Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Growth of preneoplastic mammary epithelial cells in serum-free medium

The conditions for growing preneoplastic mammary epithelial cells in primary cell cultures with serum-free factor-defined medium were established in a series of experiments. The cells, grown in Dulbecco's modified Eagle's medium with 0.6 mM Ca2+, epidermal growth factor, transferrin, insulin, and Na2SeO3, were fibroblast free, exhibited saturation densities similar to those of cells grown in serum-containing medium, and actively incorporated [3H]thymidine into DNA, as demonstrated by biochemical and autoradiography assays. The total growth fraction over a seven-day experiment was 77%. Fetuin was sufficient as a serum substitute for attachment of cells to the dish, although it was apparently not necessary for continued growth of the cells. The possible benefits of growing preneoplastic mammary epithelial cells in serum-free factor-defined medium are discussed.     

Production of basic fibroblast growth factor-like factor by cultured human cholangiocellular carcinoma cells

An extract of cultured human cholangiocellular carcinoma cells (HuCC-T1) was found to contain high mitogenic activity for BALB/c3T3 cells. The growth factor eliciting most of the mitogenic activity was purified and concluded to be identical with basic fibroblast growth factor (bFGF)-like factor on the basis of its molecular weight and heparin-Sepharose elution profile, and the results of immunoblotting and radioimmunoassay. HuCC-T1 cells also secreted bFGF-like factor into serum-free medium. A combination of insulin and transferrin or bovine serum albumin stimulated the growth of HuCC-T1 cells in serum-free medium. However, bFGF did not stimulate their growth in the presence and absence of these supplements. Neutralizing monoclonal antibody against bFGF did not inhibit growth. These results indicate that bFGF-like factor is not a growth factor for this cell line.     

脑膜瘤组织中bFGF及FGFR-1蛋白表达的研究

目的　探讨碱性成纤维生长因子 (basicfibroblastgrowthfactor ,bFGF)及成纤维生长因子受体 1(fibroblastgrowthfactorreceptor 1,FGFR 1)在脑膜瘤中的表达及其与脑膜瘤组织病理学和复发的关系。方法　用免疫组化技术检测bFGF、FGFR 1在不同类型的脑膜瘤组织中的蛋白表达 ,用组织病理学判断脑膜瘤的良恶性。结果　脑膜瘤细胞有不同程度的bFGF及FGFR 1表达 ,其表达阳性率与肿瘤的良恶性和复发有关。结论　bFGF和FGFR具有促进脑膜瘤细胞的增殖和生长的作用。脑膜瘤表达bFGF、FGFR的阳性率可作为鉴别肿瘤良恶性的有用指标 ,并对脑膜瘤的预后和术后复发起提示作用。     

Effects of growth factors and hormones on growth and morphological differentiation of human breast epithelial cells within collagen gel in serum-free medium

When human epithelial cells that had grown out from either carcinoma or histologically non-malignant breast tissues were seeded within type I collagen gels in serum-free medium, they successively grew and protruded many radial duct-like extensions with lumina. Separate deletion of each of the supplements from the medium showed that growth as well as morphological differentiation of carcinoma-derived cells were prevented in the absence of epidermal growth factor (EGF) or hydrocortisone. Removal of insulin or ethanolamine plus phosphoethanolamine caused a significant inhibition of cell growth without interfering with the morphological differentiation. Contrary to the case with carcinoma-derived cells, both growth and morphological differentiation of epithelial cells derived from non-malignant breast tissues were prevented when EGF, hydrocortisone or insulin was absent. Removal of each of the other supplements (except for transferrin), including ethanolamine plus phosphoethanolamine, prolactin, or prostaglandin, caused a significant inhibition of cell growth with no apparent inhibition of morphological differentiation. The present results suggest that human epithelial cells derived from either carcinoma or histologically non-malignant breast tissues strongly depend on the presence of EGF and hydrocortisone and there is a decreased dependence on insulin in carcinoma-derived cells with respect to their growth and morphological differentiation during culture within collagen gels.     

