Margot M. Ip - US grants

The major objective of this proposal is to study the promoting effect of dietary fat on mammary carcinogenesis. Specific aims include the following. (1) The effect of quantity and type of fat on mammary carcinogenesis induce by dimethylbenz(a) anthracene (DMBA) will be examined in female rats. In these experiments, saturated and unsaturated fat will be compared with a blend of fat simulating that found in the typical American diet. Results of this study will be most relevant to nutritionists and oncologists in their deliberations to recommend a reduction of fat intake to lower the risk of breast cancer. More importantly, these results will specifically indicate how much the fat content of the diet needs to be reduced or conversely, if anything meaningful is to be gained by only a small reduction. (2) The efficacy of dietary fat inpromoting mammary carcinogenesis at various times after the administration of DMBA will be examined to determine whether rats switched to a high fat diet several months after the administration of DMBA still develop a high incidence of tumors. (3) The effect of trans fatty acids (produced during hydrogenation in the processing of vegetable oil) on DMBA-induced mammary carcinogenesis will be studied in female rats. (4) The effect of both quantity and type of dietary fat on the DNA synthetic rate of the mammary gland will be examined by measuring the incorporation of 3H-thymidine into the DNA of isolated epithelial cells at various times after the administration of DMBA. The purpose of this experiment will be to determine if there is a correlation between the promoting effect of dietary fat on mammary carcinogenesis and an effect of fat on the DNA synthetic rate of the mammary gland. (5) The effect of quantity and type of dietary fat will be examined in rats bearing transplantable metastasing mammary tumors to determine if fat affects either the extent or the location of metastases. (6) The effect of dietary fat on the development and progression of ductal hyperplasia will be examined in the mammary gland of DMBA-treated mice. In view of the overwhelming epidmiological and experimental evidence liking dietary fat and breast cancer, it is felt that further research focused on defining the conditions under which fat is most effective in stimulating mammary neoplasia should be an area of high priority towards realizing our goal of reducing breast cancer morbidity.

The objective of this grant is to study tumor progression from hormone dependency to autonomy, using the Dunning R3327 rat prostate adenocarcinoma as a model. It is well known that human prostatic cancer, while initially hormone-dependent, eventually relapses to an androgen-insensitive state and it is the goal of our studies to elucidate those factors that control tumor progression and thus ultimately to design treatment protocols that will minimize the emergence of new cell types. Towrads this goal, the following specific aims will be pursued. (1) The mechanism of selection and/or adaptation during relapse from androgen dependence to androgen insensitivity will be studied using rats that have undergone various types of hormonal treatment or chemotherapy, and in rats whose immune system has been altered. The goal of these studies is to better understand those factors that affect tumor progression, so that more effective and rational therapies for the initial treatment of prostate cancer can be designed. (2) Subpopulations of cells will be isolated from the R3327 prostate tumor and characterized in terms of hormone-dependence and sensitivity, steroid receptor content, growth rate in vivo, DNA content, and histology. These studies are being done to gain a better understanding of the types of cells that co-exist within a tumor. (3) Interactions between various cell populations will be studied in rats inoculated with more than one cell type, in order to determine whether a particular cell population can alter the growth rate, genetic stability, or metastatic capacity of other cell populations. Such studies are of importance in the design of chemotherapeutic protocols that will minimize the emergence of new cell types with altered hormone-dependence, growth rate or metastatic capacity and thus provide a more effective treatment for human prostatic cancer.

