Tumor Biology


Research Project Awards



Breast Cancer Progression and the Extracellular Matrix

Francis S. Markland, Jr., Ph.D.
University of Southern California

The most common cause of death in breast cancer patients is the spread, or metastasis, of the cancer cells from the breast to the bones, lungs, liver and brain and the progressive growth of the cancer cells at these sites. Therefore, controlling breast cancer metastasis represents an effective method of preventing progression of the disease. We propose to use a multidisciplinary approach to not only study how the surrounding tissue effects the ability of breast cancer cells to spread, but also to evaluate a unique pharmacologic approach for combating breast cancer metastasis.

Research proposed in this application will lead to an enhanced understanding of the pathogenesis of breast cancer, one of the BCRP's priority research issues. Recent studies suggest that cell adhesion proteins on breast cancer cells interact with the extracellular matrix (ECM), the tissue surrounding the tumor cells. This interaction induces increased production by the breast cancer cells of proteins that degrade the ECM. This degradation enables the tumor cells to invade the surrounding tissue and ultimately to enter the circulatory system. Once in the circulation, tumor cells travel to other organ sites where they progressively grow. Our investigations should lead to a better understanding of the role of cell adhesion proteins in breast cancer invasion and dissemination. A clearer understanding of this process will provide a basis for developing effective therapies for this most lethal aspect of breast cancer.

Our studies are also aimed at developing more effective interventions for preventing progression of breast cancer, another priority research issue of the BCRP. Our approach will be aimed at blocking cell adhesion proteins on the breast cancer cells to prevent them from interacting with the ECM. This should also inhibit growth and dissemination of the breast cancer cells by: 1) preventing the growth of new blood vessels that provide nourishment for the cancer cells, a process called neovascularization; and 2) interfering with pathways leading to the production of proteins essential to the invasive properties of breast cancer cells.

Inhibition of cell adhesion can be achieved by treating breast cancer cells with proteins called disintegrins. The Principal Investigator has purified and characterized disintegrins and shown that they are well tolerated in animals. We will study the inhibitory effect of disintegrins on cancer growth, neovasculari-zation, and metastasis in mammary fat pads. The results from this study will be directly applicable to over 90% of women with metastatic breast cancer in the state of California. Data gathered by our studies will support an important, new, and exciting approach for the prevention of breast cancer progression.

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Model of Human Breast Cancer Development and Progression

Satyabrata Nandi, Ph.D.
University of California, Berkeley

The clinical course of human breast lesions and cancer is extremely variable. Our proposal is directly aimed at gaining insight into the variable clinical course of human breast cancer development and progression. Through a better understanding of these processes, new aproaches will hopefully be identified for the development of both the prevention and treatment of human breast cancer. Perhaps the only definitive way to study human breast cancer development and progression consists of sampling the patient's breast lesion as a function of time until the very terminal stage. Such a longitudinal study, of course, cannot be done for ethical reasons, and surgical intervention is most often undertaken. The clinical course of the undisturbed lesion in vivo in a patient therefore can no longer be followed.

The availability of a model system which could propagate and maintain human breast lesions, similar to those actually seen in surgical breast specimens, would greatly enhance our understanding of human breast cancer development and progression. We propose in this application just such a model system: the "surrogate human breast" in immunosuppressed mice. In this system the breast cells are isolated from human surgical breast specimens, embedded in collagen gel, and then transplanted subcutaneously in immunosuppressed mice. Histological sections of recovered transplants show that various breast lesions and cancer similar to those actually seen in surgical breast specimens, as well as normal cells, can be maintained and propagated in this system. In essence, the breast lesion from a single patient has now been propagated into multiple "surrogate human breasts" in an animal for study. This model system thus provides a unique opportunity to study the development and progression of different breast lesions as well as various factors influencing their biological course. The influence of various hormonal environments and gene therapy on the development and progression of these lesions can be determined. Specifically, we propose to alter the hormonal environment of the host animals to determine the conditions affecting both positively and negatively the biological course of various breast lesions. We also propose to utilize a highly efficient adenoviral mediated transfer of genes implicated to be of importance in human breast carcinogenesis. To analyze genetic influrences collectively, these results using a physiologically relevant model system, the surrogate human breast in immunosuppressed mice, will not only provide a better understanding of human breast cancer development and progression but may have far-reaching implications in both the prevention and treatment of human breast cancer.

