Research

One of the goals of our lab is to define the phosphatases dysregulated in estrogen receptor (ER)-negative breast cancer.  We have performed RNA gene profiling analysis of breast cancer samples obtained through a Susan G. Komen Promise grant (PI: Powel Brown).  In one project, we compared phosphatase expression levels in ER-positive versus ER-negative breast cancers.  Through this study, we have identified 276 phosphatase genes with differential expression levels in ER-negative breast cancers. We have determined that 20 of these phosphatases are expressed at levels ≥1.5-fold higher and 29 are expressed at levels ≤1.5-fold lower in ER-negative cancers compared to ER-positive cancers. Currently, we are focusing on those phosphatases over-expressed in ER-negative breast cancers and are testing whether knock-down of any of these phosphatases affects growth in soft agar and conventional cultures on plastic petri dishes, as well as in xenograft tumor models in nude mice. In the future, we will focus on determining those phosphatases critical for the induction and maintenance of ER-negative breast cancer by testing their ability to transform normal and pre-malignant cells and will investigate whether inhibitors of overexpressed phosphatases will be useful for the treatment of ER-negative breast cancer.

Lab members working on this project:  

Petra den Hollander, Ph.D., Abhijit Mazumdar, Ph.D., Jamal Hill, Senior Research Assistant, and Yun Zhang, Senior Research Assistant.

Inflammatory Genes Differentially Regulated in ER-negative Breast Cancer

While breast cancer remains the leading cause of cancer-related death in women, substantial progress has been made in therapies targeting estrogen receptor (ER)-positive cancer (e.g., selective estrogen receptor modulators, or SERMs) as well as in therapies targeting the overexpression of human epidermal growth factor receptor 2 (HER2).  The overexpression of HER2 and activation of hormone-related signaling are major initiating mechanisms responsible for the development and maintenance of both HER2-positive and ER-positive progesterone receptor (PR)-positive breast cancers. However, the pathways underlying the development and maintenance of ER-negative, PR-negative, HER2-negative cancer, also known as triple-negative or basal-like cancer, are unknown. Using in vitro genomic and expression profiling strategies1 in patient biopsies and basal-like breast cancer cells, we have discovered a subset of inflammatory cytokines and chemokines highly expressed and enriched in ER-negative and triple-negative tumors.  While some of these genes have been shown to play pivotal roles in other types of cancer, the significance of this cytokine/chemokine signature in human basal-like breast tumors remains unclear.  Our investigation of this pathway could provide critical understanding of the mechanisms underlying the progression of certain subtypes of basal-like cancer.  If the mechanisms that initiate these tumors and facilitate their growth, or conversely, those that prevent or hinder their inception, development and metastasis, could be defined, they could provide useful targets, diagnostics, and approaches for preventing and treating triple-negative breast cancer as well as other types of cancer with similar molecular and genetic profiles.

References:

1. Speers C, Tsimelzon A, Sexton K, Herrick AM, Gutierrez C, Culhane A, Quackenbush J, Hilsenbeck S, Chang J and Brown P. Identification of novel kinase targets for the treatment of estrogen receptor-negative breast cancer. Clinical Cancer Research 15(20): 6327-40, 2009.

Lab members working on this project:  

Zachary Hartman, Ph.D., Jamal Hill, Senior Research Assistant and Yun Zhang, Senior Research Assistant.

Targeting Death Pathways for the Prevention of ER-negative Breast Tumors

Recent studies suggest that overactivation of the Wnt pathway contributes to the pathogenesis of basal-like breast cancer, a subtype of estrogen receptor (ER)-negative breast cancer. This hormone-independent pathway leads to the nuclear localization of β-catenin where it forms a complex with T-cell factors or lymphoid enhancing factors (Tcf/Lef) and activates transcription of genes that drive proliferation.  In colon cancer, loss of the tumor suppressor gene adenomatous polyposis coli (APC) also results in nuclear localization of β-catenin.  Our collaborators have identified a chemopreventive regimen that selectively induces apoptosis in APC-deficient premalignant colon cells.  Initial treatment with retinyl acetate (RAc) in this regimen upregulates tumor necrosis-related apoptosis-inducing ligand (TRAIL) receptors.  When this is subsequently followed by treatment with TRAIL ligand, apoptosis is induced specifically in those premalignant cells with deregulated anti-apoptotic machinery.  Since loss of APC and constitutive activation of Wnt signaling both result in nuclear localization of β-catenin, we hypothesize that sequential treatment with RAc/TRAIL will induce apoptosis in Wnt-dependent premalignant breast epithelial cells.  To test this hypothesis, we are treating MMTV-Wnt1 transgenic mice which spontaneously develop both ER-positive and ER-negative mammary tumors with the RAc/TRAIL chemopreventive regimen.  This study will enable us to determine the effectiveness of this novel treatment in the prevention of ER-negative tumors.

