Marc A. Becker

The IGF Pathway Regulates ERα through a S6K1-Dependent Mechanism in Breast Cancer Cells

The IGF pathway stimulates malignant behavior of breast cancer cells. Herein we identify the mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) axis as a critical component of IGF and estrogen receptor (ER)α cross talk. The insulin receptor substrate (IRS) adaptor molecules function downstream of IGF-I receptor and dictate a specific biological response, in which IRS-1 drives proliferation and IRS-2 is linked to motility. Although rapamycin-induced mTOR inhibition has been shown to block IGF-induced IRS degradation, we reveal differential effects on motility (up-regulation) and proliferation (down-regulation). Because a positive correlation between IRS-1 and ERα expression is thought to play a central role in the IGF growth response, we investigated the potential role of ERα as a downstream mTOR target. Small molecule inhibition and targeted knockdown of S6K1 blocked the IGF-induced ERα(S167) phosphorylation and did not influence ligand-dependent ERα(S118) phosphorylation. Inhibition of S6K1 kinase activity consequently ablated IGF-stimulated S6K1/ERα association, estrogen response element promoter binding and ERα target gene transcription. Moreover, site-specific ERα(S167) mutation reduced ERα target gene transcription and blocked IGF-induced colony formation. These findings support a novel link between the IGF pathway and ERα, in which the translation factor S6K1 affects transcription of ERα-regulated genes.

Insulin receptor substrate adaptor proteins mediate prognostic gene expression profiles in breast cancer

Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer.

Abstract 535: IGF-I stimulates amino acid transporter xC- function to reduce intracellular ROS level and promote proliferation in human breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnOur laboratory identified SLC7A11/xCT as an IGF-I-induced gene specifically regulated through the adaptor protein IRS-1 in breast cancer cells. xCT encodes the functional subunit of the heterodimeric plasma membrane transport system xC-, which is critical for the cellular uptake of cystine in exchange for intracellular glutamate. xC- transporter is involved in the regulation of proliferation, metastasis, and treatment resistance in cancer. To determine the role for xC- expression in IGF-mediated breast cancer cell biology, two classes of breast cancer cell lines were studied: estrogen receptor (ER) positive cell lines (IRS-1 activated) MCF-7, ZR-75-1, and T47D; basal-like cell lines (IRS-2 activated) MDA-MB-231, BT549, and HS578T. Significantly increased xCT mRNA expression was observed only in ER positive cell lines, which was eliminated by IRS-1 specific siRNA (25 nM). IGF-I increased xCT protein expression measured by immunblots and flow cytometry only in ER positive cells in an IRS-1 dependent manner. To measure the function of upregulated xC-, extracellular levels of glutamic acid and intracellular level of total glutathione were measured in MCF-7 and MDA-MB-231. IGF-I regulated xC- transporter function only in IRS-1 activated MCF-7 cells. In MCF-7 cells, IGF-I-stimulated monolayer and anchorage-independent growth were suppressed by treating cells with xCT shRNA or the xCT chemical inhibitor sulfasalazine (SASP, 0.1 mM). Direct measurement of reactive oxygen species (ROS) and levels of phospho-p38MAPkinase showed that IGF-I reduced cellular ROS level (induced by 10 Gy of irradiation or 1 ug/ml of mitomycin C), SASP (0.1 mM) reversed this IGF-I effect. Anchorage-independent growth assays suggested that inhibiting xC- function by 0.1 mM SASP sensitized the response of MCF-7 cells to anti-IGF-IR monoclonal antibody humanized EM164 (ImmunoGen, Inc.) and tyrosine kinase inhibitor NVP-AEW-541. These data suggest that IGF-I promotes the proliferation of ER positive breast cancer cells by regulating xC- transporter function in an IRS-1 dependent manner. Furthermore, IGF-I protects cells from ROS via upregulation of xC-. Our findings also imply that combination of xC- transporter inhibitor with anti-IGF-IR agents may have synergic therapeutic effects.nnCitation Format: Yuzhe Yang, Marc A. Becker, Douglas Yee. IGF-I stimulates amino acid transporter xC- function to reduce intracellular ROS level and promote proliferation in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 535. doi:10.1158/1538-7445.AM2013-535