The objective of this proposal is to study the mechanism of the dietary fat induced promotion of mammary carcinogenesis, with special emphasis being placed on the role of prostaglandins and of certain components of the host defense system in this process. An additional aim is to gain a greater understanding of the role of the prostaglandins in the regulation of immune surveillance during tumorigenesis, tumor growth, and metastatic spread. The specific experiments that will be done are as follows. (1) The role of the prostaglandins in the stimulation of DMBA-induced mammary tumorigenesis by diets high in polyunsaturated fat will be studied by using inhibitors at several steps of prostaglandin synthesis. This will include the use of drugs which block the cyclooxygenase, lipoxygenase, thromboxane synthetase and prostacyclin synthetase pathways. (2) The role of prostaglandins in the growth and metastatic spread of certain transplantable metastasizing mammary tumors will be examined in W/Fu rats. In these studies, manipulation of dietary fat intake in conjunction with specific prostaglandin synthesis inhibitors will be used to probe the role of prostaglandin synthesis inhibitors in cancer therapy. (3) Natural killer (NK) cell activity and macrophage function will be studied in rats fed different levels and types of fat to determine their role in DMBA-induced mammary tumorigenesis and in mammary tumor metatasis. Previous studies have demonstrated that NK activity is suppressed by prostaglandins and our goal is to determine whether agents that alter NK and macrophage activities can affect the growth and development of mammary tumors. (4) The effect of dietary fat on the cell-mediated immune response will be studied in SD rats with the goal of determining whether prostaglandin synthesis inhibitors or other agents can overcome the immune suppression caused by a high intake of polyunsaturated fat. The overall goal of these studies is to develop more effective therapeutic and/or immunoprophylactic treatment strategies for breast cancer, possibly utilizing specific prostaglandin synthesis inhibitors.

The objective is to determine the effect of the lipoxygenase and cyclooxygenase products of arachidonic acid metabolism (eicosanoids) on the growth of normal and transformed mammary cells. It is our hypothesis that certain lipoxygenase products, formed in increased amounts in the presence of cyclooxygenase inhibitors by diversion of arachidonic acid into the lipoxygenase pathways, or by actual stimulation of a lipoxygenase, may plan a beneficial role in the reduction of tumorigenesis and the inhibition of growth of transformed mammary cells. The following studies will address this question. 1. To determine the role of lipoxygenase and cyclooxygenase products in the growth of normal and transformed mammary cells in vivo. Specifically, the effects of cyclooxygenase inhibitors iwll be compared with those of dual cyclooxygenase plus lipoxygenase inhibitors on DMBA-induced mammary carcinogenesis and these effects will be correlated with changes in the products of the 5-, 12- and 15-lipoxygenases, as well as cyclooxygenase, in mammary gland, mammary tumor and blood. 2. To determine if changes in arachidonate content of mammary gland phospholipids can modulate the efficacy of the cyclooxygenase inhibitor indomethacin on DMBA-induced mammary carcinogenesis. 3. To determine if the inhibitory effect of diets containing high levels of fish oil on DMBA-induced mammary carcinogenesis is correlated with changes in the fatty acid content of mammary gland membrane phospholipids and of levels of mammary gland and mammary tumor arachidonic acid metabolites. Aim 4. To determine the role of lipoxygenase and cyclooxygenase products in the growth of normal and transformed mammary cells in vitro. Specifically, production of arachidonic acid metabolites will be compared in both normal rat mammary epithelial cells and DMBA-induced rat mammary tumor cells in primary culture under basal, hormone- and Ca++ ionophore-stimulated conditions. In addition, the effects of cyclooxygenase and lipoxygenase inhibitors will be compared in terms of their ability to alter growth of both normal and transformed mammary cells and the effects will be correlated with changes in levels of arachidonic acid metabolites. Studies will also be done to determine if changes in the fatty acid composition of membrane phospholipids alter the efficacy of the cyclooxygenase and lipoxygenase inhibitors, or of eicosanoid production under basal or stimulated conditions.

