Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays an essential role in cell cycle progression and can be activated by many growth factor/mitogen pathways including estrogen. MAPK has also been implicated in ligand-independent activation of estrogen receptor-alpha (ER-alpha). The development of estrogen-independent growth in breast cancer is likely a first step in progression to hormone independence and antiestrogen resistance. We examined MAPK expression and activity in T5-PRF and T5 human breast cancer cells. T5-PRF is an estrogen-nonresponsive cell line developed from T5 cells by chronically depleting the cells of estrogen in long-term culture. MAPK activity measured in vitro was significantly higher (P < 0.05) in T5-PRF compared with T5 cells. Western blot analyses showed increased levels of active dually phosphorylated MAPK in T5-PRF cell extracts compared with T5. The increased activity and expression of MAPK may contribute to the estrogen nonresponsive growth phenotype and ligand-independent activity of ER in T5-PRF cells.     

Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-epsilon (PKC epsilon). We recently have shown that Raf-1 is constitutively activated in PKC epsilon transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKC epsilon induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKC epsilon activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKC epsilon and Raf-1. PKC epsilon, but not PKC alpha, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKC epsilon could be co-precipitated from Sf-9 insect cells and PKC epsilon transformed NIH313 cells (NIH/epsilon). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/epsilon cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/epsilon cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKC epsilon. PKC epsilon did not induce translocation of Raf-1 to the membrane. Furthermore, PKC epsilon did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKC epsilon transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFbeta, a growth factors secreted by PKC epsilon transformed Rat6 cells. In conclusion, our results suggest that PKC epsilon stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.     

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-beta 2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BR-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-beta-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2-6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells.     

The MEK1 proline-rich insert is required for efficient activation of the mitogen-activated protein kinases ERK1 and ERK2 in mammalian cells

MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.     

Pharmacokinetics and pharmacodynamics of conjugated equine estrogens: chemistry and metabolism

Raf-1, A-Raf and B-Raf comprise a small family of highly conserved serine/threonine protein kinases, whose activities play a fundamental role in the control of proliferation and differentiation. The best studied family member, Raf-1, is expressed ubiquitously and constitutively, and its activity is regulated by post-translational mechanisms. Raf-1 can be activated by many signals that include growth factors, tumor promoters, inflammatory cytokines, calcium mobilization, DNA damaging agents, and oxygen radicals. Ras-mediated translocation of Raf-1 to the plasma membrane is a crucial step in its activation process, and is thought to facilitate phosphorylation by membrane-bound kinases. Raf-1 has also been reported to undergo intracellular redistribution following its activation: to the perinuclear space in murine NIH3T3 cells and rat hepatic Ito cells, and into the nucleus in gerbil hippocampal pyramidal cells and human MO7 leukemia cells. In contrast to the translocation to the plasma membrane, the perinuclear and/or nuclear translocation of Raf-1 has not been investigated in detail. In this paper, we report an examination of the subcellular localization of endogenous Raf-1 in a fibroblastic cell line (Rat-1) commonly used in transformation assays. Using the methods of cellular fractionation as well as in situ immunofluorescence, we show that no detectable movement of Raf-1 to the perinuclear or nuclear space can be observed. Tethering of activated Raf to the plasma membrane does not interfere with its transforming activity.     

Estrogens, cathepsin D and metastasis in cancers of the breast and ovary: invasion or proliferation?

This short review presents the current stage of knowledge of our laboratory on the mechanism of action of cathepsin D and estrogens on tumor progression, mostly based on studies of human breast and ovarian cancer cell lines. Cathepsin D (cath-D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients as confirmed by a recent meta-analysis of clinical studies on node negative breast cancer patients. Transfection of a human cDNA cath-D expression vector increases the metastatic potential of a rat tumor cells line when intravenously injected into nude mice. The mechanism of cath-D induced metastasis seems to require maturation of the pro-enzyme, mostly in large acidic compartments identified as phagosomes. Cath-D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factor etc.) are proposed to mediate this activity. A mitogenic effect of the pro-enzyme on transmembrane receptor is not totally excluded. The mitogenic activity of estrogens in several estrogen receptor positive breast and ovarian cancer cell lines is well established in our and other laboratories. By contrast the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility is more controversial. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines were studied in our laboratory using a modified Boyden chamber assay. We observed, in all cases, estradiol-induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. In breast cancer cells other estrogen regulated proteins may be involved. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies, and with the better prognosis of breast cancer occurring after a prolonged treatment of menopause by estrogen as described by the collaborative group on hormonal factors in breast cancer.