Powel Brown Laboratory

Research Projects

   

   
Studies of Signal Transduction
Targeting STATs
Targeting MAPK
Tyrosine Kinase Inhibitors
Retinoids and Nuclear Hormonal Receptors
 
RXR partner receptors
Mechanisms by which Rexinoids
 
 

 

 

Identification of Novel Targets for the Treatment of Estrogen Receptor-Negative Breast Cancer

Breast cancer is the number one cause of death in women between the ages 40 and 55.  About 60% of breast cancer is estrogen receptor (ER)-positive and can be treated by selective estrogen receptor modulators (SERMs). ER-negative cancers, which comprise up to 40% of breast cancers, are currently treated with toxic chemotherapy that targets rapidly proliferating cells with minimal specificity.  It is imperative that specific, effective, and less toxic treatments be developed to effectively treat or prevent ER-negative breast cancer. The purpose of this project is to use genomic technologies, including genomic RNA profiling as well as proteomic profiling, to identify the genes and proteins responsible for ER-negative tumor growth.  Using Affymetrix microarray gene expression profiling we are currently profiling ER-negative and ER-positive human breast cancers and identifying a list of kinases and phosphatases that are differentially expressed between these two.  These experiments have provided a list of kinases differentially expressed between ER positive and negative tumors.  Studies are underway to investigate the roles these genes play in the biology of breast cancer.  Another aim of this project is to use reverse-phase protein lysate arrays to identify critical growth regulatory molecules in ER-negative breast cancer at the protein level.  The identification of these kinases will help us better understand the biology of ER-negative breast cancer and may lead to the identification of druggable targets for the treatment of this deadly disease.

We are currently recruiting postdoctoral fellows to investigate the biologic function of these novel genes and to develop and test inhibitors of these molecules for the treatment of ER-negative breast cancer.
Elevated kinases in ER-negative human breast tumors

References:

  1. Speers, et. al., “Identification of Novel Targets for the Treatment of Estrogen Receptor-Negative Breast Cancer”.  Abstract, Molecular Targets in Cancer Meeting, March 2007
  2. Lu, et. al., “Inhibition of the p38 Kinase Supresses the Proliferation of ER-Negative Human Breast Cancer Cells", April 2007, submitted

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Studies on the Cancer Preventive Mechanisms Invoked by Rexinoids

Rexinoids, the class of retinoids selectively binding to the retinoid X receptor (RXR), are promising agents for the prevention of breast cancer. We have previously shown that the RXR-selective retinoid bexarotene (LGD1069, Targretin) prevents the development of ER-negative mammary tumors in ErbB2 overexpressing transgenic mice (Figure 1.)(1). Bexarotene is currently tested in a phase I clinical trial for the prevention of breast cancer. Rexinoids activate or modulate the activity of nuclear hormone receptor heterodimers that involve an RXR component. These complexes, in turn, regulate the expression of genes of growth inhibition and apoptosis (Figure 2.).
Gene expression profiling studies revealed a number of genes (Figure 3.) that undergo concerted regulation by rexinoids both in human and mouse tissues (2). In depth investigation of the transcriptional mechanisms involved in the regulation of one of these genes, IGF binding protein 6, identified a novel synergistic mechanism between retinoid receptors and the AP-1 family of transcription factors (3).
To develop more effective chemopreventive agents the transcriptional mechanisms mediating the growth suppressive effects of currently used rexinoids need to be investigated. To identify the nuclear hormone receptors partnering RXR that are involved in mediating the cancer preventive activity of RXR-selective ligands we utilize RNA interference technology in high-throughput screening experiments coupled with high-content single cell assays in mammary epithelial cells.
Studies on the cancer preventive mechanisms invoked by rexiniods - figures 1-3

References:

  1. Wu K. et al. The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice. Cancer Res.62(22):6376-80, 2002.
  2. Kim HT. et al. Identification of biomarkers modulated by the rexinoid LGD1069 (bexarotene) in human breast cells using oligonucleotide arrays. Cancer Res. 66(24):12009-18, 2006.
  3. Liby K, Rendi M, Suh N, Royce DB, Risingsong R, Williams CR, Lamph W, Labrie F, Krajewski S, Xu X, Kim H, Brown PH, Sporn MB. The combination of the rexinoid, LG100268, and a selective estrogen receptor modulator, either arzoxifene or acolbifene, synergizes in the prevention and treatment of mammary tumors in an estrogen receptor-negative model of breast cancer. Clin Cancer Res. 12(19):5902-9, 2006.
  4. Uray I. et al. Synergistic Activity of AP-1 and RXR Mediates Retinoid-Dependent Regulation of the IGF Binding Protein-6 Gene (in preparation).
  5. 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. 15;13(20):6224-31, 2007.
  6. Li Y, Brown PH. Translational approaches for the prevention of estrogen receptor-negative breast cancer. Eur J Cancer Prev. 16(3):203-15, 2007.Review.

