EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.     

Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)

Human oncostatin M (OM) is a M(r) 28,000 glycoprotein that has been shown to regulate cell proliferation and differentiation. The biological activities of OM can be mediated by two different heterodimeric receptor complexes, the leukemia inhibitory factor (LIF)/OM shared receptor and the OM-specific receptor. In this study, we have examined the growth-regulatory effect of OM on 10 breast cancer cell lines derived from human tumors. The cellular proliferation of seven of these breast cancer cell lines was inhibited by OM. The three cell lines that did not respond to OM treatment lacked the expression of OM receptors. The growth-inhibitory activity of OM is examined further in the H3922 breast cancer cell line, which expresses the high-affinity OM receptor at a relatively higher level. We found that the cellular proliferation of H3922 cells was induced strongly by extrogenous epidermal growth factor (EGF), EGF-like factor, and basic fibroblast growth factor. The proliferative activities of these growth factors can be abolished totally by cotreatment of H3922 cells with OM. Treatment of H3922 cells with OM for 24 h did not block EGF binding or the induction of EGF receptor tyrosine phosphorylation. This finding suggests that OM interferes with the mitogenic signal at steps distal to the EGF receptor. Examination of proto-oncogene expression demonstrated that OM down-regulates the c-myc gene in H3922 cells. The biological effects reported herein are not shared by the OM-related cytokines interleukin 6 or LIF, as demonstrated by the inability of these proteins to inhibit cell growth or modulate c-myc gene expression in breast cancer cells. Additionally, the high-affinity binding of labeled OM cannot be displaced by LIF. Together, these data suggest that OM is a growth inhibitor for breast cancer cells. The inhibitory activity is mediated predominantly through the OM-specific receptor, and activation of this receptor abrogates growth factor stimulation and down-regulates the c-myc proto-oncogene.     

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and diindolylmethane (DIM) is the major acid-catalyzed condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah-responsive T47D human breast cancer cells, I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 microM, respectively. A 1 nM concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells, and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31 microM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.     

Pharmacokinetics and pharmacodynamics of candesartan after administration of its pro-drug candesartan cilexetil in patients with mild to moderate essential hypertension--a population analysis

Among the peptide growth factors active in breast glandular cell proliferation epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are thought to play a major role in tumour development. They operate through binding to and activation of a common membrane receptor, defined as EGF-R. Their production is modulated by hormones and local growth factors. After it was shown by previous investigation in this laboratory that EGF-R could be detected in 90% of the tumours, but was masked by endogenous ligand in 36% of them, the question was raised as to the level of the ligand's expression in tumour tissue biopsies. Therefore, we investigated the expression of EGF and TGF alpha mRNA in 146 breast cancer biopsies by slot blot analysis using specific 32P-labelled probes. The data were correlated with sex steroids and EGF receptor content. Our results showed that EGF and TGF alpha coexisted in all tumour samples, and that their level of mRNA expression was similar in half of the tumours. Northern blot and polymerase chain reaction (PCR) analysis validated these findings. A significant direct correlation was found between the level of TGF alpha/EGF mRNA expression and the ER/progesterone receptor (PGR) content. TGF alpha and EGF mRNA levels were significantly higher in ER+ (P = 0.0015 and P = 0.0001, respectively) and in PGR+ tumours (P < 0.005 and P = 0.0001) than in their negative counterparts. Moreover, TGF alpha mRNA expression negatively correlated with the number of EGF-R binding sites measured by the standard method (P = 0.02), and it was significantly related to the number of sites occupied by endogenous ligand. In conclusion, it was shown that TGF alpha and EGF mRNA were coexpressed in all the tumour biopsies tested and that their level was higher in the hormone receptor positive than in negative samples. The correlation between the presence of ER/PGR sites, high level of TGF alpha/EGF mRNA and EGF-R occupancy by endogenous ligand is in favour of ER mediated control of TGF alpha and EGF production.     

Platelet-derived growth factor-B increases colon cancer cell growth in vivo by a paracrine effect

PDGF-B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B-chain in cells grown in vitro and the number of factor VIII-positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF-B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c-K-ras genes (Huang et al. [1994] Oncogene, 9:3701-3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF-AB chains. An inverse correlation was found between induction of factor VIII-positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGF alpha or k-FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGF beta 1 was capable of inducing PDGF-B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGF beta 1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGF beta 1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF-B.     