Lab members working on this project:  

Karrie Wheatley, Ph.D., Jamal Hill, Senior Research Assistant and Yun Zhang, Senior Research Assistant.

Our research interests focus on the early detection and prevention of estrogen receptor (ER)-negative breast cancer. We are studying the mechanisms of breast cancer growth to support development of novel molecular-targets for cancer prevention and therapy.  Additionally, we are investigating tumor cell-signaling events in order to determine the biological pathways involved in tumorigenesis. It is our goal to define these signaling strategies using in vitro and in vivo models in order to develop future therapeutic agents.

Breast cancer is the most common form of cancer and is the second most common cause of cancer-related death in women. Extensive studies have been conducted to identify agents for breast cancer prevention. Results from recent clinical trials have demonstrated that anti-estrogens significantly prevent the development of ER-positive breast cancer by more than 50%.

Retinoids have been shown to prevent ER-negative mammary tumor development in animal models.  Bexarotene has been shown to partially prevent breast cancer development in mice, but has some toxic side effects. LG100268, a more selective rexinoid, has been developed and is expected to be a more effective chemopreventive agent for ER-negative breast cancer.

We have analyzed the effects of LG100268 in the MMTV-ErbB2 mouse model. The results of this study demonstrate that the rexinoid LG100268 effectively prevents ER-negative mammary tumorigenesis in these mice. Additionally, LG100268 prevents the development of pre-malignant lesions, supporting its clinical usefulness as a chemopreventive agent. These results identify LG100268 as a potentially critical therapeutic agent for the future prevention of breast cancers in women determined to be at high-risk for breast cancer.

References:

1. Wu K, Kim HT, Rodriquez JL, Hilsenbeck SG, Mohsin SK, Xu XC, Lamph WW, Kuhn JG, Green JE, Brown PH. Suppression of mammary tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069. Cancer Epidemiol Biomarkers Prev 2002; 11:467-74.
2. Wu K, Kim HT, Rodriquez JL, Munoz-Medellin D, Mohsin SK, Hilsenbeck SG, Lamph WW, Gottardis MM, Shirley MA, Kuhn JG, Green JE, Brown PH. 9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice. Clin Cancer Res 2000; 6:3696-704.
3. Wu K, Zhang Y, Xu XC, Hill J, Celestino J, Kim HT, Mohsin SK, Hilsenbeck SG, Lamph WW, Bissonette R, Brown PH. The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice. Cancer Res 2002; 62:6376-80.
4.  Li Y, Zhang Y, Hill J, Shen Q, Kim HT, Xu X, Hilsenbeck SG, Bissonnette RP, Lamph WW, Brown PH. The Rexinoid LG100268 prevents the development of preinvasive and invasive estrogen receptor negative tumors in MMTV-erbB2 mice. Clin Cancer Res 13(20): 6224-31, 2008.
5. Medina D, Kittrell F, Hill J, Zhang Y, Hilsenbeck SG, Bissonette R, Brown PH. Prevention of tumorigenesis in p53-null mammary epithelium by rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib. Cancer Prev Res (Phila). 2009 Feb;2(2):168-74. Epub 2009 Jan 27.

Lab members working on this project:  

Abhijit Mazumdar, Ph.D., Ivan Uray, Ph.D., Jamal Hill, Senior Research Assistant and Yun Zhang, Senior Research Assistant.

Our research is focused on critical endocrine pathways in human breast cancers regulated by nuclear hormone receptors that can be targeted for the treatment and prevention of breast cancer and other malignancies.  

RXR-selective retinoids (rexinoids) are promising candidates for the prevention of estrogen receptor (ER)-negative breast cancer, a disease not treatable by endocrine therapy.  As these RXRs have been shown to be effective at minimal toxicity1, a better understanding of the underlying molecular mechanisms involved with RXR-mediated gene regulation and the development of more effective candidate therapeutic compounds for ER-negative breast cancer is necessary. 

We are currently investigating the role of RXR and its associated nuclear receptors as well as the role of cellular energy metabolism in regulating breast cell proliferation and transformation.  Previously, we have described several mechanisms by which rexinoids regulate growth of pre-malignant mammary epithelial cells2.  In addition, we have performed high throughput screens using RNAi to identify nuclear receptor partners of RXR essential for the growth regulatory effect of rexinoids, as well as kinase-dependent pathways of rexinoid resistance.  

We are currently using high-content cell-based assays, gene expression profiling and molecular techniques to discover additional pathways essential for growth regulation and elimination of pre-cancerous cells. It is our goal to identify effective chemopreventive agents that can be used at reduced dosage, and to determine synergistic combinations of those compounds to eliminate the long-term toxic side effects associated with these drugs.

