Biomedical subjects

Sandra E Dunn

Publications and source records attributed to Sandra E Dunn.

10 recordsLinked to original sources

PubMed2006

Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor.

Although trastuzumab has been successfully used in patients with HER2-overexpressing metastatic breast cancer, resistance is a common problem that ultimately culminates in treatment failure. In light of the importance of Akt signaling in trastuzumab's antitumor action, we hypothesized that concurrent inhibition of Akt could enhance trastuzumab sensitivity and moreover reverse the resistant phenotype in HER2-positive breast cancer cells. Based on our finding that celecoxib mediates antitumor effects through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of cyclooxygenase-2 (COX-2), we used celecoxib as a scaffold to develop a COX-2-inactive PDK-1 inhibitor, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012). Here, we investigated the effect of OSU-03012 on trastuzumab-mediated apoptosis in four breast cancer cell lines with different HER2 expression and trastuzumab-resistance status, including MDA-MB-231, BT474, SKBR3, and insulin-like growth factor-I receptor-overexpressing SKBR3 (SKBR3/IGF-IR). Effects of trastuzumab and OSU-03012, individually or in combination, on cell viability and changes in pertinent biomarkers including HER2 expression, phosphorylation of Akt, p27(kip1), and the PDK-1 substrate p70(S6K) were assessed. OSU-03012 alone was able to trigger apoptosis in all cell lines with equal potency (IC(50) = 3-4 microM), suggesting no cross-resistance with trastuzumab. Medium dose-effect analysis indicates that OSU-03012 potentiated trastuzumab's antiproliferative effect in HER2-positive cells, especially in SKBR3/IGF-IR cells, through the down-regulation of PDK-1/Akt signaling. This synergy, however, was not observed in HER2-negative MDA-MB-231 cells. This combination treatment represents a novel strategy to increase the efficacy of trastuzumab and to overcome trastuzumab resistance in the treatment of HER2-positive breast cancer.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2006

Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2.

The overexpression of the epidermal growth factor receptor (EGFR) and HER-2 underpin the growth of aggressive breast cancer; still, it is unclear what governs the regulation of these receptors. Our laboratories recently determined that the Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, induced breast tumor cell growth in monolayer and in soft agar. Importantly, mutating YB-1 at Ser(102), which resides in the DNA-binding domain, prevented growth induction. We reasoned that the underlying cause for growth attenuation by YB-1(Ser(102)) is through the regulation of EGFR and/or HER-2. The initial link between YB-1 and these receptors was sought by screening primary tumor tissue microarrays. We determined that YB-1 (n = 389 cases) was positively associated with EGFR (P < 0.001, r = 0.213), HER-2 (P = 0.008, r = 0.157), and Ki67 (P < 0.0002, r = 0.219). It was inversely linked to the estrogen receptor (P < 0.001, r = -0.291). Overexpression of YB-1 in a breast cancer cell line increased HER-2 and EGFR. Alternatively, mutation of YB-1 at Ser(102) > Ala(102) prevented the induction of these receptors and rendered the cells less responsive to EGF. The mutant YB-1 protein was also unable to optimally bind to the EGFR and HER-2 promoters based on chromatin immunoprecipitation. Furthermore, knocking down YB-1 with small interfering RNA suppressed the expression of EGFR and HER-2. This was coupled with a decrease in tumor cell growth. In conclusion, YB-1(Ser(102)) is a point of molecular vulnerability for maintaining the expression of EGFR and HER-2. Targeting YB-1 or more specifically YB-1(Ser(102)) are novel approaches to inhibiting the expression of these receptors to ultimately suppress tumor cell growth.

Breast Neoplasms↗

PubMed2006

Preferential dependence of breast cancer cells versus normal cells on integrin-linked kinase for protein kinase B/Akt activation and cell survival.

