-state c-Met levels, decreased Src kinase activity, decreased vascular endothelial growth factor expression, and decreased migration and invasion versus the pU1 (control) virus. Significant inhibition of tumorigenicity (histologically confirmed tumors in only 1 of 10 mice) and consequent lymph node metastasis were observed upon ex vivo infection of Ad-c-Met. Similarly, gene therapy experiments led to complete inhibition of tumor growth in 7 of 8 mice.Reduction in c-Met expression substantially inhibits both tumor growth and lymph node metastasis of PC3-LN4 cells in orthotopic nude mouse models. Therefore, targeting the c-Met signaling pathways may be important in controlling tumor growth and metastasis in human prostate cancers.

Increased expression and/or activity of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, occurs commonly during colon tumor progression. To examine potential roles for c-Met in promoting metastasis, we compared the colon tumor cell line KM12C with low metastatic potential to the isogenic variants KM,12L4 and KM12SM with high metastatic potential. KM12C cells express c-Met with low levels of tyrosine phosphorylation in the absence of HGF. The high metastatic cells express a c-Met that is constitutively tyrosine phosphorylated, they have increased colony formation, and are minimally responsive to HGF relative to the parental cells. Tyrosine-phosphorylated beta-catenin was constitutively associated with c-Met in the more metastatic cells, but was inducible only after HGF addition in the less metastatic cells. Functions mediated by beta-catenin, including cell-cell adhesion and migration, and activation of the tcf (T-cell factor) family of transcription factors, were also elevated in the more metastatic KM12SM and L4 cells. Furthermore, analysis of the known tcf transcriptional target genes, cyclin D1, c-Myc, and uPAR, demonstrated increased expression in the high metastatic cells, correlating with the levels of tcf activity. Collectively, these results suggest that endogenous activation of c-Met in highly metastatic KM12SM CRC cells results in increased survival and growth under anchorage independent conditions, increased in vitro migration, and elevated levels of tcf target genes. Thus, beta-catenin association with activated c-Met may contribute to a more aggressive liver metastatic phenotype of these cells.

Emergence of therapeutic resistance to angiogenesis inhibitors, the mechanisms of which are poorly understood, remains a major obstacle in treatment of NSCLC patients. Previously we reported that mechanisms governing resistance to anti-angiogenic therapy may involve both tumor and stromal cells in the tumor microenvironment. In this study we investigated potential mechanisms of resistance to the multi-tyrosine kinase inhibitors cediranib (AZD2171, Recentin®) and vandetanib (ZD6474, Zactima®) using NSCLC xenografts treated either for 2 weeks (sensitive tumors) or until resistance occurred. Quantification of TUNEL+ staining using laser scanning cytometry (LSC) showed increased apoptosis in H1975 xenografts sensitive to cediranib (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 376.

Background: Chemoresistance occurs in nearly all patients with metastatic colorectal cancer (CRC), and mechanisms to reverse chemoresistance remain elusive. We tested the hypothesis that CRC cells resistant to 5-fluorouracil (5FU-R) and oxaliplatin (Ox-R) exhibit proteomic profiles that may identify previously unrecognized mediators of resistance. In prior studies, we found that conditioned media from oxaliplatin-resistant HT29 cells (Ox-R) could mediate growth and chemoresistance in chemonaive parental HT29 cells in vitro. We sought to identify soluble factors in conditioned media that are potential mediators of the paracrine cell survival mechanisms. Methods: Parental HT29 (Par) cells were grown in increasing concentrations of 5-FU and oxaliplatin to generate 5FU-R and Ox-R cells. Protein from cell lysates and conditioned media (CM) were analyzed by liquid chromatography-mass spectrometry (LC-MS), and spectral counts were compared. Antibody-conjugated bead technology and ELISAs were used to obtain cytokine profiles of CM. Reverse phase proteomic arrays (RPMA) were used to determine signal transduction pathways activated in cells treated with CM. Ox-R cells were injected into nude mice to determine the paracrine effect on Par cells growing on the opposite flank. Results: Chemoresistant cells displayed significantly different proteomic profiles. In 5FU-R cells, pathways involving oxidative phosphorylation, inositol metabolism, actin cytoskeleton signaling, regulation of actin-based motility by Rho, and ATM signaling were significantly altered vs Par cells. In Ox-R cells, pathways mediating pyruvate metabolism, integrin signaling, caveolar-mediated endocytosis signaling, and mitochondrial dysfunction were among the most altered vs Par cells. Comparison of 5FU-R and Ox-R cells revealed differences in RNA post-transcriptional modification, ERK/MAPK, RAN, and chemokine signaling. Cytokine profiling demonstrated a significant increase in stem cell factor/c-Kit ligand (p=0.02) and a decrease in TRAIL (p=0.008) and IL-10 (p=0.04). RPPA analysis demonstrated early phosphorylation of EGFR and MEK1 followed by GSK, and mTOR activation. Ox-R tumors growing in vivo induced faster and larger tumor growth of contralateral Par tumors indicating a systemic effect. Conclusions: Chemoresistant CRC cells exhibit proteomes that reflect specific survival pathways, with many previously unrecognized potential mediators of resistance. Analysis of soluble factors from chemoresistant CRC cells demonstrates the presence of numerous potential mediators of cancer cell survival that may act, not only in an autocrine/paracrine manner, but also systemically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2276.

