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MMP-3-Induced Epithelial-Mesenchymal Transition and Genomic Instability
Posted October 18, 2005
Mina Bissell, Ph.D., Lawrence Berkeley National Laboratory
Hong Liu, Lawrence Berkeley National Laboratory[*]
Celeste M. Nelson, Ph.D., Lawrence Berkeley National Laboratory[†]

In recent years, many cancer researchers have focused on identifying and studying oncogenes. But compelling evidence indicates that the microenvironment of epithelial cells plays a critical role in the responses of normal and malignant cells to stimuli (including therapeutic agents), and when the extracellular context is altered, regulatory pathways signal differently. The proteolytic enzymes known as matrix metalloproteinases (MMPs) are major contributors to these microenvironmental signals because they degrade structural components of the extracellular matrix, making tumor invasion and metastasis possible. Dr. Mina Bissell, an FY01 Innovator Award recipient, and her colleagues[* †] have previously shown that stromelysin-1/MMP-3 (MMP-3), a stromal enzyme that is upregulated in many breast tumors, can cause epithelial-mesenchymal transition (EMT). EMT causes epithelial cells to break away from neighboring cells, become mobile, and break through such barriers as the walls of lymph and blood vessels, which facilitates metastasis. In the current study, Bissell and colleagues explored the molecular pathways through which MMP-3 exerts this effect. They found that the Rho guanosine-triphosphatase proteins (GTPases), which control the proteins that define the cytoskeleton, play a key role in the link between MMP-3 and EMT. When normal cells were treated with MMP-3, the cells expressed Rac1b, an unusual form of Rho GTPase that has previously been found in breast and colon cancers. Rac1b dramatically altered the cytoskeleton, allowing epithelial cells to separate and move away from neighboring cells. The changes that Rac1b induced in the cytoskeleton increased the formation of reactive oxygen species (ROS), which are highly reactive, oxygen-containing molecules. The increased ROS had a direct impact on genomic DNA by activating key genes that control the EMT. The duplicated and missing DNA regions that resulted from the increased ROS are key characteristics of cancer development. The researchers have developed a method to block the formation of Rac1b. When they used RNA interference (RNAi) to reduce the levels of Rac1, they prevented MMP-3 from exerting its effect on the EMT. These findings offer a possibility for intervening in the tumor development pathway identified in this study. The study also suggests other potential therapeutic targets, including stopping the cytoskeleton alterations, ROS effects, and the process through which ROS activates genes that induce EMT and facilitate metastasis.

Publication:

Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, Leake D, Godden EL, Albertson DG, Nieto MA, Werb Z, and Bissell MJ. 2005. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 43617:123-127.

Links:

http://www.lbl.gov/lifesciences/labs/bissell_lab.html

Abstract: Breast Cancer in Context: New Tools and Paradigms for the Millennium

Abstract: Cytoskeletal Control of Mammary Epithelial Morphogenesis and Tumorigenesis

Abstract: Role of PI3 kinase signaling pathways in polarity determination of human mammary epithelial cells grown in three-dimensional extracellular matrix

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Chk1: The Doctor Jekyll and Mr. Hyde of the Cell Cycle
Posted September 30, 2005
Robert T. Abraham, Ph.D., The Burnham Institute