The emerging paradigm of "oncogene addiction" has been called an Achilles' heel of cancer that can be exploited therapeutically. Here, we show that integrin-linked kinase (ILK), which is either activated or overexpressed in many types of cancers, is a critical regulator of breast cancer cell survival through the protein kinase B (PKB)/Akt pathway but is largely dispensable for the survival of normal breast epithelial cells and mesenchymal cells. We show that inhibition of ILK activity with a pharmacologic ILK inhibitor, QLT-0267, results in the inhibition of PKB/Akt Ser473 phosphorylation, stimulation of apoptosis, and a decrease in mammalian target of rapamycin (mTOR) expression in human breast cancer cells. In contrast, QLT-0267 treatment has no effect on PKB/Akt Ser473 phosphorylation or apoptosis in normal human breast epithelial, mouse fibroblast, or vascular smooth muscle cells. The inhibition of PKB/Akt Ser473 phosphorylation by QLT-0267 in breast cancer cells was rescued by a kinase-active ILK mutant but not by a kinase-dead ILK mutant. Furthermore, a dominant-negative ILK mutant increased apoptosis in the MDA-MB-231 breast cancer cell line but not in normal human breast epithelial cells. The inhibitor was active against ILK isolated from all cell types but did not have any effect on cell attachment and spreading. Our data point to an "ILK addiction" of breast cancer cells whereby they become dependent on ILK for cell survival through the mTOR-PKB/Akt signaling pathway and show that ILK is a promising target for the treatment of breast cancer.

Amino Acid Sequence↗

PubMed2005

Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours.

INTRODUCTION: Phosphorylated Akt (P-Akt) is an attractive molecular target because it contributes to the development of breast cancer and confers resistance to conventional therapies. Akt also serves as a signalling intermediate for receptors such as human epidermal growth factor receptor (HER)-2, which is overexpressed in 30% of breast cancers; therefore, inhibitors to this pathway are being sought. New celecoxib analogues reportedly inhibit P-Akt in prostate cancer cells. We therefore examined the potential of these compounds in the treatment of breast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very high levels of P-Akt. METHODS: To evaluate the effect of the celecoxib analogues, immunoblotting was used to identify changes in the phosphorylation of Akt and its downstream substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP-1). In vitro kinase assays were then used to assess the effect of the drugs on Akt activity. Cell death was evaluated by poly(ADP-ribose) polymerase cleavage, nucleosomal fragmentation and MTS assays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression. RESULTS: OSU-03012 and OSU-O3013 inhibited P-Akt and its downstream signalling through 4EBP-1 and GSK at concentrations well below that of celecoxib. Disruption of P-Akt was followed by induction of apoptosis and more than 90% cell death. We also noted that the cytotoxicity of the celecoxib analogues was not significantly affected by serum. In contrast, the presence of 5% serum protected cells from celecoxib induced death. Thus, the structural modification of the celecoxib analogues increased P-Akt inhibition and enhanced the bioavailability of the drugs in vitro. To assess how many patients may potentially benefit from such drugs we screened tumour tissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35% (9/26) of normal breast tissues. Furthermore, HER-2 positive tumours expressed high levels of P-Akt (P < 0.01), supporting in vitro signal transduction. CONCLUSION: We determined that Celecoxib analogues are potent inhibitors of P-Akt signalling and kill breast cancer cells that overexpress HER-2. We also defined an association between HER-2 and P-Akt in primary breast tissues, suggesting that these inhibitors may benefit patients in need of new treatment options.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2005

Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells.