Invasion of human colorectal carcinoma cells is promoted by endogenous basic fibroblast growth factor

The growth-stimulatory and invasion-promoting effects of basic fibroblast growth factor (bFGF) were examined in 2 series of related human colon carcinoma cell lines (HCT116A, HCT116B and 20-10-1 as well as and LS180, LS174T and ARK1A) that exhibit different invasive potentials. The invasive cell lines 20-10-1 and ARK1A grew more rapidly than their non-invasive counterparts; exogenously added bFGF stimulated the proliferation of all the cells. When extracts of the cells were fractionated on columns of heparin-Sepharose, bFGF-like activity was found in extracts from each cell line. The amount of bFGF-like growth-stimulatory activity was greater in the more invasive cells: the invasive cells 20-10-1 contained 35-fold more activity than the non-invasive HCT116A cells, and the ARK1A cells contained 15-fold more activity than LS180 cells. Relatively small amounts of bFGF-like activity were recovered from medium conditioned by the invasive cells. The bFGF-like growth-stimulatory activity from the cell extracts was neutralised by an antibody to bFGF, and immunoblotting revealed the presence of an 18 kDa immunoreactive polypeptide, consistent with the presence of bFGF in the cell extracts. Exogenously added bFGF caused the usually non-invasive HCT116A cells to invade collagen gels. The HCT116B and 20-10-1 cells that were naturally invasive in a collagen gel assay also showed increased levels of invasiveness in the presence of bFGF, but an antibody that neutralised the activity of bFGF reduced the constitutive invasiveness of these cells. Our results suggest a causal relationship between the endogenous production of bFGF and the invasive potential of human colon carcinoma cells. Int. J. Cancer 71:390-395, 1997. © 1997 Wiley-Liss Inc.     

Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor

Human melanoma cells express two prominent angiogenic factors, e.g., vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF/fibroblast growth factor-2). In this study, we report on the relative contribution of these two factors to in vitro and in vivo growth of a tumorigenic melanoma cell line (WM164) and nontumorigenic, immortalized melanocytes (FM516SV). Overexpression of either cytokine significantly boosted tumorigenicity of WM164 cells in immunodeficient SCID mice. Attempting to overexpress bFGF antisense sequences produced no viable clones confirming earlier reports that autocrine bFGF is obligatory to melanoma cell survival and growth. By contrast, down-regulation of endogenous VEGF production did not affect growth of WM164 cells in vitro. In vivo expansion of WM164 cells expressing VEGF antisense was delayed but not abrogated. Forced expression of either bFGF or VEGF in immortalized but nontumorigenic melanocytes did not induce sustained tumor growth in vivo highlighting that neither of the two factors is sufficient for induction of tumorigenicity in this model system. Overexpression of either cytokine in WM164 cells led to the development of atypical large vessels but not to an increase in microvessel density. Taken together our results confirm an essential autocrine role of bFGF in human melanoma and indicate a beneficial but nonessential role of VEGF in the tumorigenic phenotype of human melanoma cells.     

Modulation of plasminogen activator activity in human endometrial adenocarcinoma cells by basic fibroblast growth factor and transforming growth factor beta

Human endometrial adenocarcinoma HEC-1-A and HEC-1-B cells produce and secrete the urokinase-type plasminogen activator and trace amounts of the tissue-type plasminogen activator. The two clones, which originate from the same tumor, possess high affinity binding sites for basic fibroblast growth factor (bFGF) and they respond to the addition of human recombinant bFGF with an increase of the synthesis and secretion of urokinase-type and tissue-type plasminogen activators and with an increase in cell proliferation. Transforming growth factor beta, (TGF beta), when added alone or 6 h before bFGF, induces an increase of plasminogen activator synthesis in both cell lines and stimulates the production of the endothelial cell-type plasminogen activator inhibitor in HEC-1-A cells, but not in HEC-1-B cells. Moreover, TGF beta inhibits basal proliferation of both cell lines and suppresses the mitogenic activity of bFGF. We have previously demonstrated that HEC-1-A and HEC-1-B cells produce significant amounts of bFGF. On the basis of the well-established coordinate modulation of solid tumor growth and plasminogen activator production, our data suggest that bFGF may contribute, in an autocrine fashion, to the development of endometrial carcinomas. Moreover, endometrial tumor cells appear also to be a target for TGF beta. Our results demonstrate also that significant qualitative and quantitative differences in the response to growth factors exist in clones derived from the same tumor, and support the view that the properties of in vivo tumors cannot be extrapolated from results obtained with a single isolate of tumor cells.     