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Role of L-Plastin in Breast Cancer

Ching-Shwun Lin, Ph.D.
University of California, San Francisco

L-plastin is a protein normally found in leukocytes (white blood cells) only. In these cells L-plastin regulates cell movement by interacting with actin, a major cellular protein that directly controls cell movement. Normal breast epithelial cells do not have L-plastin. However, we have discovered that all cultured breast carcinoma cells that were derived from breast tumors contain L-plastin. Furthermore, the amount of L-plastin contained in the cells correlates with the degree of invasiveness of these cells, as determined by matrigel invasion assay (where cell movement through a gel is measured). Therefore, it appears that L-plastin may be important for the motility of breast cancer cells. In this project we will: 1) determine whether there is a correlation between the pattern of L-plastin synthesis and the degree of malignancy of clinically defined stages of breast tumors, 2) through the use of gene transfer determine whether overproduction of L-plastin will result in enhanced invasiveness, and 3) determine how the L-plastin gene is activated in breast cancer cells. For the first aim we will screen a panel of breast tumors that have been clinically graded at the UCSF Medical Center. We will use an anti-L-plastin antibody to detect the L-plastin protein or use an L-plastin DNA probe to detect the messenger RNA in these specimens.

For the second aim we will introduce L-plastin, by a gene transfer technique called transfection, into normal breast epithelial cells. We will then perform matrigel invasion assays to see if increased L-plastin synthesis will result in enhanced invasiveness. Alternatively we will introduce a genetically engineered molecule called rybozyme into cultured breast cancer cells. We expect this molecule to specifically destroy the L-plastin messenger RNA, thereby preventing the synthesis of L-plastin. If so, the rybozyme-treated cells may lose the ability to invade. The third aim of this project is to elucidate the mechanisms by which the L-plastin gene gets turned on in breast cancer cells. We have isolated the entire L-plastin gene and identified a region that controls the synthesis of L-plastin messenger RNA. In this region, we have further recognized a few highly specialized DNA sequences that may be responsible for the activation of L-plastin gene in breast cancer cells. We will continue testing these DNA sequences by deleting or modifying the nucleotides to precisely determine how the L-plastin gene is activated in breast cancer cells.

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The Breast Tumor Suppressor Function of Hyaluronidase

Robert Stern, M.D.
University of California, San Francisco

The enzyme hyaluronidase appears to interfere with metastasic spread of breast cancer through three mechanisms. Two of the requirements for cancer cells to metastasize are that they must become motile, and that they must have a space into which to move. Hyaluronan (HA) accomplishes both of these tasks. Hyaluronan is a carbohydrate polymer on the surface of cancer cells that hydrates, expands, and opens up spaces around cancer cells, enabling them to invade surrounding tissues. It is also involved in the development of motility. Hyaluronidase, the enzyme that breaks down HA, eliminates this mechanism for cancer cell spread. Hyaluronidase appears to have other direct effects on cancer cells. The receptor for HA, CD44, functions as an anchor to bind HA to the cancer cell surface. CD44 comes in a variety of forms, a short form found on normal cells, and longer variants that occur on motile cancer cells. A portion of CD44 protrudes through the surface membrane into the cell cytoplasm, where it interacts with the cytoskleleton, the machinery for cell movement. Experimentally, inserting this longer cancer form of CD44 into benign tumor cells changes them into cancer cells that aggressively metastasize. Hyaluronidase eliminates the cancer cells' ability to move, by converting the long cancer form of CD44 to the short "normal" immobile form. Remarkably, hyaluronidase also causes established breast cancers to shrink.