Antiestrogenic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17 beta-estradiol-induced pS2 expression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits a broad spectrum of antiestrogenic activities in rodents and mammalian cells in culture. The effects of TCDD on 17 beta-estradiol (E2)-induction of pS2, a prognostic marker for breast cancer, were investigated in MCF-7, ZR-75, HeLa, and Hepa 1c1c7 wild-type and mutant cells. These effects were compared to the suppressive activities of the congener, 2,8-dichlorodibenzo-p-dioxin, and the established antiestrogens, ICI 164,384 and tamoxifen, in order to determine the relative potency of TCDD and to distinguish the mechanism of action of Ah receptor-mediated antiestrogens. Treatment of MCF-7 cells with 10 nM TCDD decreased E2-induced secreted pS2 protein levels by 50% and the induction of the transiently transfected -1100 to -86 pS2 promoter-regulated reporter gene (pS2-LUC) by 57%. Comparable effects on PS2-LUC activity were observed in HeLa and ZR-75 cells. In contrast, TCDD had minimal effects on pS2ERE(-405 to -393)-LUC induction, whereas treatment with 10 nM ICI 164,384 caused a 60% decrease in luciferase activity. In Hepa 1c1c7 wild-type and clone 1 (C1) mutant cells, TCDD also reduced E2 induction of pS2-LUC activity but had little effect in clone 4 (C4) or clone 12 (C12) mutant cells. However, suppression was reestablished following transfection of the human Ah receptor nuclear translocator (ARNT) complementary DNA expression vector into C4 cells and the mouse Ah receptor (AhR) complementary DNA expression vector into C12 cells. Induction of pS2-LUC activity by the ligand-dependent and -independent chimeric estrogen receptors (HE15, HE19, ERcVP16, and ERGR) were also used to examine the role of E2 metabolism and the mechanism of TCDD-mediated antiestrogenic activity. Induction by HE15 and ERcVP16 was suppressed by 57 and 74%, respectively, following treatment with TCDD, whereas ICI 164,384 was significantly less effective (38 and 20%, respectively). These results demonstrate a role for the Ah receptor in TCDD-mediated suppression of E2-induced pS2 expression. Data is presented demonstrating that the effect requires sequences within the pS2 promoter other than the estrogen response element and is independent of E2 oxidative metabolism.     

The effects of growth factors on human normal placental cytotrophoblast cell proliferation

The effects of growth factors were investigated on the proliferation of a normal placental cytotrophoblast cell line (NPC). Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast, TGFbeta1 was found to be a negative regulator, inhibiting EGF-induced cell proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand binding, two binding sites of different affinities were revealed in the proliferating NPC cells but only the low affinity binding site was detected in the non-proliferating cytotrophoblast cells in primary cultures. The results suggest that EGF stimulates cytotrophoblast proliferation through high affinity binding sites.     

Chronic pancreatitis is associated with increased concentrations of epidermal growth factor receptor, transforming growth factor alpha, and phospholipase C gamma

The epidermal growth factor (EGF) receptor is a transmembrane protein that binds EGF and transforming growth factor alpha (TGF alpha), and that stimulates phospholipase C gamma 1 (PLC gamma 1) activity. In this study the role of the EGF receptor in chronic pancreatitis was studied. By immunohistochemistry, the EGF receptor, TGF alpha, and PLC gamma 1 were found to be expressed at high concentrations in pancreatic ductal and acinar cells from chronic pancreatitis patients. Northern blot analysis showed that, by comparison with normal controls, 19 of 27 chronic pancreatitis tissues exhibited a 5.7-fold increase in EGF receptor mRNA concentrations, and 20 of 27 chronic pancreatitis tissues exhibited a sixfold increase in TGF alpha mRNA concentrations. In situ hybridisation confirmed that overexpression occurred in ductal and acinar cells, and showed that both mRNA moieties colocalised with their respective proteins. These findings suggest that TGF alpha may act through autocrine and paracrine mechanisms to excessively activate the overexpressed EGF receptor in the two major cell types of the exocrine pancreas, thereby contributing to the pathobiology of this disorder.     

Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells

Flavopiridol (L86-8275) is a synthetic flavone currently undergoing Phase I clinical trials. It is active against a series of human cancer cell lines and has been shown to inhibit a broad range of protein kinases, including cyclin-dependent kinases and protein kinase C (PKC). Previous studies have shown that the PKC-specific inhibitor safingol significantly enhances the induction of apoptosis by mitomycin-C (MMC) in gastric cancer cells. Because flavopiridol can potentially inhibit PKC, we elected to determine the extent to which flavopiridol would promote MMC-induced apoptosis in both gastric and breast cancer cells. For these studies, MKN-74 gastric cancer cells and MDA-MB-468 breast cancer cells were exposed to either no drug, 1 microgram/ml MMC alone, 300 nM flavopiridol alone, or a combination of chemotherapy with flavopiridol for 24 h. Sequence specificity was also examined by first exposing cells to MMC for 24 h followed by flavopiridol for 24 h or to the same drugs in the reverse order. Apoptosis was measured by quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzimide trihydrochloride. Exposure of MKN-74 cells to flavopiridol alone induced apoptosis in 12 +/- 1% of the cells, and exposure to MMC alone induced apoptosis in 10 +/- 1%. However, the combination of flavopiridol and MMC increased the induction of apoptosis to 55 +/- 3% of the cells (P < 0.005 for the drug combination versus flavopiridol alone). Pretreatment with the PKC activator 3-phorbol 12-myristate 13-acetate only partially reversed this effect (43 +/- 1%; P < 0.025). In MDA-MB-468 cells, flavopiridol alone induced apoptosis in 17 +/- 1% of the cells, and MMC alone induced apoptosis in 10 +/- 1% of the cells. The combination of flavopiridol and MMC increased the percentage of MDA-MB-468 cells undergoing apoptosis to 58 +/- 4% (P < 0.005 for the drug combination versus flavopiridol alone). Sequential treatment with MMC followed by flavopiridol induced apoptosis in 63 +/- 2% of the MKN-74 cells (P < 0.05 versus the concomitant drug combination) and in 76 +/- 2% of the MDA-MB-468 cells (P < 0.025 versus the concomitant drug combination), whereas flavopiridol followed by MMC did not increase the induction of apoptosis in either cell line. As determined by the terminal deoxynucleotidyl transferase labeling of the 3' ends of DNA fragments produced in apoptotic cells, the induction of apoptosis with the combination of flavopiridol and MMC occurred to MKN-74 cells in all phases of the cell cycle (i.e., G0-G1, S, and G2-M). These results indicate that flavopiridol potentiates the cytotoxic effect of the chemotherapeutic agent MMC by promoting drug-induced apoptosis in tumor cells. Sequencing studies suggest that MMC followed by flavopiridol or simultaneous treatment is superior to flavopiridol followed by MMC. The enhancement of MMC-induced apoptosis by flavopiridol may be partially PKC dependent and is not associated with one specific region of the cell cycle.     

Role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta 1 in c-myc overexpressing mammary epithelial cells

We previously showed that TGF alpha synergizes with c-myc in mammary tumorigenesis through inhibition of Myc-induced apoptosis. We therefore examined the effects of growth factors on apoptosis induction in several cell lines from MMTV-myc mammary tumors. When EGF was withdrawn or TGF beta 1 was added, cells became apoptotic after 15 h (by ELISA and morphology). Northern and Western analysis revealed high levels of Bax and p53, and low or undetectable levels of Bcl-2 and Bcl-xS under all treatment condition. In contrast, Bcl-xL expression was highest in the presence of EGF or TGF alpha, with a significant reduction upon removal of EGF or exposure to TGF beta. In mouse mammary tumors, the relative Bcl-xL/Bax ratio was higher in TGF alpha/Myc double transgenics than in Myc single transgenics, in agreement with the in vitro data. Our results suggest a role for Bcl-xL in the regulation of apoptosis by EGF and TGF beta in mammary epithelial cells.     

