Analysis of the Transcriptional Program Induced by Raf in Epithelial Cells

Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of the transcriptional program induced by Raf in epithelial cells Almut Schulze,1 Kerstin Lehmann,1 Harold B.J. Jefferies,1 Martin McMahon,2 and Julian Downward1,3 1Signal Transduction Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, UK; 2Cancer Research Institute, University of California at San Francisco/Mt. Zion Cancer Center, San Francisco, California 94115-0128, USA Activation of the Raf/MAP kinase pathway is a critical event in tumorigenesis induced by RAS and other oncogenes, a major role of this signaling system being the regulation of cellular transcription factors. To address the contribution of MAP kinase mediated transcriptional changes to the transformed phenotype, we used an inducible form of Raf to analyze early changes in the transcription of some 6000 genes following activation of the kinase in a normal human breast epithelial cell line. Of the more than 120 significant changes in mRNA level detected, genes promoting cell proliferation, invasiveness, and angiogenesis featured prominently. Some of the most strongly induced genes encoded growth factors of the EGF family: Autocrine activation of the EGF receptor was shown to be responsible for the ability of Raf activation to protect these cells from apoptosis induced by detachment of cells from extracellular matrix (anoikis), which is a critical component of the transformed phenotype. [Key Words: Ras; Raf; transcription; apoptosis; matrix; microarray] Received October 15, 2000; revised version accepted February 16, 2001. Tumorigenesis is a multistep process during which cells The transformed phenotype induced by oncogenic RAS acquire a characteristic set of properties that make up is caused by both the short-term consequences of acti- the malignant phenotype. The dominantly acting onco- vating the Ras effector pathways and the longer-term gene that is implicated most frequently in human cancer effects of changes in the gene expression program of the is RAS, which is activated by mutation in about 25% of cell that this causes. Much emphasis has been placed on malignancies (Bos 1989). Ras proteins exert their trans- the immediate cytoplasmic signaling induced by Ras ac- forming influence through a number of effector en- tivation, but each of the major Ras effector pathways zymes, including the serine/threonine kinase Raf, which also has profound effects on transcription. In particular, activates the ERK/MAPkinase pathway; the lipid kinase activation of the MAPkinase pathway by Raf induces phosphoinositide 3-kinase (PI 3-kinase), which activates phosphorylation and stimulation of several transcription the serine/threonine kinase PKB/Akt and the small factors, including Elk1, SRF, ATF2, and Jun (Treisman GTPase Rac; and the exchange factor Ral-GDS, which 1996), whereas PKB/Akt influences the activity of Fork- activates the Ras-related protein Ral (Marshall 1996; head transcription factors and possibly NF-␬B (Datta et Downward 1998). PI 3-kinase and PKB/Akt have been al. 1999). In this study, we have used oligonucleotide- previously implicated in mediating antiapoptotic signals based DNA-microarrays to assess the contribution of generated by growth factors, extracellular matrix, and transcriptional changes resulting from activation of just activated Ras (Downward 1998; Datta et al. 1999), the Raf/MAPkinase pathway alone to the Ras-trans- whereas the Raf/MAPkinase pathway has been found to formed phenotype. The use of an inducibly activatable play a critical role in the control of cell cycle progression Raf protein allows early transcriptional events under di- (Marshall 1999). More recently, it has become clear that rect control of this pathway to be studied, which is im- Raf/MAPkinase can also induce protection against cer- portant in the establishment of the transformed pheno- tain apoptotic stimuli (Bonni et al. 1999; Erhardt et al. type. To reflect the major cell type involved in human 1999; Kazama and Yonehara 2000; Le Gall et al. 2000) cancer, a normal, spontaneously immortalized human and that PI 3-kinase and PKB/Akt can contribute to cell epithelial cell line derived from breast tissue has been cycle progression (Diehl et al. 1998; Klippel et al. 1998). used. With this approach we were able to identify over 120 genes that were rapidly modulated in their expression levels by threefold or more in response to Raf acti- 3Corresponding author. vation. Among these, a significant number of genes en- E-MAIL 44-20-7269-3533; FAX 44-20-7269-3094. Article and publication are at www.genesdev.org/cgi/doi/10.1101/ coded key regulators of proliferation, survival, angiogen- gad.191101. esis, and invasiveness. Furthermore, the ability of Raf GENES & DEVELOPMENT 15:981–994 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org 981 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Schulze et al. activation to very strongly induce autocrine expression cells in this