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance

Introduction

Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods

Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results

TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion

These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.     

Inhibitory effect of antibody against basic fibroblast growth factor on androgen- or glucocorticoid-induced growth of Shionogi carcinoma 115 cells in serum-free culture

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. However, we and others recently found that the growth of SC115 cells is also stimulated by high doses of glucocorticoids. We already reported the following findings. In a serum-free medium [Ham's F-12: Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin], greater than or equal to 10(-9) M testosterone and greater than or equal to 10(-8) M dexamethasone significantly stimulated the growth of SC-3 cells (a cloned cell line from a SC115 tumor) through androgen and glucocorticoid receptors, respectively. In the present study, we have demonstrated that higher concentrations (10(-7)-10(-6) M) of weak androgens such as 4-androstene-3,17-dione or weak glucocorticoids such as corticosterone also significantly stimulate the growth of SC-3 cells and that their relative potency is found to be in parallel with their binding affinity for their receptors, respectively. Furthermore, DNA synthesis of SC-3 cells induced by 0.1 ng/ml basic fibroblast growth factor (FGF), 10(-8) M testosterone, 10(-6) M 4-androstene-3,17-dione, 10(-7) M dexamethasone, or 10(-6) M corticosterone was found to be similarly and significantly inhibited by the addition of basic FGF neutralizing antibody IgG in the present study; approximately 70% inhibition of the basic FGF, androgen, or glucocorticoid effects was attained. We already reported findings which suggest that SC-3 cells produce FGF-like peptide for their testosterone-induced growth. Therefore, the present study presents new additive information to demonstrate that the growth-stimulatory activity of various androgens or possibly glucocorticoids on SC-3 cells is mediated through a FGF-like peptide in an autocrine mechanism.     

Correlation between basic fibroblast growth factor immunostaining of stromal cells and stromelysin-3 mRNA expression in human breast carcinoma.

We examined the localization of basic fibroblast growth factor (bFGF) in a series of human breast carcinomas using immunohistochemistry. Staining was observed in tumour cells in 15 out of 54 (28%) tumours and in the adjacent stroma in 34 out of 54 (63%) tumours examined. No correlation was observed between positive staining of these two compartments. The relationship between bFGF staining and expression of the metalloprotease stromelysin-3, and between bFGF and microvessel density, was examined. A statistically significant correlation (P < 0.003) was observed between bFGF staining of the stromal compartment and high expression of stromelysin-3 (ST-3; MMP-11) metalloprotease mRNA by stromal cells. In contrast, no correlation was observed between bFGF and intratumour microvessel density (IMD). These results raise the possibility that bFGF may be involved in the induction of stromelysin-3 mRNA expression in breast cancer stroma.     

Overexpression of basic fibroblast growth factor and autocrine stimulation in acute myeloid leukemia

Basic fibroblast growth factor (bFGF) is known to play a critical role in tumorigenesis of solid tumors. The importance of bFGF in hematological malignancies such as acute myeloid leukemia (AML) remains to be elucidated. Therefore, we determined bFGF protein expression by immunohistochemical analyses in bone marrow biopsies of patients with newly diagnosed, untreated AML. The expression of bFGF was significantly increased in AML patients [n = 81; median, 3.0 (interquartile range, 1.8-3.9) arbitrary units (AU)] as compared with controls [n = 18; 1.9 (1.5-2.3) AU]. The degree of bFGF expression did not correlate with microvessel density. bFGF/FGF receptor mRNA and bFGF protein were detected in different AML cell lines. To study autocrine growth stimulation of AML blasts, the AML cell lines HL-60, M-07e, and KG-1 were incubated with bFGF. A significant dose-dependent increase in proliferation and colony formation was observed. These effects were abrogated by the addition of a polyclonal anti-bFGF antibody. In conclusion, increased expression of bFGF in the bone marrow of AML patients seems to play an important role in the pathophysiology of AML by promoting autocrine growth stimulation of leukemic blasts.     