The major objective of this proposal is to study the promoting effect of dietary fat on mammary carcinogenesis. Specific aims include the following. (1) The effect of quantity and type of fat on mammary carcinogenesis induce by dimethylbenz(a) anthracene (DMBA) will be examined in female rats. In these experiments, saturated and unsaturated fat will be compared with a blend of fat simulating that found in the typical American diet. Results of this study will be most relevant to nutritionists and oncologists in their deliberations to recommend a reduction of fat intake to lower the risk of breast cancer. More importantly, these results will specifically indicate how much the fat content of the diet needs to be reduced or conversely, if anything meaningful is to be gained by only a small reduction. (2) The efficacy of dietary fat inpromoting mammary carcinogenesis at various times after the administration of DMBA will be examined to determine whether rats switched to a high fat diet several months after the administration of DMBA still develop a high incidence of tumors. (3) The effect of trans fatty acids (produced during hydrogenation in the processing of vegetable oil) on DMBA-induced mammary carcinogenesis will be studied in female rats. (4) The effect of both quantity and type of dietary fat on the DNA synthetic rate of the mammary gland will be examined by measuring the incorporation of 3H-thymidine into the DNA of isolated epithelial cells at various times after the administration of DMBA. The purpose of this experiment will be to determine if there is a correlation between the promoting effect of dietary fat on mammary carcinogenesis and an effect of fat on the DNA synthetic rate of the mammary gland. (5) The effect of quantity and type of dietary fat will be examined in rats bearing transplantable metastasing mammary tumors to determine if fat affects either the extent or the location of metastases. (6) The effect of dietary fat on the development and progression of ductal hyperplasia will be examined in the mammary gland of DMBA-treated mice. In view of the overwhelming epidmiological and experimental evidence liking dietary fat and breast cancer, it is felt that further research focused on defining the conditions under which fat is most effective in stimulating mammary neoplasia should be an area of high priority towards realizing our goal of reducing breast cancer morbidity.

The objective of the proposed studies is to investigate the effect of lipid on the proliferation and differentiation of normal rat mammary epithelial cells (rmec), with the long-term goal of understanding the mechanism by which diets high in polyunsaturated fat stimulate mammary tumorigenesis. It is proposed that alterations of membrane phospholipid composition modulate the receptor transducing mechanism for hormones and growth factors, thus affecting cell proliferation and differentiation. Specific emphasis will be placed on protein kinase C, since this enzyme, which plays a major role in intracellular signal transduction, requires lipid for its activity. These studies will be carried out using a primary culture model system recently developed in our laboratory in which rat mammary epithelial cells obtained from immature virgin rats are grown within a reconstituted basement membrane matrix in a serum-free defined medium. This model allows for both extensive proliferation of the mammary epithelial cells (15-30 fold within 14-21 days) as well as functional and morphological differentiation equivalent to that of the lactating mammary gland. AIM 1. To carry out a comprehensive study of the effects of specific hormones and growth factors on morphological and functional differentiation of normal rat mammary epithelial cells in primary culture, with the goal of completing characterization of this unique model system. These studies will serve as the foundation upon which the effects of lipid as well as other protein kinase C modulators can be investigated. AIM 2. To continue studies on the effects of lipid on proliferation as well as morphological and functional differentiation of normal rat mammary epithelial cells. These studies will focus on the hormonal and growth factor conditions under which fatty acids and other lipids modulate specific mammary epithelial cell functions, with the goal of identifying those growth factors whose activity can be regulated by changes in membrane lipid composition and/or by the lipid in the incubation medium, and the mechanism by which this action is mediated. AIM 3. To determine the role of specific protein kinase C isoenzymes in the proliferation and differentiation of normal rat mammary epithelial cells. In these studies, protein kinase C will be monitored during development of both morphological and functional differentiation, as well as in response to specific combinations of hormones and growth factors known to favor either the differentiation or proliferation of the mammary cells. Considerable emphasis will be placed on defining the effect of individual hormones and growth factors on the activity and isoenzyme composition of protein kinase C and the effect of protein kinase C inhibitors and activators on proliferation and differentiation. AIM 4. To determine if changes in membrane phospholipid composition affect receptor transducing mechanisms, with specific emphasis on protein kinase C. In this aim, the effect of membrane lipid on hormone- or growth factor-induced translocation of protein kinase C, as well as on its ability to phosphorylate specific protein(s) will be assessed.

