Isere Kuiatse

Mammary Tumorigenesis and Metastasis Caused by Overexpression of Insulin Receptor Substrate 1 (IRS-1) or IRS-2

ABSTRACT Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prognosis. We found that overexpressed IRS-1 was constitutively phosphorylated in vitro and in vivo and that transgenic mice overexpressing IRS-1 or IRS-2 in the mammary gland showed progressive mammary hyperplasia, tumorigenesis, and metastasis. Tumors showed extensive squamous differentiation, a phenotype commonly seen with activation of the canonical β-catenin signaling pathway. Consistent with this, IRSs were found to bind β-catenin in vitro and in vivo. IRS-induced tumorigenesis is unique, given that the IRSs are signaling adaptors with no intrinsic kinase activity, and this supports a growing literature indicating a role for IRSs in cancer. This study defines IRSs as oncogene proteins in vivo and provides new models to develop inhibitors against IRSs for anticancer therapy.

The growth hormone receptor antagonist pegvisomant blocks both mammary gland development and MCF-7 breast cancer xenograft growth

SummaryMammary gland development is dependent upon the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis, this same axis has also been implicated in breast cancer progression. In this study we investigated the effect of a GH antagonist, pegvisomant (Somavert®, Pfizer), on normal mammary gland development and breast cancer xenograft growth. Intraperitoneal administration of pegvisomant resulted in a 60% suppression of hepatic IGF-I mRNA levels and upto a 70–80% reduction of serum IGF-I levels. Pegvisomant administration to virgin female mice caused a significant delay of mammary ductal outgrowth that was associated with a decrease in the number of terminal end buds and reduced branching and complexity of the gland. This effect of pegvisomant was mediated by a complete inhibition of both GH and IGF-IR-mediated signaling within the gland. In breast cancer xenograft studies, pegvisomant caused shrinkage of MCF-7 xenografts, with an initial 30% reduction in tumor volume, which was associated with a 2-fold reduction in proliferation and a 2-fold induction of apoptosis. Long-term growth inhibition of MCF-7 xenografts was noted. In contrast, pegvisomant had no effect on MDA-231 or MDA-435 xenografts, consistent with primary growth of these xenografts being unresponsive to IGF-I both in vitro and in vivo. In MCF-7 xenografts that regressed, pegvisomant had only minor effects upon GHR and IGF-IR signaling. This data supports previous studies indicating a role for GH/IGF in mammary gland development, and suggests that pegvisomant maybe useful for the prevention and/or treatment of estrogen receptor positive breast cancer.

Sustained c-Jun-NH2-Kinase Activity Promotes Epithelial-Mesenchymal Transition, Invasion, and Survival of Breast Cancer Cells by Regulating Extracellular Signal-Regulated Kinase Activation

The c-Jun NH2-terminus kinase (JNK) mediates stress-induced apoptosis and the cytotoxic effect of anticancer therapies. Paradoxically, recent clinical studies indicate that elevated JNK activity in human breast cancer is associated with poor prognosis. Here, we show that overexpression of a constitutively active JNK in human breast cancer cells did not cause apoptosis, but actually induced cell migration and invasion, a morphologic change associated with epithelial-mesenchymal transition (EMT), expression of mesenchymal-specific markers vimentin and fibronectin, and activity of activator protein transcription factors. Supporting this observation, mouse mammary tumor cells that have undergone EMT showed upregulated JNK activity, and the EMT was reversed by JNK inhibition. Sustained JNK activity enhanced insulin receptor substrate-2–mediated ERK activation, which in turn increased c-Fos expression and activator protein activity. In addition, hyperactive JNK attenuated the apoptosis of breast cancer cells treated by the chemotherapy drug paclitaxel, which is in contrast to the requirement for inducible JNK activity in response to cytotoxic chemotherapy. Blockade of extracellular signal-regulated kinase activity diminished hyperactive JNK-induced cell invasion and survival. Our data suggest that the role of JNK changes when its activity is elevated persistently above the basal levels associated with cell apoptosis, and that JNK activation may serve as a marker of breast cancer progression and resistance to cytotoxic drugs. Mol Cancer Res; 8(2); 266–77