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Mechanisms by Which Rexinoids Prevent Cancer

ER-negative breast cancer, which accounts for one third of total breast cancers, has a poor prognosis and is poorly understood.  To prevent the development of ER-negative breast cancer, a number of chemopreventive agents targeting on non-endocrine signaling pathways are being studied.  Among the numerous chemopreventive agents for ER-negative breast cancer tested in preclinical studies to date, no agents are more effective than rexinoids, which prevent the ER-negative breast cancer by 95% in our preclinical studies.  Distinct from retinoids, which bind primarily to RAR, rexinoids bind specifically to RXR, a promiscuous protein that is able to form dimmers with RAR and many other nuclear receptors.  This allows rexinoids to prevent cancer though a multidisciplinary mechanism.   Our current research is focused on understanding the molecular mechanism of rexinoids.  We are investigating how rexinoids prevent cancer and regulate target genes.   Elucidating these mechanisms will help us identify novel target to develop more effective and less toxic anti-cancer agents.  

These studies are funded by two R01 grants.  We are currently recruiting for Postdoctoral fellows to investigate the molecular mechanisms by which rexinoids prevent cancer.
mechanisms by which rexiniods prevent cancer, figures 1-2

Reference:

  1. Wu, K., et al., Receptor-selective retinoids inhibit the growth of normal and malignant breast cells by inducing G1 cell cycle blockade. Breast Cancer Res Treat, 2006. 96(2): p. 147-57.
  2. Wu, K., et al., Suppression of mammary tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069. Cancer Epidemiol Biomarkers Prev, 2002. 11(5): p. 467-74.
  3. Wu, K., et al., 9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice. Clin Cancer Res, 2000. 6(9): p. 3696-704.
  4. Wu, K., et al., The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice. Cancer Res, 2002. 62(22): p. 6376-80.
  5. 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. 15;13(20):6224-31, 2007.
  6. Li Y, Zhang Y, Hill J, Kim HT, Shen Q, Bissonnette RP, Lamph WW, Brown PH. The rexinoid, bexarotene, prevents the development of premalignant lesions in MMTV-erbB2 mice. Br J Cancer. 2008 Mar 25; [Epub ahead of print]

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Targeting Transcription Factors for the Treatment and Prevention of Breast Cancer

Long term goal of our research is to develop effective therapies to prevent and treat breast cancer.  Over the last few years we have focused on identification of promising molecular targets for the prevention and treatment of breast cancer.  We have demonstrated that the AP-1 transcription factor is an important regulator of breast cell growth.  We found that AP-1 blockade by a specific AP-1 inhibitor (Tam67, a cJun dominant negative mutant) suppresses growth factor-induced breast cancer cell proliferation and arrests cell cycle progression at G1 phase.  We also demonstrated that AP-1 factors cross-talks with ER, which is also critical for breast cell proliferation.  Cyclin D1 is an important cell cycle regulator controlling proliferation of both normal and malignant breast cells, and we recently found that AP-1 activates cyclin D1 expression by direct and indirect mechanisms.  We will next conduct studies of activators and repressors of the AP-1 complexes in gene regulation and tumorigenesis.

To determine whether AP-1 regulates mammary gland development, we created transgenic mouse models that have mammary-specific induction of an AP-1 inhibitor, Tam67.  We studied the effect of in vivo blockade of AP-1 in mammary gland and found that AP-1 regulates mammary gland development at the prepubertal, pubertal, adult, and hormone-stimulated mammary glands.  These developmental alterations by AP-1 blockade are caused by reduced proliferation and expression of many AP-1-regulated genes.  Our next goal is to develop strategies to prevent ER-positive and ER-negative breast cancers.  We created triple transgenic mouse models to determine whether AP-1 blockade in the mammary glands blocks mammary tumorigenesis.  We have completed the MMTV-erbB2 model and found that AP-1 blockade delayed and partially prevented ER-negative tumor formation.  We are now investigating the effect of AP-1 blockade on transformation induced by other oncogenes such as cMyc and Wnt-1.  Dedicated postdoctoral fellows are being recruited to study the role of AP-1 in gene regulation and transformation, in in vitro and in vivo systems.
Targeting transcription factors for the treatment and prevention of breast cancer, figures 1-4

References:

  1. Liu, Y., Lu, C., Shen, Q., Munoz-Medellin, D., Kim, H., and Brown, P. H. 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.
  2. Liu, Y., Ludes-Meyers, J., Zhang, Y., Munoz-Medellin, D., Kim, H. T., Lu, C., Ge, G., Schiff, R., Hilsenbeck, S. G., Osborne, C. K., and Brown, P. H. Inhibition of AP-1 transcription factor causes blockade of multiple signal transduction pathways and inhibits breast cancer growth. Oncogene, 21: 7680-7689, 2002.
  3. DeNardo, D. G., Kim, H. T., Hilsenbeck, S., Cuba, V., Tsimelzon, A., and Brown, P. H. 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, I. P., Hill, J. L., Kim, H. T., Lu, C., Young, M. R., Gunther, E. J., Hilsenbeck, S. G., Chodosh, L. A., Colburn, N. H., and Brown, P. H. The AP-1 transcription factor regulates postnatal mammary gland development. Dev Biol, 295: 589-603, 2006.

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Mechanisms & Estrogen-regulated Gene Expression

Estrogen (E2) is a critical mitogen for breast cancer cells that modulates gene expression, mostly through regulation of transcription by interaction with estrogen receptors (ERα and β). Determining the mechanisms by which estrogen regulates gene expression is important for understanding the role of estrogen in breast cancer development and progression.  Identification of the genes that are modulated by E2 may lead to new therapeutic targets for breast cancer prevention and treatment. Using genomic microarray analysis we and others have shown that over two-thirds of the genes modulated by E2 are down-regulated. Little is known about the mechanisms by which estrogen down regulates these target genes. We hypothesize that estrogen down-regulates gene expression by one of several mechanisms, including: 1) Recruitment of co-repressors:  ER, when bound by estrogen, binds DNA and recruits co-repressors 2) Transcription factor interference:  ER, when bound by estrogen, interacts with other transcription factors (such as AP-1, Sp1) and interferes with their ability to recruit co-activators, or induces recruitment of co-repressors 3) Co-activator sequestration:  ER, when bound by estrogen, binds co-activators making them unavailable for other transcription factors. Using microarray analysis we have already identified a set of estrogen-down-regulated genes. Currently, we are elucidating the mechanisms by which these genes are suppressed by estrogen. Using chromatin immunoprecipitation experiments, we are identifying the important regulators that shut off transcription. Most of these mechanisms involve transcription factor cross-talk and HDACs are necessary for de-acetylation of these promoters.  Preliminary results demonstrate that the loss of cell cycle regulators, apoptotic regulators, and signal transduction proteins following estrogen induced down-regulation is critical for the basic biology of breast cancer. These studies have led to additional projects studying the mechanism by which estrogen and other steroid hormones regulate gene expression. Available projects include studies of specific estrogen down-regulated genes or proteins in regulating breast cell growth, transformation, or metastasis.
Mechanisms & Estrogen-regulated Gene Expression, figures 1-2

References:

  1. Denardo DG, Kim HT, Hilsenbeck S, Cuba V, Tsimelzon A, Brown PH. Global gene expression analysis of ER transcription factor cross-talk in breast cancer:  Identification of estrogen-induced/AP-1-dependent genes.  Molecular Endocrinology, 19: 362-378, 2005
  2. DeNardo DG, Cuba VL, Kim HT, Wu K, Lee AV, Brown PH. Estrogen receptor DNA binding is not required for estrogen-induced breast cell growth. Molecular and Cellular Endocrinology, submitted.

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Targeting STATs for the Treatment and Prevention of Breast Cancer

STATs (Signal Transducers and Activators of Transcription) are a family of transcription factors involved in the regulation of early embryonic development, the immune response, cell proliferation, differentiation and apoptosis.  In the mammary gland STAT1 regulates remodeling of the mammary gland during involution, STAT3 regulates lobuloalveolar apoptosis during involution, and STAT5 regulates lobuloalveolar proliferation, differentiation and expansion.  A sizable body of data also implicates STATs in the oncogenesis of breast cancer.  STAT3 in particular is constitutively activated in 30 to 60% of primary human breast cancers.  In an ongoing project we are investigating the role of STAT3 in the development and progression of estrogen receptor (ER) negative breast cancer.  Recently, increased STAT3 transcriptional activity was found to correlate with ER negativity in breast cancer cells in primary human invasive ductal carcinomas.  This suggests that high STAT3 transcriptional activity may be important to the development and or progression of ER negative human breast cancers.  In our laboratory we are currently investigating the necessity of STAT3 transcriptional activity to the tumorogenicity of ER negative breast cancer cells, the correlation between high STAT3 transcriptional activity in human breast carcinomas and its association with other prognostic markers, and the importance of STAT3 transcriptional activity to the development and progression of ER negative breast cancer in mouse models.  Projects are available for interested students or postdoctoral fellows to investigate the role of this and other STAT proteins in regulating breast cell growth, transformation, and metastasis.
STAT3 Expression and Transcriptional Activation on Human Breast Cancer Cells