Akt/PKB is a serine/threonine kinase that promotes tumor cell growth by phosphorylating transcription factors and cell cycle proteins. There is particular interest in finding tumor-specific substrates for Akt to understand how this protein functions in cancer and to provide new avenues for therapeutic targeting. Our laboratory sought to identify novel Akt substrates that are expressed in breast cancer. In this study, we determined that activated Akt is positively correlated with the protein expression of the transcription/translation factor Y-box binding protein-1 (YB-1) in primary breast cancer by screening tumor tissue microarrays. We therefore questioned whether Akt and YB-1 might be functionally linked. Herein, we illustrate that activated Akt binds to and phosphorylates the YB-1 cold shock domain at Ser102. We then addressed the functional significance of disrupting Ser102 by mutating it to Ala102. Following the stable expression of Flag:YB-1 and Flag:YB-1 (Ala102) in MCF-7 cells, we observed that disruption of the Akt phosphorylation site on YB-1 suppressed tumor cell growth in soft agar and in monolayer. This correlated with an inhibition of nuclear translocation by the YB-1(Ala102) mutant. In conclusion, YB-1 is a new Akt substrate and disruption of this specific site inhibits tumor cell growth.

Breast Neoplasms↗

PubMed2005

Celecoxib inhibits urokinase-type plasminogen activator (uPA) production in MDA-MB-231 breast cancer cells.

Elevated urokinase-type plasminogen activator (uPA) expression in breast tumors predicts poor survival. We found celecoxib (25 microM) significantly reduced uPA protein and mRNA in MDA-MB-231 breast cancer cells following 72 h of treatment. Celecoxib also inhibited cell viability (12.5 and 25 microM) and induced G2M arrest (25 microM). Therefore, celecoxib therapy for uPA positive breast cancer should be explored.

Breast Neoplasms↗

PubMed2004

Hierarchical clustering analysis of tissue microarray immunostaining data identifies prognostically significant groups of breast carcinoma.

Prognostically relevant cluster groups, based on gene expression profiles, have been recently identified for breast cancers, lung cancers, and lymphoma. Our aim was to determine whether hierarchical clustering analysis of multiple immunomarkers (protein expression profiles) improves prognostication in patients with invasive breast cancer. A cohort of 438 sequential cases of invasive breast cancer with median follow-up of 15.4 years was selected for tissue microarray construction. A total of 31 biomarkers were tested by immunohistochemistry on these tissue arrays. The prognostic significance of individual markers was assessed by using Kaplan-Meier survival estimates and log-rank tests. Seventeen of 31 markers showed prognostic significance in univariate analysis (P < or = 0.05) and 4 markers showed a trend toward significance (P < or = 0.2). Unsupervised hierarchical clustering analysis was done by using these 21 immunomarkers, and this resulted in identification of three cluster groups with significant differences in clinical outcome. chi2 analysis showed that expression of 11 markers significantly correlated with membership in one of the three cluster groups. Unsupervised hierarchical clustering analysis with this set of 11 markers reproduced the same three prognostically significant cluster groups identified by using the larger set of markers. These cluster groups were of prognostic significance independent of lymph node metastasis, tumor size, and tumor grade in multivariate analysis (P=0.0001). The cluster groups were as powerful a prognostic indicator as lymph node status. This work demonstrates that hierarchical clustering of immunostaining data by using multiple markers can group breast cancers into classes with clinical relevance and is superior to the use of individual prognostic markers.

Adult↗

PubMed2004

Expression of the insulin-like growth factor I receptor and urokinase plasminogen activator in breast cancer is associated with poor survival: potential for intervention with 17-allylamino geldanamycin.

Urokinase plasminogen activator (uPA) expression in breast cancer is associated with relapse and a reduction in disease-specific survival. Thus, efforts are under way to identify uPA inhibitors. By screening a chemical library of >1000 compounds, 17-allyaminogeldanamycin (17AAG) was identified as a potent inhibitor of uPA by the National Cancer Institute and is now in Phase I clinical trials. At this time, it remains unclear how 17AAG blocks uPA; one possibility is through disruption of the insulin-like growth factor I receptor (IGF-IR) pathway. This would be consistent with studies from our laboratory showing that activation of IGF-IR results in the induction of uPA protein. In the study described herein, we observed that IGF-IR and uPA were highly expressed in 87 and 55% of breast cancer by screening tumor tissue microarrays representing 930 cases. A significant proportion (52.1% = 354 of 680 cases, P < 0.0001) of the patients had tumors expressing both proteins. uPA alone (P = 0.033) or in combination with IGF-IR (P = 0.0104) was indicative of decreased disease-specific survival. Next, we demonstrated that treating MDA-MB-231 cells with increasing concentrations of 17AAG resulted in IGF-IR degradation (IC(50) = 1.0 micro M) and blocked signal transduction through the Akt and mitogen-activated protein kinase pathways. Finally, we found that 17AAG had a robust inhibitory effect on the production of uPA mRNAand protein in the presence of IGF-I. Thus, our study raises the possibility that 17AAG could prove to be an effective therapeutic agent for a large number of breast cancer patients by inhibiting the IGF-IR and ultimately uPA.