BMS-536924 Reverses IGF-IR-induced Transformation of Mammary Epithelial Cells and Causes Growth Inhibition and Polarization of MCF7 Cells

Purpose: This study aimed to test the ability of a new insulin-like growth factor receptor (IGF-IR) tyrosine kinase inhibitor, BMS-536924, to reverse the ability of constitutively active IGF-IR (CD8-IGF-IR) to transform MCF10A cells, and to examine the effect of the inhibitor on a range of human breast cancer cell lines. Experimental Design:CD8-IGF-IR-MCF10A cells were grown in monolayer culture, three-dimensional (3D) culture, and as xenografts, and treated with BMS-536924. Proliferation, cell cycle, polarity, and apoptosis were measured. Twenty-three human breast cancer cell lines were treated in monolayer culture with BMS-536924, and cell viability was measured. MCF7, MDA-MB-231, and MDA-MB-435 were treated with BMS-536924 in monolayer and 3D culture, and proliferation, migration, polarity, and apoptosis were measured. Results: Treatment of CD8-IGF-IR-MCF10A cells grown in 3D culture with BMS-536924 caused a blockade of proliferation, restoration of apical-basal polarity, and enhanced apoptosis, resulting in a partial phenotypic reversion to normal acini. In monolayer culture, BMS-536924 induced a dose-dependent inhibition of proliferation, with an accumulation of cells in G0/G1,, and completely blocked CD8-IGF-IR–induced migration, invasion, and anchorage-independent growth. CD8-IGF-IR-MCF10A xenografts treated with BMS-536924 (100 mg/kg/day) showed a 76% reduction in xenograft volume. In a series of 23 human breast cancer cell lines, BMS-536924 inhibited monolayer proliferation of 16 cell lines. Most strikingly, treatment of MCF7 cells grown in 3D culture with BMS-536924 caused blockade of proliferation, and resulted in the formation of hollow polarized lumen. Conclusions: These results show that the new small molecule BMS-536924 is an effective inhibitor of IGF-IR, causing a reversion of an IGF-IR–mediated transformed phenotype.

Novel egg white–based 3-D cell culture system

Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.

Abstract 4133: Comparative gene expression and proteomic analysis of IGF-I and insulin signaling in a large panel of breast cancer cell lines

IGF-I signaling is involved in tumor progression and drug resistance and many anti-IGF-IR therapeutic agents are currently in clinical trials. Moreover, growing evidence also suggests that insulin signaling may play an important role in cancer development and progression. However, little is known regarding similarities and difference in IGF-I and insulin signaling in cancer, and there are currently no biomarkers to predict patient response to IGF targeted therapy. To gain a better understanding of these signaling pathways in human breast cancer, we measured the levels of insulin receptor (IR) and IGF-IR, and the activity of insulin and IGF-I, in a large panel of human breast cancer cell lines. Comparative gene expression analysis was performed using publicly available gene expression data and IR and IGF-IR mRNA levels in 17 breast cancer cell lines were measured by Q-RT-PCR. IGF-IR and IR levels were found to be highly variable in breast cancer cell lines. MCF7 and MDA-MB-134 have high levels of IGF-IR expression and MDA-MB-468 and ZR-75-1 have high levels of IR expression, relative to other breast cancer cell lines. Reverse phase protein array (RPPA) analysis using 134 different antibodies was performed on MCF7 cells stimulated with increasing doses of insulin or IGF-I. One Way ANOVA was performed to identify proteins affected by insulin and/or IGF stimulation. Maximal response to insulin and IGF-I stimulation was observed at a 10nM concentration of both ligands. Therefore, we further performed RPPA on 21 breast cancer cell lines treated with 10nM of insulin and IGF-I for 6 time points (5min, 10min, 30 min, 6hrs, 24hrs, and 48hrs). Spearman rank correlation was performed on IR and IGF-IR protein levels to mRNA levels from Q-RT-PCR. There was a significant correlation between IGF-IR protein and mRNA levels (r=0.559, p=0.024), but not between IR protein and mRNA levels. Principle component analysis showed that MCF7, MDA-MB-134, T47D and ZR-75-1 are insulin and IGF-I responsive. Results from ANOVA with contrast in MCF7 cells showed that insulin and IGF-I affected similar set of proteins at 10 minute and 24 hours time points, however, there were several proteins affected only by IGF-I or insulin stimulation. For example, phospho-estrogen receptor (p-ER) was found to be increased by 10 minutes IGF-I stimulation, but not by insulin stimulation. Beta-catenin and c-Myc were significantly suppressed by IGF stimulation while PARP was significantly suppressed by insulin stimulation at the 24 hour time point. Moreover, IGF affected twice the number of proteins compared to insulin at the 48 hour time point. For instance, Bcl2 and SMAD3 were suppressed and Raf and FOXO3a were increased by IGF stimulation after 48 hours of stimulation. Further statistical and system biology analysis of the temporal activation of signaling by IGF-I and insulin in the 21 cell lines is ongoing and will be presented. 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 4133.