The mammary gland is a complex organ which is under a myriad of regulatory controls. These include circulating hormones and growth factors, as well as local trophic factors produced by the mammary epithelial cells (MEC) or by the stroma within which the MEC are embedded. We have made the unique observation that the cytokine tumor necrosis factor-alpha (TNF-alpha) can stimulate the proliferation and morphological differentiation of rat MEC and inhibit casein production. Intriguingly, other investigators have reported that TNF-alpha inhibits the growth of malignant breast epithelial cells. Since this cytokine is produced by some of the types of cell that form the mammary stroma, we propose to determine whether TNF-alpha is a physiological regulator of growth and differentiation in normal rat MEC, to undertake preliminary studies to assess the mechanism of its action and to determine whether the activity of TNF-alpha is altered in transformed cells. Specific aims are as follows. AIM 1. To assess whether TNF-alpha is a potential paracrine or autocrine regulator of mammary epithelial cell function. Studies will be undertaken to determine if TNF-alpha mRNA and its protein product are produced in mammary epithelial and stromal cells. If TNF-alpha is found to be synthesized by one or more cell types in the mammary gland, its hormonal regulation will be investigated. AIM 2. To determine whether the effects of TNF-alpha on the proliferation and differentiation of the MEC are direct or are mediated by another cytokine. These studies will focus on how quickly MEC respond to TNF-alpha by examining the time course of its effect on early response genes such as c- myc, c-fos and c-jun, as well as on overall DNA synthesis. Additionally, potential mediation by other cytokines or growth factors will be assessed by determining whether they are synthesized in response to TNF-alpha, the time course of such a response, and if neutralizing antibodies to these cytokines block and the cytokine itself mimics, the effects of TNF-alpha on proliferation and/or differentiation. AIM 3. To determine if TNF-alpha stimulates mammary gland development in vivo. In these studies, Elvax pellets of TNF-alpha will be implanted in the mammary fat pad of immature or of ovariectomized mature rats, and development of the implanted gland compared with that of the contralateral gland. AIM 4. To determine the mechanism of the inhibitory effect of TNF-alpha on casein synthesis by examining the effect of TNF-alpha on stability of beta-casein mRNA and on the casein protein family. A determination will also be made whether the inhibitory effect of TNF-alpha is specific for casein, or whether other markers of functional differentiation (lipid synthesis, expression of WAP and transferrin) are inhibited as well. AIM 5. The effect of TNF-alpha on proliferation and differentiation of transformed rat mammary epithelial cells will be investigated both in primary culture as well as in vivo.

The objective of the proposed studies is to investigate the effect of lipid on the proliferation and differentiation of normal rat mammary epithelial cells (rmec), with the long-term goal of understanding the mechanism by which diets high in polyunsaturated fat stimulate mammary tumorigenesis. It is proposed that alterations of membrane phospholipid composition modulate the receptor transducing mechanism for hormones and growth factors, thus affecting cell proliferation and differentiation. Specific emphasis will be placed on protein kinase C, since this enzyme, which plays a major role in intracellular signal transduction, requires lipid for its activity. These studies will be carried out using a primary culture model system recently developed in our laboratory in which rat mammary epithelial cells obtained from immature virgin rats are grown within a reconstituted basement membrane matrix in a serum-free defined medium. This model allows for both extensive proliferation of the mammary epithelial cells (15-30 fold within 14-21 days) as well as functional and morphological differentiation equivalent to that of the lactating mammary gland. AIM 1. To carry out a comprehensive study of the effects of specific hormones and growth factors on morphological and functional differentiation of normal rat mammary epithelial cells in primary culture, with the goal of completing characterization of this unique model system. These studies will serve as the foundation upon which the effects of lipid as well as other protein kinase C modulators can be investigated. AIM 2. To continue studies on the effects of lipid on proliferation as well as morphological and functional differentiation of normal rat mammary epithelial cells. These studies will focus on the hormonal and growth factor conditions under which fatty acids and other lipids modulate specific mammary epithelial cell functions, with the goal of identifying those growth factors whose activity can be regulated by changes in membrane lipid composition and/or by the lipid in the incubation medium, and the mechanism by which this action is mediated. AIM 3. To determine the role of specific protein kinase C isoenzymes in the proliferation and differentiation of normal rat mammary epithelial cells. In these studies, protein kinase C will be monitored during development of both morphological and functional differentiation, as well as in response to specific combinations of hormones and growth factors known to favor either the differentiation or proliferation of the mammary cells. Considerable emphasis will be placed on defining the effect of individual hormones and growth factors on the activity and isoenzyme composition of protein kinase C and the effect of protein kinase C inhibitors and activators on proliferation and differentiation. AIM 4. To determine if changes in membrane phospholipid composition affect receptor transducing mechanisms, with specific emphasis on protein kinase C. In this aim, the effect of membrane lipid on hormone- or growth factor-induced translocation of protein kinase C, as well as on its ability to phosphorylate specific protein(s) will be assessed.