IGF system and breast cancer

The IGF system components have been implicated in breast cancer progression. IGF-I and IGF-II are mitogenic and anti-apoptotic peptides that influence the proliferation of various cell types including normal and transformed breast epithelial cells. The IGF system is a key growth regulatory pathway in breast cancer. As various components of the IGF system have been directly or indirectly implicated in breast cancer, it is essential to gain a better understanding of these in order to develop more specific therapeutic strategies for treating breast cancer.

P2-03-03: An Insulin-Like Growth Factor I (IGF-I)-Induced Gene, Solute Carrier Family 7 Member 11 (SLC7A11)/xCT, Mediates IGF-I-Induced Biological Behaviors in Breast Cancer Cells.

Our laboratory studied the gene expression profiles of a series of T47D variant cell lines with differential insulin receptor substrate (IRS) adaptor protein expression to develop predictive IGF-I pathway biomarkers. We identified an IGF-I-induced gene, SLC7A11 (or xCT), which is specifically regulated through IRS-1. xCT encodes the cystine/glutamate transporter subunit of the heterodimeric amino acid transport system xc- which is a major plasma membrane transporter for the cellular uptake of cystine in exchange for intracellular glutamate. xCT is involved in the regulations of proliferation, metastasis, and drug resistance in various cancers. However, to date, the linkage between xCT and the IGF-I signaling pathway has not been described. To study the role for xCT in mediating IGF-I-induced biology in breast cancer cell lines, we examined xCT mRNA expression upon IGF-I stimulation in two breast cancer cell lines; the MCF-7 (IRS-1 activated) and MDA-MB-231 (IRS-2 activated) cells. Significant increased xCT expression was observed only in MCF-7 cells after IGF-I treatment. Immunoblots showed that xCT protein expression was elevated after IGF-I treatment and induced glutamate/cystine exchange in MCF-7 cells. shRNA was used to downregulate xCT in MCF-7 and MDA-MB-231 cells. In MCF-7, IGF-I-stimulated cell monolayer growth was suppressed by xCT shRNA or by the xCT inhibitor sulfasalazine (SASP). In MDA-MB-231 cells, xCT downregulation did not affect IGF-mediated Boyden chamber migration. Thus, IGF-I induction of cellular xCT levels is associated with cell growth in the IRS-1 activated MCF-7 cells, while MDA-MB-231 cells were not affected by downregulation of this gene. Therefore, our data imply that xCT may mediate IGF-I induced biological functions in breast cancer cell lines through an IRS-1 dependent pathway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-03.

Abstract CN07-02: Disrupting insulin‐like growth factor signaling with monoclonal antibodies

The insulin‐like growth factor (IGF) system is composed of a network of ligands, receptors, and binding proteins. The type I IGF receptor (IGF1R) is a transmembrane tyrosine kinase receptor and shares a high degree of homology with the insulin receptor (InsR). Data from many sources implicate the IGF system in cancer biology. Because of the role for InsR in glucose homeostasis, specific targeting of IGF1R with monoclonal antibodies has been deemed desirable. Numerous monoclonal antibodies have been developed against this receptor and share similar properties in preclinical model systems. To date, all of the antibodies have been shown to bind IGF1R and cause its internalization and downregulation. Early clinical studies have shown that IGF1R antibodies have activity as single agents in some diseases (sarcoma) and have activity in combination with cytotoxic chemotherapy. These early promising results suggest that inhibition of this receptor will emerge as a new cancer therapy. In addition, these early clinical results raise additional questions regarding optimizing their use in cancer treatment. In a xenograft model, monoclonal antibodies to IGF1R do not suppress tumor growth of the MDA‐435/LCC6 cell line yet inhibit tumor metastasis. These data suggest that a commonly measured clinical phenotype, tumor growth, may not be detected in early phase II clinical trials. Clinical trials have also shown that IGF1R monoclonal antibody therapy results in elevation of growth hormone, IGF‐I, and insulin levels. Since InsR may also mediate signaling in the IGF system, it is possible that InsR is also a target for cancer therapy. To address this issue, we have used shRNA to downregulate InsR expression while leaving IGF1R levels intact. In the MDA‐435/LCC6 cells, disruption of InsR decreases tumor growth and inhibits pulmonary metastases. We have also selected cells in increasing concentrations of IGF1R antibodies. Finally, long term incubation of cells with IGF1R monoclonal antibodies results in cells with low levels of receptor expression, yet the receptor remains biochemically functional and can be inhibited by IGF1R tyrosine kinase inhibitors. Thus, disruption of IGF1R signaling has promise as a new cancer therapy. Future consideration of the role of the ligands, the InsR, and combination therapies with other signaling disruptors will allow us to optimize this therapeutic strategy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-02.