Nuclear accumulation of basic fibroblast growth factor in human astrocytic tumors

BACKGROUND: The authors recently reported that nuclear accumulation of basic fibroblast growth factor (bFGF) demonstrated a significant correlation with recurrence of pituitary adenomas. The current study sought to determine whether nuclear bFGF accumulation was a predictor of survival in patients with astrocytic tumors. METHODS: The authors examined 52 patients with primary astrocytic tumors. Immunohistochemical assays for bFGF, fibroblast growth factor receptor 1 (FGFR1), and proliferating cell nuclear antigen were performed. Immunoreactivity of bFGF in nuclei was recorded in terms of the bFGF nuclear index (NI), which was calculated as the percentage of tumor cells with nuclear immunoreactivity. Western blot analysis of bFGF in nuclear fractions was performed. RESULTS: The bFGF NI had a mean value of 35.1% and was < 30% (low NI) in 27 patients and >or= 30% (high NI) in 25 patients. In all cases, FGFR1 immunoreactivity was observed in the cytoplasm but not in the nucleus. Western blot analysis indicated that the nuclear fractions from tumor specimens with high NI contained high-molecular-weight bFGF. Univariate analyses showed that age, tumor histology, gender, and bFGF NI were significantly correlated with patient survival. Multivariate analyses demonstrated that NI had the greatest influence (P = 0.0073) on survival rate, compared with age (P = 0.0083) and gender (P = 0.0492). Compared with low NI, high NI was associated with a relative risk of 3.292. CONCLUSIONS: The findings of the current study suggest that bFGF NI may be a useful predictor of survival in patients with astrocytic tumors.     

Nipple fluid basic fibroblast growth factor in patients with breast cancer.

PURPOSE: It has been shown that early detection of breast cancer could save lives. Recently, there has been increasing interest in nipple fluid as a potential supplemental avenue for breast cancer diagnosis. EXPERIMENTAL DESIGN: In this study, we determined the levels of an angiogenic factor basic fibroblast growth factor (bFGF) in the nipple fluid of healthy subjects as well as patients with benign breast conditions, those at high risk for breast cancer, and patients with active breast cancer. ELISAs were used to measure bFGF. RESULTS: Nipple fluid bFGF levels were as follows (mean +/- SE): 158 +/- 17 pg/mL from benign breasts, 561 +/- 277 pg/mL from high-risk breasts, and 1,343 +/- 441 pg/mL from cancerous breasts. One-way ANOVA showed that the bFGF levels from cancerous breasts were significantly higher than those from benign and high-risk breasts (P = 0.0001 and P = 0.0193, respectively). After logarithmic transformation was applied to the data, high-risk breast bFGF levels were higher than those from benign breasts (P = 0.0028). With a cutoff level of 250 pg/mL, the sensitivity was 79.2%, specificity was 82.5%, and correct diagnosis was 66.4%. The area under the receiver operating characteristic curve was 0.86. CONCLUSIONS: We conclude that nipple fluid bFGF levels are progressively elevated in high-risk and cancerous breasts compared with benign breasts. The sensitivity and specificity of this test are promising compared with current breast cancer screening methods, and this test deserves further studies with larger clinical trials. Potential areas of usefulness include the detection of breast cancer risk or breast cancer, as well as the monitoring and/or prediction of the antiangiogenic effect of preventive therapies.     

Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain

Primary cultures of epithelial cells from human prostate acini proliferate in defined medium. However, the limited availability of human tissue and the lack of knowledge of the conditions required for clonal growth and serial culture of epithelial cells have limited progress in the study of human prostate cell biology. Here we report conditions that permit the proliferation of single epithelial cells from normal, benign hyperplastic, and carcinomatous prostate through three to four serial passages, which represents at least seven to nine cumulative doublings of the cell populations. Primary cultures were prepared from prostatic acini. Monolayers resulting from the outgrowth of epithelial cells from acini were harvested and dissociated into suspensions of single cells which gave rise to discrete colonies in subsequent culture. The requirements for successful serial culture were (a) a low calcium concentration, (b) the presence of a growth factor that is concentrated in bovine neural tissue, (c), detachment of the epithelial cells with collagenase, and (d) harvest of cells before the cell concentration reached 6000 cells/cm2 of culture surface. Suspensions of single cells were successfully stored between subcultures in 10% dimethylsulfoxide with 5% fetal bovine serum and revived after storage for up to 2 months in liquid nitrogen.     