The mammary gland is a complex organ which is under a myriad of regulatory controls. These include circulating hormones and growth factors, as well as local trophic factors produced by the mammary epithelial cells (MEG) or by the stroma within which the MEC are embedded. In addition, the components of the extracellular matrix (ECM) have been found to exert a profound effect on both the morphological and functional development of the mammary gland. The objective of the studies proposed herein is to investigate the mechanism by which the ECM exerts its effect on morphological and functional differentiation of the mammary gland, with specific emphasis on the interaction of ECM proteins with their cellular receptors (integrins and non integrins), and the mechanism by which this interaction alters cytoskeletal remodeling and thus milk protein gene expression. The overall goal of these studies is to determine the mechanism by which communication between the extracellular and intracellular matrices directs gene expression in normal cells, and to determine the mechanism(s) by which this regulation is disrupted in malignant cells. AIM 1. To study the role of the ECM in regulating the proliferation and differentiation of normal rat MEC (RMEC). This aim will focus on the effect of agents which disrupt the binding of fibronectin and laminin to their cellular receptors (with emphasis on the alpha5beta1 and alpha6beta4 integrins and the non integrin 32/67 kDa laminin receptor [LR]), with the goal of determining the importance of each of these receptors on cellular function. AIM 2. To determine the mechanism by which the ECM proteins laminin and fibronectin stimulate morphological and functional differentiation of RMEG. This aim will focus on the cytoskeletal changes that occur in response to binding of laminin or fibronectin to their receptors, or in response to disruption of this interaction, and assess whether TGF and/or protein tyrosine phosphorylation (especially of the actin attachment protein vinculin) are essential mediators of this signal transduction pathway. AIM 3. To investigate the regulation of integrin expression of normal RMEC. The integrin family of cell surface receptors are critical components in the pathway by which EGM proteins modulate proliferation and differentiation. In order to determine if changes in integrin expression and/or activity can modify EGM signalling, the functional significance of changes in expression will be assessed in normal RMEG, with focus on the integrins alpha5beta1 and alpha6beta4, as well as on LR AIM 4. To investigate the role of protein kinase C (PKC) (PKC) in modulating the response of normal RMEC to EGM proteins. In this aim, the effect of phorbol esters on (i) cytoskeletal redistribution of specific PKC isoenzymes and (ii) on the interaction of the RMEG with the EGM will be assessed, with the goal of determining whether a PKC-mediated phosphorylaUon event is required for a functional ECM-cellular interaction. AIM 5. To compare regulation of integrin and LR expression and function in normal and transformed MEC. The goal of these studies is to assess the importance of the integrins alpha5beta1 and alpha6beta4 and the non integrin LR in regulating the interaction of the cells with their ECM as well as to probe the significance of these proteins in altering the tumorigenic and metastatic potential of mammary tumor cells.