References:

1. Uray IP, Shen Q, Seo HS, Kim H, Lamph WW, Bissonnette RP, Brown PH. Rexinoid-induced expression of IGFBP-6 requires RARbeta-dependent permissive cooperation of retinoid receptors and AP-1. J Biol Chem 284(1):345-353, 1/2009. e-Pub 10/2008. PMCID: PMC2610495.
2. Uray IP, Brown PH. Chemoprevention of Hormone Receptor-negative Breast Cancer: New Approaches Needed. Recent Results Cancer Res 188:147-62, 2011. PMID: 21253797.

Lab members working on this project:

Ivan Uray, M.D., Ph.D. and Abhijit Mazumdar, Ph.D.

Our lab is interested in targeting cancer stem cells for the prevention of breast cancer.  Recent research in breast biology supports the cancer stem cell hypothesis, which asserts that malignancies arise in tissue stem cells through dysregulation of processes involved in self-renewal.  This hypothesis carries important implications for the early detection, prevention and treatment of breast cancer.  

Previous studies have demonstrated the importance of retinoid signaling in the regulation of self-renewal and differentiation of normal breast stem cells. Furthermore, several molecularly targeted agents, including RXR-selective retinoids (rexinoids), have been shown to prevent the development of estrogen receptor (ER)-negative breast cancer in transgenic mouse models. Bexarotene and LG100268 are two such rexinoids that have been shown to selectively bind RXR receptors.  Based on the effects of these molecularly targeted agents, we hypothesize that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population.  This work is supported by a grant from the Breast Cancer Research Foundation.

Lab members working on this project:  

Jing Zhao, Karrie Wheatley, Ph.D., Jamal Hill, Senior Research Assistant and Yun Zhang, Senior Research Assistant.

Our lab has discovered that kinase activation defines a number of clinically prognostic ER-negative patient subgroups. This study focuses on a specific kinase subset, the mitogen-activated protein kinase (MAPK) family of proteins.

MAPKs are an essential group of molecules that transduce both extracellular and intracellular stimuli. These signals are passed to the nucleus where they regulate cell growth and cell death. Since MAPKs are critical for cellular development, malignant cells commonly exhibit alterations within this pathway that significantly increase their growth. We hypothesize that there is a set of MAPKs critical for the growth of ER-negative breast cancers.

Using RNAi and small molecule inhibitors, we are investigating whether the targeting of these kinases affect biological characteristics including proliferation, apoptosis, migration, and invasion. We also utilize mouse models in order to understand the in vivo effects of kinase inhibition. By defining novel targets and using combinatorial strategies, we seek to uncover effective treatments for this deadly form of breast cancer.

References:

1. Speers C, Tsimelzon A, Sexton K, Herrick AM, Gutierrez C, Culhane A, Quackenbush J, Hilsenbeck S, Chang J and Brown P. Identification of novel kinase targets for the treatment of estrogen receptor-negative breast cancer. Clinical Cancer Research 15(20): 6327-40, 2009.

Lab members working on this research:

Graham Poage, Ph.D.

The long term goal of our research is to develop novel and effective therapies to prevent and treat breast cancer. Over the last few years we have focused on the identification of promising molecular targets for this purpose. One of these potential targets is the activator protein 1 (AP-1) transcription factor, which we have demonstrated to be an important regulator of breast cell growth. Further investigation showed that loss of AP-1 activity by a specific AP-1 inhibitor (Tam67, a cJun dominant negative mutant) can suppress growth factor-induced breast cancer cell proliferation and arrest cell cycle progression at the G1 phase.

Estrogen receptors, members of the nuclear receptor super family, mediate most of the effects of estrogen in breast cancer. Estrogen stimulation of gene expression is complex and involves several different mechanisms. Our lab focuses on non-classical ER pathways that use TF crosstalk.

We have demonstrated that ER-AP-1 crosstalk is critical for breast cell proliferation, and through microarray analysis we have identified genes dependent upon AP-1 and estrogen signaling. Currently, we are investigating the mechanism by which ER and AP-1 regulate expression of one of these genes, c-myc.

c-myc is an oncogene that encodes a nuclear transcription factor. The cMyc protein regulates expression of a variety of target genes which control cell cycle progression, apoptosis, and cellular transformation. Deregulated expression of c-myc is observed in many cancers and is associated with poor prognosis. c-myc is frequently amplified and overexpressed in breast cancer and previous studies have shown that c-myc is a potential target for breast cancer prevention and treatment.  We have identified a novel pathway in which ER and AP-1 transcription factor crosstalk at a distant enhancer element is involved in estrogen induction of c-myc expression. This alternative pathway of estrogen-regulated gene expression may be targeted in the future to improve prevention and treatment strategies of breast cancer.