In mice with human breast cancer, inoculation of hyaluronidase i.v. shrinks the tumor to half its size in four days. If malignant cells cannot maintain motility and thus invasiveness, and if the tumor cannot progress or grow, it apparently must undergo regression. This third function of hyaluronidase is not understood, but may follow from the first two functions. Hyaluronidase may also prevent progression of human breast cancer, and it does not have the devastating toxic side effects of other anticancer agents. We will vary injection schedules, and modify the enzyme chemically, to stabilize activity, in order to maximize the anticancer effect in mice, in preparation for clinical trials. Simultaneously, we will examine breast cancer cells in culture using molecular and cell biology to determine precisely how hyaluronidase regulates this alternative splicing of CD44. We postulate that HA regulates its own receptor. When HA bound to CD44 is present outside the cell, tags are placed on the intracellular portion of CD44. We have identified these specific tagging enzymes or phosphokinases, and when they are inactivated by techniques involving antisense or mirror image DNA, CD44 converts to its short form, as CD44 does in response to hyaluronidase. Understanding these events in the regulation of CD44 will make it possible to develop a new generation of anticancer drugs that prevent the metastases associated with breast cancer progression.

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Postdoctoral Fellowship Awards

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Cell Microenvironment and Progression of Breast Cancer

André H. Lochter, Ph.D.
Lawrence Berkeley National Laboratory

Progression of breast cancer from benign cells to a life-threatening disease is characterized by acquisition of migratory and invasive properties of tumor cells as they move through the breast tissue in search of blood vessels. It is this process and the subsequent infiltration of blood vessels and colonization of other organs which constitutes metastasis. From our own past and ongoing research, and from published reports, we have concluded that a major factor in tumor progression is the interaction of tumor cells with their surrounding microenvironment. This microenvironment consists of stroma, cells that create a supporting framework which is often composed of connective tissue for a gland or organ, and extracellular matrix (ECM), the interwoven network of proteins which provides additional support and vital information to surrounding cells.

It is known that for normal functioning, cells require the surrounding structure of the ECM. Although attempts have been made to understand the interaction of the cell with its microenvironment, the lack of suitable tissue culture models has hampered the precise interpretation of the data obtained and raised questions about their physiological relevance. We have now begun to reconstitute the cellular and ECM microenvironment of tumor cells in three dimensions by taking into culture a cell line which recapitulates the different stages of tumor progression. With this designer microenvironment in hand, we are able to analyze the role of tumor cell-stroma interactions and molecules of the ECM in tumor progression and to test our hypotheses by experimental manipulation. In our investigation, we will focus on a class of matrix-degrading enzymes, metalloproteinases, which are frequently found in large amounts in breast tumors, and are believed to promote tumor cell invasion by breaking down the components of ECM.

The proposed study is expected to enable a better understanding of the development of breast cancer and to identify molecular changes associated with the early stages of tumor progression in the breast. Detection of these changes in tumor patients would thus provide a valuable tool for the diagnosis of breast cancer. Furthermore and foremost, we expect to identify those molecules which are indispensable for tumor migration and invasion. By interfering with their function we hope to block tumor progression. We will also search for signals inside the ECM which attenuate or abolish tumor progression. Upon identification of these inhibitory signals we will engineer small peptides (chains of amino acids) which can be administered to breast tissues and block tumor progression.

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Changes in Transport into the Nucleus in Breast Cancer

Deborah J. Sweet, Ph.D.
The Scripps Research Institute

The aim of the research described in this proposal is to characterize the relationship between changes in movement of proteins between the nucleus and cytoplasm and the development of breast cancer. Exchange of proteins between the nucleus and cytoplasm occurs continuously and at a rapid rate, and is a fundamental aspect of normal cellular activity. It is also an important element of cell growth regulation, particularly for transmission of growth signals such as steroid hormones to the genome to produce changes in gene expression that alter the growth rate. Characterization of differences in the transport capacity of normal breast cells compared to those at different stages of conversion to a cancerous state will provide new insights into a previously unexplored aspect of the development of breast cancer. Improved understanding of these changes will provide additional information about the characteristics of breast tumors and could be used to enhance hormone based strategies.

Transport of proteins from the cytoplasm to the nucleus across the nuclear envelope, i.e., nuclear protein import, is a signal- and energy-dependent process that requires the activity of both nuclear envelope-associated and cytoplasmic factors. The nuclear import capacity of cells has been shown to vary depending on their growth state: rapidly growing cells have significantly higher nuclear import rates than cells that are not actively growing. The proposed research will characterize differences in the nuclear import capacity of human breast cell lines representing normal, proliferating and malignant tumor cells, with the aim of identifying the changes that occur during the development of cancer.