IGF-Induced Gene Profiles Are Dependent on IRS Expression and Convey Prognostic Value in Human Breast Cancer.

The insulin-like growth factor (IGF) pathway mediates aberrant function during the initiation and progression of primary tumors and secondary metastases in cancer. As a result, a host of tyrosine kinase inhibitors and monoclonal antibodies directed against the type 1 IGF receptor (IGF-1R) have entered clinical trials with early positive results. However, no predictive biomarkers have yet emerged from these initial studies. We propose that expanding IGF biomarkers beyond IGF-1R alone may identify the most appropriate candidates for anti-IGF therapy. Previous work has shown that the insulin receptor substrate (IRS) proteins serve as the functional link between IGF-I-induced IGF-1R phosphorylation and downstream signaling linked to cellular behavior. Our work has demonstrated that IRS isoforms differentially mediate IGF-I action, whereby IRS-1 drives proliferation and IRS-2 triggers motility. Here we employed the T47D-YA (IRS null) breast cancer cell line and T47D-YA/IRS clones stably transfected with IRS-1 or IRS-2. In response to acute (4h) and chronic (24h) IGF-I stimulation, global gene expression patterns were assessed by Affymetrix U133 Plus 2.0 microarray analysis. Analysis revealed that IGF-1R activation alone was insufficient to affect gene expression as no genes were regulated by IGF-I in T47D-YA cells. Conversely, ligand stimulation of IRS-1 and IRS-2 clones induced or repressed hundreds of transcripts in both overlapping and distinct fashions. Direct comparison of IRS-1 to IRS-2 clones revealed a number of early (4h) IRS-2 genes linked to metastasis and late (24h) IRS-1 proliferative genes. Interestingly, a 10-fold upregulation in the transforming growth factor beta 2 (TGFβ2) gene by IGF-I in IRS-2 cells suggested a link between the IRS-derived gene signatures and the TGFβ pathway known to regulate breast cancer metastasis. We then compared our arrays with published IGF-I (MCF-7) and TGFβ-derived (MCF10A, MDA-231, HaCaT, HPL1) microarrays to find a list of commonly regulated genes and performed cluster analysis to reveal consistent patterns of gene expression (Creighton el al 2008 & Padua et al 2008). We found 75 genes that were regulated in common between these signatures. To explore the clinical relevance of the signatures we developed, we examined the NKI-295 dataset used to establish the 70-gene profile of prognosis and found 71 genes regulated in common between all four datasets (van de Vijver et al 2002). Strikingly, we discovered that patient survival was heavily influenced by the degree to which tumor expression correlated to the conserved signatures. A high degree of correlation resulted in the poorest disease free survival and an inverse correlation resulted in an improved disease free survival. Our data suggest that IGF stimulation of breast cancer cells results in distinct profiles of gene expression that are dependent on IRS adaptor protein expression. In addition, some of the “IRS-regulated” genes are shared in common with other gene signatures of poor prognosis. With the use of anti-IGF therapies in breast cancer, attention should focus on the use of these profiles as prognostic and predictive biomarkers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3031.