JNK, essential for anticancer therapy-induced apoptosis, promotes breast cancer progression.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5075 The c-Jun N-terminal kinase (JNK) is a critical mediator of stress-induced apoptosis and is required for the cytotoxic effect of anticancer therapies. Interestingly, recent clinical studies showed that high JNK activity is associated with poor prognosis in human breast cancer. The molecular basis of this potential dual function of JNK in breast cancer is poorly understood. Here we report that overexpression of a constitutively active JNK in breast cancer cells increased cell migration and invasion. In agreement with this, fibroblast-specific markers like the intermediate filament vimentin and the extracellular matrix protein fibronectin were up-regulated. AP-1 transcription factor, which induces expression of these two proteins, was markedly increased by JNK overexpression. In addition, hyperactive JNK reduced Akt1 activation but enhanced Erk activation in breast cancer cells. In relevance, we also found that JNK levels are significantly higher in invasive breast cancers including those with distant metastases (n = 235, p < 0.0001, Wilcoxon rank sum test) than in normal breast tissues (n = 69). Most surprisingly, an increase of JNK attenuated the apoptosis of breast cancer cells treated with chemotherapy drugs doxorubicin and taxol. This suggests that the role of JNK changes when its activity/expression increases above the basal levels associated with apoptosis. Our data, together with a recent finding that cells undergoing cytotoxic agent-induced apoptosis can induce compensatory hyper-proliferation of proximal cancer cells, suggest a novel mechanism of breast cancer therapeutic resistance. Therapy-elicited apoptosis of tumor cells with basal JNK activity may release mitogens that induce hyperactive JNK in neighboring cells to promote growth and invasion. Thus JNK activation may serve as a marker of breast cancer progression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5075.

Estrogen and IGF-I Independently Down-Regulate Critical Repressors of Breast Cancer Growth.

Background: Estrogen receptor and insulin-like growth factor (IGF) signaling pathways are important for both normal mammary gland development and breast cancer pathogenesis. Despite this evidence, cross-talk between these two critical mitogenic and survival pathways remains poorly understood, particularly at the level of downstream gene transcriptional networks. Methods: We performed microarray analysis on ER-positive MCF-7 breast cancer cells following stimulation with either estradiol (10nM) or IGF-I (10nM) for 3hr or 24hr. We defined an E2-IGF gene expression signature and examined the effect of the gene signature on time to recurrence in ER+ tumors in the publicly available NKI dataset. Q-RT-PCR was used to validate the effects of IGF-I and E2 on mRNA of repressed genes. To examine gene regulation further, MCF-7 cells were incubated with various signaling inhibitors and gene expression measured. Results: We found 183 probesets to be co-regulated by E2 and IGF-I after 3hr of stimulation and 454 probesets to be co-regulated at the 24hr time point. Patients with tumors that had the same set of genes up- and down-regulated had a poor outcome (p=7.04E-07). E2-IGF co-regulated genes showed a significant enrichment for down-regulated genes (p Conclusions: E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome. E2 and IGF-I down-regulate several critical tumor suppressor genes. While the down-regulation is independent of each other at the level of ER and IGF-IR, for some genes, there is convergence on the PI3K pathway for repression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1126.