Adrian V. Lee

Expression of Autotaxin and Lysophosphatidic Acid Receptors Increases Mammary Tumorigenesis, Invasion, and Metastases

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Crosstalk between the insulin-like growth factors and estrogens in breast cancer.

Once it was recognized that breast tumor growth was stimulated by estrogens, successfultherapeutic strategies based on depriving the tumor of this hormone were developed. Sincethe growth stimulatory properties of the estrogens are governed by the estrogen receptor (ER),4understanding the mechanisms that activate ER are highly relevant. In addition to estrogens,peptide growth factors can also activate the ER. The insulin-like growth factors (IGFs) arepotent mitogens for ER-positive breast cancer cell lines. This review will examine the evidencefor interaction between these two pathways. The IGFs can activate the ER, while ERtranscriptionally regulates genes required for IGF action. Moreover, blockade of ER function can inhibitIGF-mediated mitogenesis and interruption of IGF action can similarly inhibit estrogenicstimulation of breast cancer cells. Taken together, these observations suggest that the twogrowth regulatory pathways are tightly linked and that a further understanding of the mechanismof this crosstalk could lead to new therapeutic strategies in breast cancer.

AIB1/SRC-3 Deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice

Although the amplified in breast cancer 1 (AIB1) coactivator is amplified and overexpressed in breast cancers, its role in mammary carcinogenesis remains unknown. We demonstrate that during mammary development and tumorigenesis, the elevation of AIB1 level and its nuclear localization correlate with normal and transformed mammary epithelial proliferation, whereas its lower expression and cytoplasmic localization correlate with mammary epithelial quiescence and differentiation. In this study, the role of AIB1 in breast tumor initiation, progression, and metastasis was studied by generating AIB1+/+, AIB1+/−, and AIB1−/− mice harboring the mouse mammary tumor virus/v-Ha-ras (ras) transgene that induces breast tumors. Breast tumor incidence was reduced dramatically in the intact AIB1−/−-ras virgin mice and inhibited completely in the ovariectomized AIB1−/−-ras mice. Breast tumor latency was delayed significantly in AIB1−/−-ras virgin mice with natural estrous cycles, multiparous mice with cyclically elevated reproductive hormones, and virgin mice bearing pituitary isografts with persistently elevated hormones. Although AIB1 deficiency significantly suppressed mammary tumorigenesis under all of the concentrations of ovarian hormones, it did not affect the promotional role of ovarian hormones on mammary tumorigenesis, suggesting that AIB1 and ovarian hormones contribute to mammary carcinogenesis through different pathways. AIB1 deficiency did not alter the expression of estrogen and progesterone-responsive genes in the mammary gland, but it caused partial resistance to the insulin-like growth factor I because of a significant reduction in the insulin receptor substrates. The impaired insulin-like growth factor I pathway in AIB1−/−-ras mammary epithelium and tumor cells was responsible in part for the suppression of mammary tumorigenesis and metastasis caused by inhibition of cell proliferation and migration. These results suggest that a more effective strategy to control breast cancer is to target AIB1-mediated and ovarian hormone-initiated pathways.

Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-κB and Snail

ABSTRACT Type I insulin-like growth factor receptor (IGF-IR) can transform mouse fibroblasts; however, little is known about the transforming potential of IGF-IR in human fibroblasts or epithelial cells. We found that overexpression of a constitutively activated IGF-IR (CD8-IGF-IR) was sufficient to cause transformation of immortalized human mammary epithelial cells and growth in immunocompromised mice. Furthermore, CD8-IGF-IR caused cells to undergo an epithelial-to-mesenchymal transition (EMT) which was associated with dramatically increased migration and invasion. The EMT was mediated by the induction of the transcriptional repressor Snail and downregulation of E-cadherin. NF-κB was highly active in CD8-IGF-IR-MCF10A cells, and both increased levels of Snail and the EMT were partially reversed by blocking NF-κB or IGF-IR activity. This study places IGF-IR among a small group of oncogenes that, when overexpressed alone, can confer in vivo tumorigenic growth of MCF10A cells and indicates the hierarchy in the mechanism of IGF-IR-induced EMT.

Proteomic and transcriptomic profiling reveals a link between the PI3K pathway and lower estrogen-receptor (ER) levels and activity in ER+ breast cancer.

IntroductionAccumulating evidence suggests that both levels and activity of the estrogen receptor (ER) and the progesterone receptor (PR) are dramatically influenced by growth-factor receptor (GFR) signaling pathways, and that this crosstalk is a major determinant of both breast cancer progression and response to therapy. The phosphatidylinositol 3-kinase (PI3K) pathway, a key mediator of GFR signaling, is one of the most altered pathways in breast cancer. We thus examined whether deregulated PI3K signaling in luminal ER+ breast tumors is associated with ER level and activity and intrinsic molecular subtype.MethodsWe defined two independent molecular signatures of the PI3K pathway: a proteomic (reverse-phase proteomic array) PI3K signature, based on protein measurement for PI3K signaling intermediates, and a PI3K transcriptional (mRNA) signature based on the set of genes either induced or repressed by PI3K inhibitors. By using these signatures, we scored each ER+ breast tumor represented in multiple independent expression-profiling datasets (four mRNA, n = 915; one protein, n = 429) for activation of the PI3K pathway. Effects of PI3K inhibitor BEZ-235 on ER expression and activity levels and cell growth were tested by quantitative real-time PCR and cell proliferation assays.ResultsWithin ER+ tumors, ER levels were negatively correlated with the PI3K activation scores, both at the proteomic and transcriptional levels, in all datasets examined. PI3K signature scores were also higher in ER+ tumors and cell lines of the more aggressive luminal B molecular subtype versus those of the less aggressive luminal A subtype. Notably, BEZ-235 treatment in four different ER+ cell lines increased expression of ER and ER target genes including PR, and treatment with IGF-I (which signals via PI3K) decreased expression of ER and target genes, thus further establishing an inverse functional relation between ER and PI3K. BEZ-235 had an additional effect on tamoxifen in inhibiting the growth of a number of ER+ cell lines.ConclusionsOur data suggest that luminal B tumors have hyperactive GFR/PI3K signaling associated with lower ER levels, which has been correlated with resistance to endocrine therapy. Targeting PI3K in these tumors might reverse loss of ER expression and signaling and restore hormonal sensitivity.