Regulation of prostatic growth and function by peptide growth factors

Polypeptide growth factors are positive and negative regulators of prostatic growth and function. Expression and biological effects of epidermal growth factor (EGF), transforming growth factors (TGFs) alpha and beta, fibroblast growth factors (FGFs), and insulin-like growth factors (IGFs) in the prostate have been extensively studied. EGF and TGF alpha, which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells. Their paracrine mode of action in normal tissue and early-stage tumors is apparently altered towards an autocrine stimulation in hormone-independent tumors, which gain the ability to produce TGF alpha by themselves. TGF beta has a dual role in the regulation of prostatic growth. It inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to the inhibitory effect of TGF beta. Several members of the FGF family have been identified in the prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF (bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether prostatic cancer cells gain the ability to produce IGF-I by themselves and thus shift to an autocrine mode of IGF-I stimulation. Growth factors also interact with the androgen-signaling pathway. IGF-I in particular, other growth factors as well, can activate the androgen receptor.     

Short-term effects of pure anti-oestrogen ICI 182780 treatment on oestrogen receptor, epidermal growth factor receptor and transforming growth factor-alpha protein expression in human breast cancer

Expression of oestrogen receptor (ER), epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF alpha) proteins was assessed by immunocytochemistry on primary breast cancer specimens obtained before and following short-term (7-day) presurgical exposure to pure anti-oestrogen (7 alpha- [9- (4,4,5,5,5-pentafluoropentylsulphinyl) nonyl] estra-1,3,5, (10)-triene-3,17 beta-diol, ICI 182780) treatment and compared with no-treatment controls. Paired needle-core and mastectomy samples were obtained from 21 patients. Effects of ICI 182780 (10(-7)M) on MCF7 breast cancer cell ER, EGFR and TGF alpha expression were also examined over 14 days. ER protein was significantly suppressed by ICI 182780 in vivo (P = 0.009) and comparative analysis of short term ICI 182780 effects in vitro, using ER-positive MCF7 cells, gave largely equivalent results. EGFR and TGF alpha protein levels were unaltered by treatment. ICI 182780 suppresses ER without a concomitant rise in either EGFR or TGF alpha.     

Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.     

Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells

This report describes the isolation and characterization of a new human breast cancer cell line, SUM-102PT, obtained from a minimally invasive human breast carcinoma. SUM-102PT cells have a near diploid karyotype, and early-passage cells had minor chromosomal abnormalities including a 5, 12 and a 6, 16 reciprocal translocation. The cells were isolated and have been continually cultured in three defined media, one of which contains exogenous epidermal growth factor (EGF). SUM-102PT cells have also been carried in an EGF-free medium supplemented with progesterone. All SUM-102PT cells require EGF receptor (EGFR) activation for continuous growth, because incubation of the cells with EGFR-neutralizing antibodies or with EGFR kinase inhibitors blocks growth of these cells. Southern analysis indicates that the EGFR gene is not amplified in these cells; however, these cells express high levels of EGFR mRNA. Thus, SUM-102PT is representative of a class of human breast cancers characterized by high level EGFR expression in the absence of gene amplification. SUM-102PT cells cultured in EGF-free, progesterone-containing medium express high levels of constitutively active EGFR. Conditioned medium from SUM-102PT cells contains an EGF-like mitogen that binds to a heparin-agarose affinity matrix with high affinity. Northern analysis for various EGF family members indicates that SUM-102PT cells synthesize heparin binding (HB)-EGF mRNA. HB-EGF protein is detectable on the surface of these cells by immunohistochemistry, and SUM-102PT cells are killed by diphtheria toxin, which acts by binding to HB-EGF. Furthermore, HB-EGF antibodies partially neutralize the mitogenic activity of the conditioned medium. Thus, EGFR activation in SUM-102PT cells is mediated, at least in part, by autocrine/juxtacrine stimulation by HB-EGF. SUM-102PT cells also express constitutively active STAT-3 homodimers. Constitutively tyrosine-phosphorylated STAT-3 homodimers were also detected in another breast cancer cell line, MDA468, which has an EGFR amplification and also has constitutive EGFR activity. Thus, SUM-102PT is a new human breast cancer cell line that expresses activated EGFR as a result of an autocrine/juxtacrine interaction with HB-EGF which, in turn, results in activation of STAT-3.