The cell cycle is controlled by an elaborate system of interacting proteins. This network of proteins can halt the cell cycle at specific checkpoints when it detects that something such as damage to the DNA has gone wrong. Frequently, a tumor growing in normal tissue creates an oxygen-starved (hypoxic) and nutrient-deprived environment when tumor growth exceeds the ability of local blood vessels to adequately supply oxygen and nutrients. Under normal circumstances, checkpoint mechanisms can halt cellular proliferation in this type of stressful situation. However, a breakdown in this checkpoint can lead to genomic instability, which is a hallmark of cancer development. Some cells adapt to high stress situations such as hypoxia and nutrient-deprivation by negotiating their way through the checkpoint, leading to a selective growth advantage. In studies that served as the basis for a fiscal year 2001 Breast Cancer Research Program (BCRP) Idea Award, Dr. Robert Abraham and colleagues demonstrated that hypoxic conditions caused proliferating cancer cells to undergo growth arrest that was accompanied by the activation of the ATR-hChk1 checkpoint pathway, a major player in the cellular response to DNA damage. Using funds from his BCRP Idea Award, Dr. Abraham and his colleagues studied the interactions among hypoxia, checkpoint activation, and genomic instability in human breast cancer cells. The data demonstrated that hypoxia caused degradation of checkpoint kinase 1 (Chk1), a protein that plays an important role in maintaining the viability of normal cells. He also found that hypoxic conditions sensitized breast cancer cells to cell death induced by the anticancer drug campthothecin (CPT) and that treatment with CPT also resulted in a loss of Chk1 protein. Ironically, ATP-dependent phosphorylation of Chk1 at Serine 345 marks Chk1 for both degradation as well as activation of the protein. Although this finding may seem counterintuitive, Dr. Abraham hypothesizes that a balance of activation and degradation of Chk1 exists under normal cellular conditions, whereas this balance is disrupted under stressful situations, such as hypoxia or treatment with chemotherapeutic agents, leading to degradation of Chk1, extensive DNA damage, and cell death. The relevance of this finding to breast cancer therapy was established when Dr. Abraham showed that this mechanism of Chk1 regulation is abolished in certain breast cancer cell lines that are resistant to CPT therapy. In drug resistant cells, treatment with CPT did not result in degradation of Chk1 and cancer cells were able to resume proliferating following termination of CPT therapy. Taken together, these findings establish a novel mechanism of Chk1 regulation and are significant for understanding breast cancer development and therapy. These results were recently published in the September 2005 edition of Molecular Cell and discussed in a press release issued by the Burnham Institute (http://www.burnham.org/newsandinformation/news.asp).

Publication:

Zhang Y-W, Otterness DM, Chiang GG, Liu Y-C, Mercurio F, and Abraham RT. 2005. Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway. Molecular Cell 19:607-618.

Link:

Abstract: Hypoxia as a Driving Force for Genetic Instability During Breast Tumorigenesis

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Single Cell Protein Profiles for Improved Diagnosis and Treatment of Breast Cancer
Posted August 16, 2005
Norman Dovichi, Ph.D., University of Washington, Seattle, Washington

Widespread use of mammography as well as breast self-exams accompanied by the detection of prognostic biomarkers, such as p53, c-erbB-2, and estrogen and progesterone receptors, during the screening phase, have made it possible to discover breast tumors at their earliest stages. However, despite the success of prognostic biomarkers, more detailed information is still required for guiding therapy. Fine needle aspirate biopsies of breast tumors are often obtained for the detection of cancer; however, these samples typically contain a limited number of breast epithelial cells mixed in a heterogenous pool of various cell types. Dr. Dovichi, the recipient of a fiscal year 2002 Exploration Award, developed a two-dimensional (2D) fingerprinting method that can produce protein profiles from a single cell, compared to 100,000 cells normally required for the detection of protein with 2D gel electrophoresis. The generation of single-cell proteome fingerprints from cancer biopsies will potentially be a powerful prognostic tool in the characterization of breast cancers. "Single cell protein fingerprinting allows study of the cell-to-cell variation in response to treatment, which can provide insight into those factors that determine the response of a particular cell to treatment" (Hu S et al. 2004). The data from an individual's 2D protein profile may greatly accelerate the discovery of relevant biomarkers representing various stages of breast cancer, which will aid in the diagnosis and treatment of the disease.

Publication:

Hu S, Michels DA, Fazal MA, et al. 2004. Capillary sieving electrophoresis/ micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells. Anal Chem 67:4044-4049.

Link:

Abstract: The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in a Breast Cancer Cell Line

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Preventing the Angiogenic Switch in Human Breast Cancer
Posted June 28, 2005
Judah Folkman, M.D., Children's Hospital & Harvard Medical School