assay (data not shown), indicating that Ras of the EGF-like growth factors HB-EGF, TGF␣, and am- uses pathways in addition to Raf to transform cells (for phiregulin was directly implicated in the ability of sus- review, see Shields et al. 2000). tained Raf/MAPkinase pathway stimulation to protect these cells from matrix detachment-induced apoptosis, a major feature of the transformed phenotype. Activation of Raf leads to protection from detachment-induced apoptosis Results Although Raf is not able to transform MCF-10A cells by itself, we asked whether it could provide an important Activation of Raf-ER in MCF-10A cells induces component of the Ras-transformed phenotype, resis- sustained activation of ERK/MAP kinase and changes tance to detachment-induced apoptosis (anoikis; Frisch in cell morphology but not growth and Francis 1994). Although a major effector pathway factor independence mediating Ras-induced survival is thought to involve activation of protein kinase B (PKB/Akt) in a PI 3-kinase– To investigate the effect of activation of Raf on epithelial dependent manner (Khwaja et al. 1997), we asked cell biology, we used MCF-10A cells, a normal, sponta- whether the sustained activation of Raf that was ob- neously immortalized human lumenal mammary epi- served in MCF-10A ⌬Raf-ER cells after 4-OHT treatment thelial cell line (Soule et al. 1990). These cells were could be sufficient to induce survival from detachment- infected with retroviruses encoding a Raf/estrogen induced apoptosis. When attachment of the cells to sub- receptor/green fluorescent protein fusion, the kinase strate was prevented by plating cells on poly-HEMA activity of which is stimulatable by 4-hydroxy tamoxifen coated dishes in minimal medium, parental MCF-10A (4-OHT) but not natural estrogens (Bosch et al. 1997). cells infected with empty vector and uninduced MCF- After drug selection and repeated fluorescence-activated 10A ⌬Raf-ER cells showed a six- to sevenfold increase in cell sorting, a population highly enriched for cells ex- apoptosis within 24 h, measured by DNA fragmentation pressing the fusion protein was obtained (MCF-10A (Fig. 2A). When Raf was activated by treating the cells ⌬Raf-ER). The addition of 4-OHT to MCF-10A ⌬Raf-ER with 4-OHT for 8 or 48 h before culture in suspension, cells that had been grown in growth factor–poor medium induction of apoptosis was abolished (Fig. 2A). In addi- for 24 h induced rapid and sustained stimulation of ERK/ tion, MCF-10A cells expressing V12 Ras were also pro- MAPK phosphorylation (Fig. 1A), indicating activation tected from detachment-induced apoptosis (Fig. 2A), as of the fusion protein. has been reported previously (Rytömaa et al. 1999). MCF-10A cells are dependent on the presence of a As shown in Figure 2B, the ⌬Raf-ER protein is capable mixture of supplements, including EGF and insulin, for of activating p42/p44ERK phosphorylation in response to proliferation in culture. Figure 1B shows growth curves 4-OHT, both in attached cells and in cells grown in sus- of MCF-10A ⌬Raf-ER cells in the presence or absence of pension. Although physiological signaling from Raf to 4-OHT in medium supplemented with different growth ERK has been found to be attenuated following loss of factors. In medium containing 5% horse serum and all adhesion in fibroblasts (Renshaw et al. 1997), the artifi- supplements (full medium, left panel), activation of Raf cial Raf construct used here is potent enough to over- led to an initial but transient decrease in proliferation, come this level of dependence on matrix, as has also consistent with the previous findings that Raf can in- been found for oncogenic mutant Ras proteins (Khwaja duce growth inhibitory pathways (Sewing et al. 1997). In et al. 1997). a growth factor–poor medium, 5% horse serum without The mechanisms by which apoptosis is triggered in supplements (minimal medium), these cells show no in- response to loss of adhesion to the extracellular matrix crease in numbers regardless of whether 4-OHT is pres- are not completely understood. We investigated whether ent (right panel), indicating that Raf activation does not detachment leads to cytochrome c release from mito- induce complete growth factor independence. chondria into the cytoplasm, a key event in many forms Parental MCF-10A cells and uninduced MCF-10A of apoptosis, and whether Raf activation effects this. Us- ⌬Raf-ER cells displayed typical morphological character- ing differential lysis of the plasma membrane by digito- istics of epithelial cells, growing in monolayer culture as nin and subsequent detection of cytoplasmic cyto- islands of cells in close contact. Induction of Raf kinase chrome c by immunoblotting, we could show that pre- activity by addition of 4-OHT led to disruption of cell– activation of Raf by 4-OHT treatment for 48 h was cell contacts, cell scattering, and establishment of a sufficient to protect cells from detachment-induced more fibroblastoid morphology (Fig.

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