Transformation of human breast epithelial cells by 7, 12, dimethylbenz(a)anthracene, but not by N-methyl-N-nitrosourea, is accompanied by up-regulation of basic fibroblast growth factor

Normal human breast epithelial cells (HBL100) are immortalized by endogenous SV40 genome and are not tumorigenic in nude mice if injected in the first 50 passages in culture. This cell line also depends on the expression of basic fibroblast growth factor (bFGF) for its viability in culture. The present study was designed to transform these cells with chemical carcinogens to establish malignant HBL100 cell lines in order to evaluate the eventual modulation in the expression of bFGF. In the first experiment, HBL100 cells were treated with 2 mu g/ml of 7, 12, dimethylbenz(a)anthracene (DMBA) for two 24 h periods. In a second experiment, HBL100 cells were transformed by exposing them to 20 mu g/ml of N-methyl-N-nitrosourea (MNU) twice a day for two days. Unlike the HBL100 parental cell line, the two newly derived cell lines (named respectively HBLTDMBA and HBLTMNU) were tumorigenic when injected in nude mice. The expression of bFGF, as measured by immunocytochemistry and Western blot analysis, was higher in the DMBA-transformed cell line than in the parental HBL100 and MNU-transformed lines. These results differentiate between transformation selectivity by two different carcinogens. The direct-acting carcinogen MNU, which induces point mutation, does not require enhanced expression of bFGF, whereas bFGF may be necessary for early events leading towards transformation by carcinogens requiring metabolism for their action, such as DMBA.     

Growth and continuous passage of COMMA-D mouse mammary epithelial cells in hormonally defined serum-free medium

Growth of the mouse mammary epithelial cell line designated COMMA-D has been studied in serum-free medium (SFM) formulated with Ham's F12 and Dulbecco's modified Eagle's medium (1/1) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM glutamine, gentamicin (50 micrograms/ml; basal medium) and supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), selenous acid (10 ng/ml), epidermal growth factor (20 ng/ml; EGF), 10 nM 3,5,3'-triiodothyronine, 50 microM ethanolamine, 1.0 nM 17 beta-estradiol, 65 microM glutathione, and ovalbumin (100 micrograms/ml). COMMA-D cells were able to undergo serial passage and continued to exhibit dome formation after 20 passages in SFM. Cells seeded at low density in SFM underwent four population doublings at low passage number in 1 week compared to six doublings for cells grown in medium containing insulin, transferrin, selenium, EGF, and 1% fetal bovine serum. After many passages in SFM, the growth rates of cells were similar to those in serum-supplemented medium used for stock culture. Deletion of insulin or EGF from SFM resulted in cell growth similar to that of cells seeded in basal medium alone. When cells were seeded in basal medium without added supplements, addition of insulin or EGF resulted in 29 and 22%, respectively, of the number of cells grown in SFM for 5 days. However, when insulin and EGF were combined in basal medium, the cell number at 5 days was 83% of that in SFM. When insulin was deleted from SFM, COMMA-D cells became responsive to insulin-like growth factors I and II. The growth-promoting characteristics of EGF and transforming growth factor alpha were compared in SFM and were not distinguishable, showing identical dose-response curves. When incorporation of [3H]thymidine was used as an assay of cell growth, saturating levels of basic fibroblast growth factor (20 ng/ml) showed a stimulation 1.35 times greater than EGF (20 ng/ml). When EGF and fibroblast growth factor were combined, the stimulation was 1.75 times greater than EGF alone suggesting that COMMA-D cells are responsive to multiple classes of growth factors. COMMA-D cells seeded in basal medium supplemented with insulin, transferrin, and selenous acid have been used to detect mitogenic activity present in extracts of hypothalamus, uterus, and pituitary. The results show that COMMA-D cells can be grown long term in a hormonally defined serum-free medium and that maximal mitogenic effects were seen only with the addition of two or more growth factors.