The mammary gland is a complex organ which is under a myriad of regulatory controls. These include circulating hormones and growth factors, as well as local trophic factors produced by the mammary epithelial cells (MEC) or by the stroma within which the MEC are embedded. In addition, components of the extracellular matrix (ECM) exert a profound effect on both morphological and functional development of the mammary gland. During progression of MEC towards malignancy, many of these regulatory influences are lost. Considerable evidence has accumulated that stromal - epithelial interaction is altered in breast cancer, however, the time-dependent progression of the events that contribute to this change during transformation have yet to be elucidated. The long-term objective of the studies proposed herein is to determine the functional, biochemical and molecular events that comprise this altered communication between the MEC and it surrounding stroma, with the goal of developing new leads for the preVention and/or therapy of breast cancer. AIM 1. To test the hypothesis that there are alterations in stroma and/or stromal-epithelial interactions during mammary carcinogenesis, stroma will be isolated from the intact or epithelium-free ("cleared") mammary gland of rats at several times after administration of the carcinogen DMBA and the ability of isolated adipocytes and fibroblasts, evaluated separately, to induce proliferation and/or differentiation of normal, initiated and malignant MEC evaluated. The complementary possibility that normal, initiated or transformed MEC can alter stromal mitogenesis, adherence or migratory capacity will also be examined. AIM 2. It is hypothesized that growth factors or inhibitors synthesized by the stroma, or by the MEC, affect the proliferation, function, and/or differentiation of the other cell type, and that the synthesis of such modulators is progressively altered during rat mammary carcinogenesis. This will be assessed by measurement of mRNA levels by Northern blot analysis or in situ hybridization, and protein levels by Western blot analysis, immunocytochemistry, or bioactivity assays, with the main focus on TGFbeta and TNFalpha. Follow-up studies will assess the cellular consequences of this alteration in growth modulator secretion, in an attempt to assess its physiological significance and to determine whether it may play a mechanistic role during carcinogenesis. AIM 3. A second, not mutually exclusive, hypothesis that may explain an altered stromal - epithelial interaction during carcinogenesis is that the known ability of the stroma and the MEC to elaborate ECM macromolecules and/or ECM remodelling enzymes is progressively altered during carcinogenesis in such a way as to favor the growth and invasion of the transformed MEC into the mammary fat pad. To test this, the ability of stroma and MEC isolated at various times during the course of mammary carcinogenesis to synthesize and secrete ECM proteins and matrix-degrading metalloproteinases will be evaluated. Follow-up studies will assess the functional consequences of this change in cellular/ECM interaction by determining whether invasiveness, motility, adherence, or transduction of the proliferative signal is altered in the MEC or stromal cells.

The mammary gland is a complex organ which is under a myriad of regulatory controls. These include circulating hormones and growth factors, as well as local trophic factors produced by the mammary epithelial cells (MEC) or by the stroma within which the MEC are embedded. We have made the unique observation that the cytokine tumor necrosis factor-alpha (TNF-alpha) can stimulate the proliferation and morphological differentiation of rat MEC and inhibit casein production. Intriguingly, other investigators have reported that TNF-alpha inhibits the growth of malignant breast epithelial cells. Since this cytokine is produced by some of the types of cell that form the mammary stroma, we propose to determine whether TNF-alpha is a physiological regulator of growth and differentiation in normal rat MEC, to undertake preliminary studies to assess the mechanism of its action and to determine whether the activity of TNF-alpha is altered in transformed cells. Specific aims are as follows. AIM 1. To assess whether TNF-alpha is a potential paracrine or autocrine regulator of mammary epithelial cell function. Studies will be undertaken to determine if TNF-alpha mRNA and its protein product are produced in mammary epithelial and stromal cells. If TNF-alpha is found to be synthesized by one or more cell types in the mammary gland, its hormonal regulation will be investigated. AIM 2. To determine whether the effects of TNF-alpha on the proliferation and differentiation of the MEC are direct or are mediated by another cytokine. These studies will focus on how quickly MEC respond to TNF-alpha by examining the time course of its effect on early response genes such as c- myc, c-fos and c-jun, as well as on overall DNA synthesis. Additionally, potential mediation by other cytokines or growth factors will be assessed by determining whether they are synthesized in response to TNF-alpha, the time course of such a response, and if neutralizing antibodies to these cytokines block and the cytokine itself mimics, the effects of TNF-alpha on proliferation and/or differentiation. AIM 3. To determine if TNF-alpha stimulates mammary gland development in vivo. In these studies, Elvax pellets of TNF-alpha will be implanted in the mammary fat pad of immature or of ovariectomized mature rats, and development of the implanted gland compared with that of the contralateral gland. AIM 4. To determine the mechanism of the inhibitory effect of TNF-alpha on casein synthesis by examining the effect of TNF-alpha on stability of beta-casein mRNA and on the casein protein family. A determination will also be made whether the inhibitory effect of TNF-alpha is specific for casein, or whether other markers of functional differentiation (lipid synthesis, expression of WAP and transferrin) are inhibited as well. AIM 5. The effect of TNF-alpha on proliferation and differentiation of transformed rat mammary epithelial cells will be investigated both in primary culture as well as in vivo.