The Breast Cancer Research Program Innovator Award was designed to fund talented individuals to pursue creative, potentially breakthrough research that could accelerate the eradication of breast cancer. In his first year of funding under this award, Dr. Judah Folkman has met this expectation. He has accumulated substantial evidence for his hypothesis that microscopic tumors can be held in check by a molecular switch that turns on angiogenesis. When human breast cancer cells were separated into angiogenic and nonangiogenic subpopulations and implanted into immune-deficient mice, there was a large and significant difference in the elapsed time before palpable tumors arose in the mice receiving nonangiogenic tumor lines. In addition, 5% to 10% of nontumorigenic subpopulations reproducibly switched to the angiogenic phenotype. Some clues about the molecular angiogenesis switch are given by analyzing the balance between the endogenous angiogenesis inhibitor thrombospondin-1 and its negative regulator c-Myc in these cell lines. Dr. Folkman and colleagues showed that two breast cancer cell lines that formed nonangiogenic, dormant tumors in mice expressed very high levels of thrombospondin-1 and very low levels of c-Myc. Conversely, two breast cancer cell lines that formed angiogenic tumors in mice expressed very low levels of thrombospondin-1 and high levels of c-Myc. Dr. Folkman's group has evidence that angiogenic breast cancer cell lines repress the expression of thrombospondin-1 in surrounding stromal fibroblasts, and breast cancer cell lines that produce nonangiogenic dormant tumors stimulate thrombospondin-1 expression in stromal fibroblasts. The biochemical changes associated with the angiogenesis switch likely happen after the switch is flipped, but before a tumor is palpable. Dr. Folkman showed that in mice bearing different types of human cancer, specific angiogenic proteins produced by a tumor are taken up by platelets and sequestered. The peak concentration of a given angiogenic protein is in direct proportion to the time the tumor has been present. This "platelet angiogenic profile" can detect an early microscopic tumor as small as 1 cubic millimeter, offering exciting possibilities for early cancer detection. The corollary to detecting tumors at this early stage is that treatment with angiogenesis inhibitors could prevent the development of cancer from these microscopic tumors essentially indefinitely, turning cancer into a "chronic manageable disease" in Dr. Folkman's words.

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An Antibiotic That Kills Breast Cancer Cells
Posted May 27, 2005
Shaomeng Wang, Ph.D., University of Michigan, Ann Arbor, Michigan

More than 60% of breast tumors have inappropriate activation of a protein called Signal Transducers and Activators of Transcription 3 (Stat3). This Stat3 activation is not observed in normal breast epithelial cells. It is believed that activation of Stat3 confers a growth advantage for cancer cells as well as resistance to conventional chemotherapeutic agents. Therefore, Stat3 is an attractive tumor-specific molecular target for new therapeutic agents. The goal of Dr. Shaomeng Wang of the University of Michigan was to discover small molecule inhibitors of Stat3 that were effective at killing breast cancer cells while having potentially low toxicity to normal breast cells. With funding from a fiscal year 2002 Breast Cancer Research Program Concept Award, Dr. Wang's laboratory and colleagues from the Ohio State University identified a promising compound. They analyzed the protein structures and binding sites of more than 429,000 compounds by virtual database screening. The goal was to find a compound that would be expected to bind to Stat3 in a region that would prevent Stat3 proteins from binding to each other, or homodimerizing. Homodimerization of Stat3 proteins is required for its activation. One hundred compounds were selected and tested in cell-based assays. From these 100 compounds, the STA-21 compound was selected as the strongest inhibitor of Stat3 activity. STA-21 is a natural product and a member of the angucycline class of antibiotics. STA-21 inhibited the growth and survival of breast cancer cell lines that have constitutively activated Stat3; however, it had no effect on the growth of other breast cancer cells. Studies of STA-21 mechanisms of action showed that STA-21 blocked the ability of Stat3 proteins to homodimerize, move to the nucleus, and bind to DNA. The next steps are to test the efficacy of STA-21 in animal models of human cancer and to make the STA-21 compound more potent. STA-21 is a promising candidate for drug development, and it represents a new class of anticancer drugs for treating human breast tumors that contain activated Stat3.

Publications:

Song H, Wang, R, Wang S, and Lin J. 2005. A low molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proceedings of the National Academy of Sciences USA 102:4700-4705.

Link:

Abstract: Structure-Based Discovery And Testing Of Non-Peptide, Cell-Permeable Small Molecule Inhibitors Of Stat-3 As A Potential Novel Therapy For Breast Cancer

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The Wealth of the Rain Forest
Posted April 8, 2005
Deborah A. Lannigan, Ph.D., University of Virginia, Charlottesville, VA

