Growth regulation of human breast carcinoma occurs through regulated growth factor secretion

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine and paracrine growth regulation of human breast cancer

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.     

Alterations in phosphoinositide metabolism associated with 17 beta-estradiol and growth factor treatment of MCF-7 breast cancer cells

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.     

Growth stimulation and differential regulation of transforming growth factor-beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 messenger RNA levels by norethindrone in MCF-7 human breast cancer cells.

Transforming growth factor-beta (TGF beta) is a potent growth inhibitor in most epithelial cells. We evaluated the effects of norethindrone (which in combination with estrogen is commonly used in oral contraceptives) and other progestins [medioxyprogesterone acetate (MPA) and R5020, which are not used in oral contraceptives] on cell growth and the expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs in MCF-7 human breast cancer cells. Growth of MCF-7 cells was stimulated by norethindrone (10(-8)-10(-5) M), with maximal growth stimulation at 10(-7) M norethindrone after 7 days of treatment. However, the growth of MCF-7 cells was not affected by MPA (10(-8) M) or R5020 (10(-8) M). Treatment with the antiestrogen 4-hydroxytamoxifen at a concentration of 10(-7) M blocked the growth stimulation induced by norethindrone. The norethindrone-induced growth stimulation was accompanied by a dramatic decrease in TGF beta 2 and TGF beta 3 mRNA levels, whereas the level of TGF beta 1 mRNA was not affected by any of the compounds tested. In addition, treatment with MPA or R5020 did not affect TGF beta 2 and TGF beta 3 mRNA levels. The inhibitory effect of norethindrone on TGF beta 2 and TGF beta 3 mRNA levels could be blocked by the addition of 10(-7) M 4-hydroxytamoxifen. Norethindrone as well as estradiol decreased estrogen receptor mRNA levels and increased progesterone receptor mRNA levels. This is the first report which demonstrates that norethindrone stimulates estrogen-responsive human breast cancer cell growth and inhibits the expression of TGF beta 2 and TGF beta 3 mRNAs. These results suggest that the differential regulation of TGF beta expression by norethindrone may be at least partly responsible for the growth stimulation induced by norethindrone. Thus, the norethindrone component of some oral contraceptives may be sufficiently estrogenic to facilitate the development of breast cancer.     

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer cells.     

Epidermal growth factor partially reverses the inhibitory effects of antiestrogens on T 47D human breast cancer cell growth

When T 47D human breast cancer cells were treated with 10 nM of the potent antiestrogen, 4-hydroxyclomiphene, growth rate was reduced to about 50% of control. Simultaneous treatment with epidermal growth factor (EGF) and 4-hydroxyclomiphene led to a partial reversal of the growth inhibitory effect of the antiestrogen. The effect of EGF was concentration-dependent being half-maximal at 0.10 ng/ml (0.02 nM) and maximal at concentrations greater than 0.5 ng/ml (greater than 0.08 nM). Furthermore, EGF partially reversed the growth inhibitory effects of several other antiestrogens including tamoxifen, 4-hydroxytamoxifen, and LY 117018. These results are compatible with the hypothesis that part of the growth inhibitory effects of antiestrogens on breast cancer cell proliferation are mediated by inhibition of autocrine secretion of growth stimulatory peptides acting through the EGF receptor.     

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.     

Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells.

Retinoids are known to inhibit the growth of hormone-dependent but not that of hormone-independent breast cancer cells. We investigated the involvement of retinoic acid (RA) receptors (RARs) in the differential growth-inhibitory effects of retinoids and the underlying mechanism. Our data demonstrate that induction of RAR beta by RA correlates with the growth-inhibitory effect of retinoids. The hormone-independent cells acquired RA sensitivity when the RAR beta expression vector was introduced and expressed in the cells. In addition, RA sensitivity of hormone-dependent cells was inhibited by a RAR beta-selective antagonist and the expression of RAR beta antisense RNA. Introduction of RAR alpha also restored RA sensitivity in hormone-independent cells, but this restoration was accomplished by the induction of endogenous RAR beta expression. Furthermore, we show that induction of apoptosis contributes to the growth-inhibitory effect of RAR beta. Thus, RAR beta can mediate retinoid action in breast cancer cells by promoting apoptosis. Loss of RAR beta, therefore, may contribute to the tumorigenicity of human mammary epithelial cells.     

Transforming growth factor-beta 1 overproduction in prostate cancer: effects on growth in vivo and in vitro.

We found previously that transforming growth factor-beta 1 (TGF beta 1) mRNA levels are markedly elevated in rat prostate cancer (Dunning R3327 sublines) compared to levels in normal prostate. Our goal was to determine whether elevated expression of TGF beta 1 is biologically relevant to prostate cancer growth in vivo. We chose as our model the R3327-MATLyLu prostate cancer epithelial cell line, which produces metastatic anaplastic tumors when reinoculated in vivo. Our approach was to stably transfect MATLyLu cells with an expression vector that codes for latent TGF beta 1 and to isolate subclones of cells that over-expressed TGF beta 1 mRNA. We also isolated a subclone of MATLyLu cells transfected with a control vector lacking the TGF beta 1 cDNA insert. We then studied the growth of these cells in vivo and in vitro. Twenty days after sc inoculation of 10(6) cells in vivo, TGF beta 1-overproducing MATLyLu tumors were 50% larger, markedly less necrotic, and produced more extensive metastatic disease (lung metastases in 73% of all lobes and lymph node metastases in 88% of animals) compared to control MATLyLu tumors (lung metastases, 21%; lymph node metastases, 7%). Thus, TGF beta 1 produced in vivo is biologically active and can promote prostate cancer growth, viability, and aggressiveness, perhaps via effects on the host and/or on the tumor cells themselves. When followed in vitro, TGF beta 1-overproducing cells became growth inhibited, but this effect was transient as cells subsequently resumed proliferating. Growth inhibition was due to TGF beta, because it could be prevented by TGF beta-neutralizing antibody. Therefore, prostate cancer cells can activate and respond to secreted latent TGF beta 1, and although the cells are transiently inhibited in vitro, there is no net inhibition of growth. The ability of the cells to respond to endogenously produced TGF beta 1 suggests that TGF beta 1 overexpression enhances tumor growth in vivo at least in part via an effect of TGF beta 1 on the tumor cells themselves.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.     

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.     

Modulation of hepatic lipocyte proteoglycan synthesis and proliferation by Kupffer cell-derived transforming growth factors type beta 1 and type alpha.

Soluble mediators elaborated by activated Kupffer cells have been implicated in the activation of liver fat-storing cells. In the present study some of these factors were identified as TGF beta and TGF alpha affecting disparate reactions in the activation process. TGF beta is secreted in an inactive, latent form by Kupffer cells. It is activated after addition to primary FSC cultures and stimulates dose-dependently sulfated proteoglycan synthesis especially that of chondroitin sulfate, whereas the incorporation of [3H] thymidine is reduced significantly. These effects were neutralized completely by anti-TGF beta antibodies which ultimately converted the proliferation inhibitory effect of Kupffer cell medium in a proliferation stimulatory action. The latter is at least partially due to TGF alpha. Both cytokines are preferentially expressed in activated Kupffer cells. We conclude that Kupffer cells modulate the mitogenic activity of FSC in culture depending on the ratio of activated TGF beta and TGF alpha and affect chondroitin sulfate synthesis mainly by TGF beta. The results suggest a paracrine activation of FSC in injured liver by both transforming growth factors secreted by activated Kupffer cells.