Antineoplastic Agents↗

PubMed2003

Microarray analysis and identification of novel molecules involved in insulin-like growth factor-1 receptor signaling and gene expression.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF-1R) are members of the same subfamily of receptor tyrosine kinases. The two receptors phosphorylate many of the same substrates and activate the same signaling modules, including the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3' kinase (PI3K) signaling pathways. Although the IR and IGF-1R share some redundant functions in metabolism, cell growth, differentiation, and apoptosis, they also exhibit distinct physiological roles. Some of these may be due to differences in tissue distribution, receptor structure, formation of hybrid receptors, or mechanisms of ligand binding. However, the divergent effects of insulin and IGF-1 also may be explained by specificity in the intracellular signals generated by insulin and IGF-1. In particular, the IR and IGF-1R are capable of triggering their own biological responses by using specific or preferential substrates, molecular adapters, or signaling pathways. In a recent study, we used cDNA microarray analysis to identify genes differentially regulated by insulin and IGF-1. Mouse NIH-3T3 fibroblasts expressing either the wild-type human IGF-1R or IR were stimulated with either IGF-1 or insulin, respectively. We identified 39 genes differentially regulated by insulin and IGF-1. Most of these genes had not been reported previously to be responsive to insulin or IGF-1. The genes induced by IGF-1 generally were involved in mitogenesis or differentiation, while the genes found to be induced by insulin did not conform to any particular category. In a separate study, immortalized breast epithelial cells were stimulated with IGF-1 and a cDNA microarray analysis was used to generate a profile of IGF-1-regulated genes. A number of genes known to be involved in angiogenesis were found to be regulated by IGF-1. These results strongly suggest that this technology may be extremely useful in identifying groups of genes that are specifically regulated by different ligands and their activated receptors.

Animals↗

PubMed2003

Role of IGF-1R in mediating breast cancer invasion and metastasis.

In this review we bring forward what is currently known about the role of type I insulin-like growth factor receptor (IGF-1R) in mediating breast cancer invasion and metastasis. We begin by addressing how activated IGF-1R could allow pre-cancerous cells to become invasive. To this effect, we discuss clinical reports suggesting that activation of IGF-1R could stimulate ductal carcinoma in situs to become invasive. In the same light, we review basic research from our laboratory showing that IGF-1R differentially regulates the expression of breast cancer progression genes when pre-malignant breast epithelial cells were stimulated with insulin-like growth factor-I (IGF-I) over time. The discussion then turns toward the ability of IGF-1R to stimulate invasion of breast cancer cells that have acquired a malignant phenotype. At this stage of breast cancer, it appears that IGF-I stimulates cells to invade in part by inducing urokinase plasminogen activator. Finally, we consider the potential role of IGF-1R in regulating breast cancer metastases by facilitating angiogenesis and lymphangiogenesis. In support of this idea, there is evidence for IGF-1R in both of these processes through the induction of vascular endothelial growth factors (VEGF(165) and VEGF(121)). Thus, IGF-1R affords breast cancer cells many opportunities to become invasive and eventually metastatic. We conclude that disrupting IGF-1R signaling has many important implications in the treatment and management of breast cancer.

Journal Article↗