IGF-I mediated phosphorylation of ERαS167 occurs downstream of mTOR/p70S6K1 to impact nuclear localization and chromatin interaction.

Abstract #6012 Crosstalk between the insulin-like growth factor (IGF) I receptor (IGF-IR) and estrogen receptor alpha (ERα) enhances breast cancer cell growth and survival. Interaction between these receptors has been extensively characterized in numerous in vitro and in vivo cell models. Activation of the IGF-IR results in the recruitment of the serine/threonine kinase Akt/PKB, which has been shown to directly phosphorylate ERα at Serine167. These findings were demonstrated in transient transfection model systems. To study if IGF-IR activation of Akt/PKB results in ERα phosphorylation, we used the MCF-7L breast cancer cell line expressing both IGF-IR and ERα. In these cells, IGF-I-induced activation of the IGF-IR/PI3K/Akt axis resulting in the subsequent phosphorylation of ERα at serine 167 as early as 10 minutes and was seen up to 24 hours indicating that this was not a transient phenomenon. In addition, this event was site-specific for ser167, as IGF-I exposure did not result in the phosphorylation of the estradiol (E2)/mitogen activated protein kinase (MAPK) regulated serine 118 site of ERα. Contrary to prior reports, we observed that IGF-I induced ERαS167 phosphorylation was abolished by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). As expected, rapamycin failed to block E2 stimulated ERαS118 phosphorylation. These events were further confirmed in two additional strains of MCF-7 cells (MVLN & MCF-7ATCC). In an effort to confirm that the events responsible for IGF-I induced ERαS167 phosphorylation were distal to mTOR activation, we targeted multiple additional signaling molecules in the IGF-IR/PI3K/Akt/mTOR pathway. Blockade of PI3K (LY294002), mTOR (rapamycin) and the downstream mTOR kinase p70S6K1 (H89) resulted in a dose-dependent ablation of ERαS167 phosphorylation. In addition to blocking ERα phosphorylation, H89 specifically also inhibited eukaryotic initiation factor (eIF) 4B (eIF4B), a known immediate downstream target of activated p70S6K1. Phosphorylation of ERαS118 did not require MAPK activation, as the MEK inhibitor U0126 ablated MAPK activation and did not affect ERαS167 phosphorylation. Furthermore, we find that siRNA-mediated knockdown of p70S6K1 results in a concordant attenuation of IGF-mediated ERαS167 phosphorylation independent of the ERαS118 site. Thus, we find that phosphorylation of ERαS167 in MCF-7L cells is regulated downstream of IGF-IR by p70S6K1, a kinase known to initiate the translation of multiple mRNA transcripts. Since modification of ERα by serine phosphorylation has been shown to impact DNA binding, transcription, dimerization and coactivator recruitment, we hypothesize that IGF-IR/PI3K/Akt initiation induces ERαS167 phosphorylation through an PI3K/mTOR-specific pathway and may alter classical nuclear ERα function. More importantly, IGF/ERα crosstalk occurs at the level of post-translational modification of ERα and may serve to promote the malignant phenotype in breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6012.

Crosstalk Between Insulin-like Growth Factor (IGF) and Epidermal Growth Factor (EGF) Receptors

Growth factors induce a multitude of responses integral to development and sustained physiological function in most normal tissues. Binding of growth factors to the epidermal growth factor (EGF) and insulin-like growth factor (IGF) transmembrane tyrosine kinase receptors activates downstream intracellular signaling pathways vital to both the normal and malignant cellular phenotype. Dysregulation of either one of these receptor-regulated pathways has been linked to aberrant modulations in proliferation, motility, and protection from apoptosis. More importantly, crosstalk between EGFR and IGF-IR has been implicated in a number of cancers and correlates with tumor grade and disease progression. The following review addresses the most recent findings involving EGF and IGF receptor crosstalk and how this interaction may impact clinical therapeutic efficacy.