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Targeting Signaling Transduction Molecules for the Treatment of ER-negative Breast Cancer

Targeting Siganlling transduction Molecules for the treatment of ER-negative Breast CancerThere is an urgent need to identify and test agent that will effectively treat ER-negative breast cancer.  While ER-negative breast cells do not respond to estrogen, they do require various growth factors.  Therefore, molecules that transduce growth factor signals can be promising targets for the treatment of ER-negative breast cancer.  p38s are members of the mitogen-activated protein kinase (MAPK) family that transduce signals from various environmental stresses, growth factors and steroid hormones that control gene regulation and cell growth.  Studies have shown that p38s are highly expressed in aggressive, invasive breast cancers, and that increased levels of activated MAPKs are markers of poor prognosis.  It is our hypothesis that blockade of p38 signaling will inhibit breast cell growth and suppress tumorigenesis.

We are currently investigating whether blockade of p38 signaling will suppress ER-negative breast cancer cell growth in vitro and in vivo.  In addition, we are conducting global genomic studies to identify biomarkers that predict responses to p38 inhibitors.  These studies will help us evaluate the role of p38 in ER-negative breast cancer cell growth, understand the mechanisms by which p38 signaling regulates cell growth, and provide the foundation for the development of p38 inhibitors for the treatment of ER-negative breast cancers.

These studies are funded by a Breast Cancer SPORE grant, a grant from the Breast Cancer Research Foundation, and a grant from AstraZeneca.  We are currently recruiting post-doctoral fellows to work on these projects.

Reference:

  1. Chen L., Krisko T.I., and Brown P.H. Blockade of mitogen-activated protein kinase (MAPK) signaling inhibits the proliferation of normal and malignant breast cells. Proceedings of the American Association for Cancer Research, 2006
  2. Chen L., Krisko T.I., Speers C.W., and Brown P.H. Inhibition of the p38 Kinase Suppresses the Proliferation of ER-Negative Human Breast Cancer Cells.  Manuscript in prep.

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Prevention of Breast Cancer Using Tyrosine Kinase Inhibitors

We are investigating whether signal transduction inhibitors are useful for the prevention of
ER-negative breast cancer. In our studies we have previously shown that gefitinib, an oral
single inhibitor of ErbB1 (EGFR) and Lapatinib, an oral dual inhibitor of ErbB1 and ErbB2
(HER2) tyrosine kinases, both prevent the development of ER-negative breast cancer in
MMTV-ErbB2 mice (1,2,3). The results of our current studies  indicate that Lapatinib
effectively blocks EGF-induced signaling through the ErbB1 and ErbB2 receptors and
inhibits cell cycle progress, suppresses cyclin D and epiregulin expression and stimulates
P27 expression. These studies demonstrate that TKIs are promising agents for the
prevention and treatment of ER-negative breast cancer. This project has lead our lab to
open a clinical trial to assess whether Lapatinib will be an effective agent in the prevention
of breast cancer in patients with DCIS breast cancer.  This work is supported by a Breast
Cancer SPORE grant, and a Breast Cancer Research Foundation grant.   Available
projects in this area involve characterizing new signal transduction molecules that are
critical for ER-negative breast tumorigenesis and development and testing of inhibitors to prevent breast cancer.
Pevention of Breast Cancer Using Tyrosine Kinase Inhibitors

References:

  1. Lu C, Speers C, Zhang Y, Xu X, Hill J, Steinbis E, Celestino J, Shen Q, Kim H, Hilsenbeck S, Mohsin SK, Wakeling A, Osborne CK, Brown PH.  Effect of epidermal growth factor receptor inhibitor on development of estrogen receptor-negative mammary tumors.  JNCI. 2003 Dec 17; 95(24):1825-33
  2. Lu C et. al. JNCI. J Natl Cancer Inst. 2004 May 5;96(9): 715-6. Author Reply
  3. Strecker TE, Zhang Y, Hill JL, Kim H, Hilsenbeck SG, Gilmer T, and Brown PH . Lapatinib, an oral dual inhibitor of ErbB1 and ErbB2, blocks signal transduction pathways and suppresses the development of Estrogen-Receptor-negative breast cancer. SABCS 2006

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Research questions? Contact Dr. Brown by email.


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