Growth Hormone and Insulin-Like Growth Factor-I in the Transition from Normal Mammary Development to Preneoplastic Mammary Lesions

Adult female mammary development starts at puberty and is controlled by tightly regulated cross-talk between a group of hormones and growth factors. Although estrogen is the initial driving force and is joined by luteal phase progesterone, both of these hormones require GH-induced IGF-I in the mammary gland in order to act. The same group of hormones, when experimentally perturbed, can lead to development of hyperplastic lesions and increase the chances, or be precursors, of mammary carcinoma. For example, systemic administration of GH or IGF-I causes mammary hyperplasia, and overproduction of IGF-I in transgenic animals can cause the development of usual or atypical hyperplasias and sometimes carcinoma. Although studies have clearly demonstrated the transforming potential of both GH and IGF-I receptor in cell culture and in animals, debate remains as to whether their main role is actually instructive or permissive in progression to cancer in vivo. Genetic imprinting has been shown to occur in precursor lesions as early as atypical hyperplasia in women. Thus, the concept of progression from normal development to cancer through precursor lesions sensitive to hormones and growth factors discussed above is gaining support in humans as well as in animal models. Indeed, elevation of estrogen receptor, GH, IGF-I, and IGF-I receptor during progression suggests a role for these pathways in this process. New agents targeting the GH/IGF-I axis may provide a novel means to block formation and progression of precursor lesions to overt carcinoma. A novel somatostatin analog has recently been shown to prevent mammary development in rats via targeted IGF-I action inhibition at the mammary gland. Similarly, pegvisomant, a GH antagonist, and other IGF-I antagonists such as IGF binding proteins 1 and 5 also block mammary gland development. It is, therefore, possible that inhibition of IGF-I action, or perhaps GH, in the mammary gland may eventually play a role in breast cancer chemoprevention by preventing actions of both estrogen and progesterone, especially in women at extremely high risk for developing breast cancer such as BRCA gene 1 or 2 mutations.

Insulin-Like Growth Factor I-Induced Degradation of Insulin Receptor Substrate 1 Is Mediated by the 26S Proteasome and Blocked by Phosphatidylinositol 3′-Kinase Inhibition

ABSTRACT Insulin receptor substrate 1 (IRS-1) is a critical adapter protein involved in both insulin and insulin-like growth factor (IGF) signaling. Due to the fact that alteration of IRS-1 levels can affect the sensitivity and response to both insulin and IGF-I, we examined the ability of each of these ligands to affect IRS-1 expression. IGF-I (10 nM) stimulation of MCF-7 breast cancer cells caused a transient tyrosine phosphorylation of IRS-1 that was maximal at 15 min and decreased thereafter. The decrease in tyrosine phosphorylation of IRS-1 was paralleled by an apparent decrease in IRS-1 levels. The IGF-mediated decrease in IRS-1 expression was posttranscriptional and due to a decrease in the half-life of the IRS-1 protein. Insulin (10 nM) caused tyrosine phosphorylation of IRS-1 but not degradation, whereas high concentrations of insulin (10 μM) resulted in degradation of IRS-1. IGF-I (10 nM) stimulation resulted in transient IRS-1 phosphorylation and extracellular signal-related kinase (ERK) activation. In contrast, insulin (10 nM) caused sustained IRS-1 phosphorylation and ERK activation. Inhibition of 26S proteasome activity by the use of lactacystin or MG132 completely blocked IGF-mediated degradation of IRS-1. Furthermore, coimmunoprecipitation experiments showed an association between ubiquitin and IRS-1 that was increased by treatment of cells with IGF-I. Finally, IGF-mediated degradation of IRS-1 was blocked by inhibition of phosphatidylinositol 3′-kinase activity but was not affected by inhibition of ERK, suggesting that this may represent a direct negative-feedback mechanism resulting from downstream IRS-1 signaling. We conclude that IGF-I can cause ligand-mediated degradation of IRS-1 via the ubiquitin-mediated 26S proteasome and a phosphatidylinositol 3′-kinase-dependent mechanism and that control of degradation may have profound effects on downstream activation of signaling pathways.

A sequence-level map of chromosomal breakpoints in the MCF-7 breast cancer cell line yields insights into the evolution of a cancer genome

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.

Thioredoxin-like 2 regulates human cancer cell growth and metastasis via redox homeostasis and NF-κB signaling

Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.

High IGF-IR Activity in Triple-Negative Breast Cancer Cell Lines and Tumorgrafts Correlates with Sensitivity to Anti–IGF-IR Therapy

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.