Mislocalization of steroid hormone receptors is a feature of some forms of breast cancer, so this study will also include a focus on the nuclear import of these receptors. It has recently become clear that steroid hormone receptors move continuously between the nucleus, where they affect gene expression, and the cytoplasm. Specific or general changes in the nuclear import pathway could therefore have significant effects on receptor localization within the cell and be a contributory factor in the development of cancer.

In the last few years, rapid progress has been made in understanding the molecular mechanism of nuclear protein import. Differences observed between the various cell types will therefore be investigated in more detail to determine whether they can be attributed to known transport factors, to better understand the molecular basis of breast cancer.

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HSP27 Regulation of Breast Tumor Blood Vessel Growth

Randolph S. Piotrowicz, Ph.D.
The Scripps Research Institute

The growth and spread of breast cancer is dependent on the establishment of a blood supply to the cancerous tumor. This is accomplished by the extension of existing blood vessels into the tumor mass by the migration and growth of the endothelial cells which line the intruding blood vessels. Highly vascularized tumors are generally larger and more likely to spread to other organs. The extent of tumor vascularization, therefore, is correlative with the risk of morbidity. Understanding how the generation of a tumor blood supply is regulated is essential to the understanding of the progression of the disease and may provide future avenues of prevention. Work in our laboratory has identified a small heat shock protein (HSP27) as a potential regulator of vessel growth. Increasing HSP27 levels by placing the human gene into bovine arterial endothelial cells results in enhanced growth and migration. HSP27 has been of interest to cancer researchers and oncologists because estrogen treatment of certain breast cancer cells, which causes accelerated growth of these cells, also causes increased HSP27 production. Since endothelial cell HSP27 is also responsive to estrogen, we propose that endothelial cell HSP27 regulates the vascularization of tumors which generate estrogen and/or grow in a milieu where estrogen is a key regulatory factor, e.g., breast carcinoma. To test this hypothesis, the following is proposed. First, the effects of estrogen treatment or the co-culture of breast cancer cells on the growth and migration of endothelial cells producing elevated HSP27 levels or mutant HSP27 will be measured. In addition, the effect of lowering endothelial cell HSP27 levels (through the infection of adenovirus vectors carrying anti-sense DNA) on these processes will be tested. Second, culture conditions will be modified to support the formation of capillary-like structures. The effect of estrogen or co-cultured breast tumor cells on the generation of these structures will be measured. Finally, mice expressing human HSP27 in addition to mouse HSP27 will be used to test the effect of elevated endothelial cell HSP27 levels on the formation of new blood vessels. The effect of elevated circulating and local estrogen levels (the latter approximating the effect of tumor-generated estrogen) will also be assessed. These studies address the complex communications which occur between breast tumor cells and invading vascular cells and will delineate the role of a key player in breast tumor vascularization.

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Sabbatical Awards


Malignant Transformation in Breast Epithelium

Heinz Furthmayr, M.D.
Stanford University

Several mechanisms are involved in the invasion of tissues by, and metastatic spread, of tumor cells. Among these are loss of differentiation, differences in communication between cells and with the extracellular environment, activation of proteolytic enzymes and changes in intracellular transfer of signals. The abnormal cell behavior of invasive tumor cells is not the result of a single change but rather likely is caused by several interdependent events. The research during this sabbatical period will focus on mechanisms breast cancer cells use to communicate with each other and to sense their environment.

The extracellular matrix plays a critical role in differentiation in the breast gland and in growth regulation, motility and death of the cells. In response to signals from the extracellular matrix, cells change shape as a result of precisely regulated rearrangements of proteins within the cell. These involve modifying enzymes, which act upon internal cytoskeletal proteins and regulate protein-protein interactions. It is believed that such signals are received by delicate and transiently formed extensions, called pseudopodia, which specifically recognize the extracellular matrix and transmit information for processing. These cellular structures are critical for cell movement and hence, for invasion of tissues and the ability of tumor cells to metastasize. Fluorescent derivatives of several relevant proteins will be prepared for introduction into mammary cells by microinjection and other methods. These cells will then be studied by time-lapse video and fluorescence microscopy.

The ultimate goal of this research program is the ability to interfere with the invasive property of breast cancer cells.

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