During the 1960s a federal initiative was implemented to systematically collect botanicals for the use in discovery, identification and characterization of pharmaceutical agents to treat and cure diseases. Dr. Deborah A. Lannigan and collaborators at the University of Virginia focused on this library of plant extracts to find a new treatment for breast cancer. In order to screen the extracts , a high through-put ELISA that used luminescence as an indicator of substrate phosphorylation was developed. It was the investigators intention to look for inhibitors of the p90 ribosomal S6 kinase (RSK), a critical member of the mitogen-activated protein kinase pathway. It was shown that one extract specifically inhibited the constitutively active RSK by 90&#37 while not inhibiting other kinases such as FAK, PKA, p70, S6K or MSK1. The extract originated in the South American rain forest from a species of the dogbane family named Forsteronia refracta. The extract was fractionated and the inhibitor, a kaempferol glycoside called SL0101, was isolated. Studies demonstrated that RSK enzymatic activity did not change with the addition of the inhibitor, but alterations of the binding constant for ATP showed that SL0101 is a competitive inhibitor. Site-specific studies delineated the residues that are important for SL0101 binding. In growth inhibition experiments, SL0101 repressed the growth of MCF-7 cells, a human breast cell line, but not that of the normal human breast cell line, MCF-10A. Cell cycle analysis confirmed that the anti-prolifrative effect is due to G1a cell cycle block. Additional studies including siRNA experiments corroborated the supposition that RSK1 and RSK2 are required for MCF-7 proliferation. Dr Lannigan and her team extended their research to include a survey of breast cancer tissues; these analyses established that 50&#37 of breast cancer tissues have increased levels of both isoforms of RSK compared to normal breast tissue. Taken together, these results point to a new target for breast cancer therapy and a novel agent, which may be further developed for breast cancer treatment. The inhibitor SL0101 derived, from a natural resource, offers new avenues for treatment and for future drug design and development. Natural resources provide a wealth of health care treatment.

Publications:

Smith J.A., Poteet-Smith C.E., Xu Y., Errington T.M., Hecht S.M. and Lannigan D.A. 2005. Identification of the First Specific Inhibitor of p90 Ribsomal S6 Kinase (RSK) Reveals an Unexpected Role for RSK in Cancer Cell Proliferation. Cancer Research 65: 1027-1034.

Link:

Abstract: The Protein Kinase, Rsk2, a Novel Drug Target for Breast Cancer

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Identifying the Risk of Breast Cancer Recurrence in Patients Treated with Tamoxifen
Posted February 11, 2005
Dennis Sgroi, M.D., Massachusetts General Hospital

Tamoxifen, a drug that blocks the interaction between estrogen and its receptor, is among the most widely used chemotherapeutic agents for the treatment of breast cancers that express the estrogen receptor (ER+). Approximately 30%; of patients do not respond to tamoxifen treatment and patients who respond initially to treatment may acquire tamoxifen resistance and eventually fail treatment. The ability to determine which patients will not respond well to tamoxifen therapy would allow earlier use of other, potentially more effective, therapies and could result in a better prognosis for these women. However, there is currently no reliable method of predicting which ER+ patient will not respond to tamoxifen treatment.

With funding received from a fiscal year 2002 Breast Cancer Research Program Exploration Award, Dr. Dennis Sgroi of Massachusetts General Hospital is looking at the levels of expression of certain genes in breast cancer as a way of identifying prognostic categories that may guide cancer treatment choices. In a recently published study, Dr. Sgroi and his colleagues performed genome wide microarray analysis on 60 tumor samples taken from patients who had received tamoxifen treatment for early-stage, ER+ breast cancer. Of these 60 patients, 32 remained disease-free for an average of 8 years, while 28 experienced tumor recurrence or metastasis. This study revealed that the ratio between the expression levels of two genes, HOXB13 and IL17BR, was a strong predictor of tumor recurrence. High levels of HOXB13 or low levels of IL17BR expression suggested that tamoxifen therapy would fail. Although these studies need to be validated in a larger population, it appears that this simple test will assist oncologists in deciding whether to use tamoxifen therapy or an alternative therapy in treating ER+ breast cancer patients.

Publications:

Ahlgren M, Sorensen T, Wohlfahrt J, Haflidadottir A, Holst C, Melbye M. 2003. Birth weight and risk of breast cancer in a cohort of 106,504 women. Int J Cancer. 107:997-1000.

Ahlgren M, Melbye M, Wohlfahrt J, Sorensen TI. Growth patterns and the risk of breast cancer in women. N Engl J Med. 2004 Oct 14;351(16):1619-1626.

Publications:

Ma XJ, Wang Z, Ryan PD, et al. 2004. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 5:607-616.

Link:

Abstract: Gene Expression Profiling Of Breast Cancer Stroma

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