DESCRIPTION: The overall goal of these studies is to investigate the mechanisms by which TNFalpha (TNFa) modulates the growth and differentiation of mammary epithelial cells (MEC). Dr. Ip has made the unique observation that TNFa stimulates the proliferation of normal rat MEC and induces extensive branching morphogenesis in vitro. Moreover, TNF either inhibits (through the p55 TNF receptor [TNFR]) or stimulates (through the p75 TNFR) functional differentiation. These effects of TNFa appear to be physiologically significant, since TNFa and the TNF receptors are regulated during normal mammary gland development. The following studies are proposed in order to gain an increased understanding of the mechanisms by which TNFa exerts these effects, with the overall objective of determining the role that TNFa may play in the development of breast cancer. The first specific aim is to elucidate the pathways through which TNFa stimulates proliferation of MEC, with initial emphasis on the erk1/erk2, JNK and p38 MAP kinase pathways, and the accessory role of the EGF receptor. The second is to test the hypothesis that TNFa is a physiological regulator of mammary gland branching morphogenesis in vivo, with emphasis on altered expression of MMP-9 and stromelysin-1 as possible mechanisms by which TNFa stimulates invasion of MEC into mammary fat pad. The third specific aim is to determine the mechanism by which TNFa modulates functional differentiation, with emphasis on the putative effect of this cytokine on transcription factors known to regulate whey acidic protein and beta-casein transcription. And finally, to examine the effects of TNFa on NMU-induced rat mammary tumors, when administered locally by Elvax implant within the mammary fat pad. These studies will test the hypothesis that the activity of TNFa changes as cells become transformed.

[unreadable] DESCRIPTION (provided by applicant): We have shown tumor necrosis factor (TNF) to be a multi-functional regulator of normal postnatal mammary gland development. Our data suggests that TNF, acting through the p55 TNF receptor, plays an active stimulatory role in the rapid proliferation that occurs during pregnancy, and in the proliferation and branching morphogenesis that occur during puberty. Significantly, branching morphogenesis of the mammary ductal epithelium is inhibited in TNF null mice, demonstrating the physiological relevance of this cytokine in the mammary gland. Surprisingly, in spite of numerous studies demonstrating that TNF inhibits the growth or induces apoptosis of breast cancer cell lines, we found that TNF stimulates the growth of mammary tumor epithelial cells in primary culture, suggesting that TNF therapy may be contra-indicated in breast cancer. The stimulatory effect of TNF on proliferation of normal epithelial cells (MEC) is accompanied by an increase in DNA-binding activity of the NFkB1/p50 homodimer. Moreover, in newly isolated tumor MEC in which p50 protein and NFkB1/p50 homodimeric-DNA binding are absent, TNF does not affect cell growth; however, upon p50 expression in the tumor cells, TNF induction of proliferation is observed. This suggests that NFkB1/p50 is required for TNF stimulation of growth of both normal and malignant MEC. In Aim 1, the mechanism by which NFkB1/p50 expression and activity are regulated by TNF in normal and malignant MEC will be investigated. Aim 2 will determine if there is an absolute requirement for the NFkB1/p50 protein in TNF-induced proliferation of MEC, or whether another NFkB/rel protein can functionally compensate for p50. Aim 3 will determine the sensitivity of the mammary gland from TNF null mice to carcinogenesis. Since TNF stimulates the growth of both normal and malignant MEC in primary culture, we propose that it may stimulate, rather than inhibit manmaary tumor growth. Thus, as part of this aim, the efficacy of anti-TNF therapy vs. local TNF therapy against mammary tumors will be compared.