5114 Background: SR, an oral inhibitor of Raf kinase, RET-receptor (R), VEGFR-1, 2, and 3, PDGFR-β, FLT-3, and c-KIT tyrosine kinases, has been shown to prolong progression-free survival (PFS) in mRCC after cytokine failure. In a phase II trial, mRCC pts were randomized to first line treatment with SR vs. SR plus interferon (IFN) to assess whether combination therapy improved PFS. Both treatment arms showed similar outcomes (Tannir et al, ASCO. 2008). Here we investigated predictive and prognostic biomarkers in plasma. Methods: Pts received SR 400 mg PO BID or same dose SR plus IFN 0.5 MU SC BID. Plasma was collected from 69 pts at baseline (BL; SR 34, SR+IFN 35), 59 on day (D) 28, and 57 pts on D56. We used multiplex bead suspension arrays to measure concentrations of 54 CAFs, including VEGF, PDGFbb, EGF, HGF, MMP-9, and multiple chemokines and interleukins (IL). Osteopontin (OPN), soluble carbonic anhydrase 9 (sCA9), placental growth factor (PlGF), collagen type IV (ColIV) and sVEGFR-2 were measured by ELISA. The primary objective of this analysis was to establish a CAF signature based on a set of individual markers at BL with a significant and differential impact on the association between treatment arm and PFS. Results: Among 52 CAFs available at BL, higher than median EGF concentrations associated with poor outcome independently of treatment arm, whereas low IL-2 had the opposite effect (p = 0.003 for both). Only OPN showed a significant treatment by factor interaction at BL (p < 0.01), suggesting that OPN has a differential effect on PFS in the two arms. Pts with high OPN benefitted more from single agent SR (7.74 vs. 3.93 mos for the combination; p = 0.007), but no differences were found for those with low OPN. Lower than median on-treatment increases in sCA9 (D28, p = 0.01) and GRO-alpha (D56, p = 0.04) on SR only were also associated with a better outcome. A 6-marker CAF signature at BL containing OPN, sCA9, VEGF, sVEGFR-2, ColIV, and TRAIL demonstrated predictive value on PFS. Conclusions: A CAF signature showed potential value in predicting differential benefit from single agent SR vs. SR+IFN in mRCC. Broad-based screening of circulating CAFs may identify predictive and prognostic biomarkers in the context of clinical trials. [Table: see text]

Abstract #602 Aromatase inhibitors (AIs) are rapidly becoming the first choice for the endocrine treatment of steroid receptor positive breast cancer in postmenopausal women. However, de novo and acquired resistance is a significant problem. We have previously identified, in premalignant and invasive breast cancers, a somatic mutation at nucleotide 908 of ERα, which was significantly associated with postmenopausal status, larger tumor size, and axillary lymph node positivity. This mutation results in a lysine to arginine transition at residue 303 (K303R). We demonstrated that the K303R ERα mutation generated an estrogen hypersensitive phenotype in vitro and in vivo. Thus, we hypothesized that the mutant could provide a continuous mitogenic stimulus to the breast even during phases of low circulating hormone, such as in postmenopausal women, affording a proliferative advantage, especially during treatment with AIs. As preclinical models to directly test whether the mutation confers resistance to AIs, we generated MCF-7 parental and MCF-7-K303R-overexpressing cells stably transfected with the aromatase gene. Cells were stimulated with the aromatase substrate, androstenedione (AD), with or without the AI anastrazole (Ana). We found using MTT assays that Ana decreased AD-stimulated growth of WT vector control cells by 47%, but had no effect on AD-stimulated growth of MCF-7-K303R cells. Furthermore, in soft agar assays, the basal non-stimulated colony number of the mutant cells was increased 10-fold, and the AI was unable to inhibit the formation of mutant colonies. Aromatase activity of both cells was reduced by 95% by Ana, suggesting that this drug resistance cannot be explained by an intrinsic insensitivity of the aromatase enzyme itself to Ana. To examine the molecular mechanisms associated with the resistance of the mutant-expressing cells, we examined short-term nongenomic signaling, and found constitutively increased levels of phospho-IGF1R, IRS1 and AKT in the mutant cells. Interestingly, the hypersensitive mutant cells showed increased growth as well as pAkt activation with IGF1 treatment. Blockade of the IGF1R or PI3K/AKT pathways completely reversed the resistant mutant phenotype. To examine for effects on proliferation, immunoblot analyses were performed. K303R cells exhibited decreased levels of the CDK inhibitor p21 with AI treatment. Accordingly, apoptosis was decreased in the mutant cells with an increased ratio of bcl-2/Bax and decreased PARP cleavage. We conclude that the mutation indeed confers resistance to an AI. Potential mechanisms of resistance include cellular strategies to evade apoptosis and increase proliferation, possibly through enhanced cross-talk with growth factor pathways and reception of downstream signaling networks, such as AKT. Our results suggest that the mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and highlight the importance of novel approaches toward the development of signal transduction inhibitors for treating patients with tumors resistant to hormone therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 602.