The results from these studies provide the scientific rationale to develop drugs targeting inhibition of AP-1 or its downstream molecules for the prevention or treatment of breast cancer.

References:

1. Liu Y, Ludes-Meyers J, Zhang Y, Munoz-Medellin D, Kim HT, Lu C, Ge G, Schiff R, Hilsenbeck SG, Osborne CK, Brown PH. Inhibition of AP-1 transcription factor causes blockade of multiple signal transduction pathways and inhibits breast cancer growth. Oncogene, 21: 7680-7689, 2002.
2. Liu Y, Lu C, Shen Q, Munoz-Medellin D, Kim H, Brown PH. AP-1 blockade in breast cancer cells causes cell cycle arrest by suppressing G1 cyclin expression and reducing cyclin-dependent kinase activity. Oncogene, 23: 8238-8246, 2004.
3. DeNardo DG, Kim HT, Hilsenbeck S, Cuba V, Tsimelzon A, Brown PH. Global gene expression analysis of estrogen receptor transcription factor cross talk in breast cancer: identification of estrogen-induced/activator protein-1-dependent genes. Mol Endocrinol, 19: 362-378, 2005.
4. Shen Q, Zhang Y, Uray IP, Hill JL, Kim HT, Lu C, Young MR, Gunther EJ, Hilsenbeck SG, Chodosh LA, Colburn NH, Brown PH. The AP-1 transcription factor regulates postnatal mammary gland development. Dev Biol, 295: 589-603, 2006.
5. Shen Q, Uray IP, Li Y, Zhang Y, Hill J, Xu XC, Young MR, Gunther EJ, Hilsenbeck SG, Colburn NH, Chodosh LA, Brown PH. Targeting the activator protein 1 transcription factor for the prevention of estrogen receptor-negative mammary tumors. Cancer Prev Res (Phila Pa). 2008 Jun;1(1):45-55.
6. Wang C, Mayer JA, Mazumdar A, Fertuck K, Kim H, Brown M, Brown PH. Estrogen induces c-myc gene expression via an upstream enhancer activated by estrogen receptor and activating protein-1 transcription factor. Mol Endocrinol, 25(9):1527-38, 2011.

Lab members working on this research:

Chunyu Wang, Ph.D., and Jonathan Shepherd.

RNA transcription profiling has enabled  breast tumors to be defined in distinct subtypes (basal, luminal and HER2) which possess unique prognoses and treatment options. Key regulators, such as estrogen receptor alpha and HER2, have been identified for the luminal and HER2 subtypes, while master regulators for the basal tumors remain unknown.  Recent studies have shown inherent distinctions between basal and luminal breast cancers at the transcriptional level. Since transcription factors (TFs) are able to directly regulate RNA transcription of multiple genes by binding cis-regulatory elements of DNA, resulting in the coordinated response of an entire gene set, TFs are promising candidates as master regulators of basal breast cancer. 

We hypothesize that specific TFs regulate the transcriptional profile related to basal breast cancer and the aggressive proliferation associated it. To test this hypothesis, we are using three independent screening approaches to identify specific TFs critical for the regulation of basal breast cancer.  

First, we are identifying TF binding motifs which are over-represented among promoters of genes over-expressed in basal tumors. Second, we are querying breast cancer protein to identify TFs which bind labeled DNA probes in basal cell lines to a higher degree than in luminal cell lines. Finally, due to the open chromatin conformations characteristic of the regulatory regions bound by TFs, we are using formaldehyde-assisted isolation of regulatory elements (FAIRE) to identify regions of open chromatin in basal cells as well as the TFs which bind these regions. Candidate TFs identified through these screening techniques will be selected for further studies to define their roles in the regulation of basal gene expression and to ascertain which TFs are critical for the proliferation of basal breast cancer cells.

Identifying TFs which act as master regulators of the basal subtype will increase our understanding of the biology of basal-like tumors and, more importantly, will identify potential targets for future therapeutic strategies to treat this aggressive type of breast cancer.

References:

1. Liu Y, Ludes-Meyers J, Zhang Y, Munoz-Medellin D, Kim HT, Lu C, Ge G, Schiff R, Hilsenbeck SG, Osborne CK, Brown PH. Inhibition of AP-1 transcription factor causes blockade of multiple signal transduction pathways and inhibits breast cancer growth. Oncogene, 21: 7680-7689, 2002.
2. Liu Y, Lu C, Shen Q, Munoz-Medellin D, Kim H, Brown PH. AP-1 blockade in breast cancer cells causes cell cycle arrest by suppressing G1 cyclin expression and reducing cyclin-dependent kinase activity. Oncogene, 23: 8238-8246, 2004.

Lab members working on this project:

Jonathan Shepherd, Chunyu Wang, Ph.D., Jamal Hill, Senior Research Assistant and Yun Zhang, Senior Research Assistant.