3106 Research Article Mammary tumorigenesis induced by fibroblast receptor 1 requires activation of the receptor

Lindsey K. Bade1, Jodi E. Goldberg2, Hazel A. DeHut3, Majken K. Hall3 and Kathryn L. Schwertfeger3,* 1Graduate Program in Molecular, Cellular, Developmental Biology, and Genetics, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455, USA 2Hamline University, Biology Department, 1536 Hewitt Avenue, Saint Paul, MN 55104, USA 3Department of Lab Medicine and Pathology, Masonic Cancer Center, 420 Delaware St. SE, MMC 609, University of Minnesota, Minneapolis, MN 55455, USA *Author for correspondence ([email protected])

Accepted 27 May 2011 Journal of Cell Science 124, 3106–3117 ß 2011. Published by The Company of Biologists Ltd doi: 10.1242/jcs.082651

Summary receptor 1 (FGFR1) is an oncoprotein with known involvement in mammary tumorigenesis. To understand how FGFR1 signaling promotes mammary tumorigenesis, an inducible FGFR1 (iFGFR1) system was created previously. Previous studies have demonstrated that upon iFGFR1 activation in vivo, the epidermal growth factor (EGF) ligands amphiregulin (AREG) and epiregulin (EREG) are upregulated. Both AREG and EREG interact with the EGF receptor (EGFR). Here, we investigated whether the FGFR1-induced increase in AREG and EREG expression might coordinately increase EGFR signaling to promote mammary tumorigenesis. Treatment of mouse mammary epithelial cells with either AREG or EREG conferred a greater migratory potential, increased cellular proliferation and increased extracellular regulated kinase 1/2 (ERK1/2) activation. These effects could be blocked with the EGFR-specific inhibitor erlotinib, suggesting that they are EGFR-dependent. In transgenic mice with iFGFR1 under the control of the mouse mammary tumor virus (MMTV) promoter, iFGFR1 activation also led to increased mammary epithelial cell proliferation that was inhibited with erlotinib. Taken together, these data suggest that AREG and EREG mediate tumorigenic phenotypes by activating EGFR signaling, and that the oncogenic potential of FGFR1 requires EGFR activation to promote mammary tumorigenesis.

Key words: EGFR, FGFR1, Breast cancer, Mammary tumor Journal of Cell Science

Introduction removed, dimerization and activation of iFGFR1 is stimulated The fibroblast growth factor receptor (FGFR) family consists of not by FGF ligands but by the synthetic molecule AP20187 (AP). four receptor tyrosine kinases that are known regulators of cellular Binding of AP to iFGFR1 results in homodimerization and processes, such as proliferation, migration, survival and subsequent activation of the same signaling pathways as angiogenesis (reviewed by Dailey et al., 2005; Eswarakumar et endogenous FGFR1. Extensive characterization of the iFGFR1 al., 2005; Ornitz, 2000; Presta et al., 2005). Anomalous expression system in vitro revealed that, following AP treatment of mouse or uncontrolled activation of these receptors or their ligands has mammary epithelial cells expressing iFGFR1, there was an increase been correlated with progression of various types of cancer, in cellular migration, proliferation and signaling through the including breast cancer (Dickson et al., 2000; Grose and Dickson, extracellular regulated kinase 1/2 (ERK1/2, hereafter referred to as 2005; Koziczak et al., 2004). Specifically, the chromosomal locus ERK) pathway, similar to endogenous FGFR1 activation (Welm et for FGFR1, 8p11-12, is aberrantly amplified in ,10% of patients al., 2002; Xian et al., 2009; Xian et al., 2005). The inducible system diagnosed with breast cancer (Andre et al., 2009; Elbauomy was also analyzed in vivo. For these previous studies, a transgenic Elsheikh et al., 2007; Gelsi-Boyer et al., 2005; Penault-Llorca et mouse line was created in which the iFGFR1 construct was placed al., 1995). Patients who harbor an FGFR1 amplification do not under the control of the mouse mammary tumor virus (MMTV) respond well to current therapies and develop resistance to promoter, thereby restricting iFGFR1 expression to mammary hormone-based therapies (Chin et al., 2006; Turner et al., 2010). epithelial cells (Welm et al., 2002). In the study by Welm et al., Therefore, understanding the molecular mechanisms of how MMTV-iFGFR1 transgenic animals develop neoplasias within as FGFR1 overexpression promotes tumorigenesis might provide little as 3 days of AP treatment, which are characterized by aberrant insights into better targets for novel, more effective therapies. budding of the mammary gland ductal epithelial cells due to Because of the lack of binding specificity among the FGF ligands increased cellular proliferation. Extended AP treatment leads to and their receptors, it is difficult to specifically activate FGFR1. progressively more severe phenotypes, and by 4 weeks of iFGFR1 Therefore, an inducible FGFR1 (iFGFR1) system was previously activation, the mammary glands of transgenic animals contain large engineered to mimic endogenous FGFR1 signaling (Welm et al., locally invasive growths. These results verify that iFGFR1 2002). Because the extracellular ligand-binding domain was activation promotes tumorigenic phenotypes both in vivo and in EGFR promotes FGFR1-driven tumors 3107

vitro. To identify mechanisms by which FGFR1 promotes treatment of HC11 mouse mammary epithelial cells with either tumorigenesis, previously published studies have described the AREG or EREG promotes increased migration, proliferation and results of microarray studies that were performed on mammary ERK activation in vitro. Importantly, the ability of these ligands to glands from MMTV-iFGFR1 transgenic mice following iFGFR1 stimulate migration and proliferation in vitro requires activation of activation (Schwertfeger et al., 2006). In the current study, we EGFR, as inhibiting the EGFR kinase blocks these processes. We explore the ability of two targets identified in the screen, also show that inhibiting EGFR in vivo blocks iFGFR1-mediated amphiregulin (AREG) and epiregulin (EREG), to promote cellular proliferation of mouse mammary epithelial cells. These FGFR1-induced mammary tumorigenesis. studies demonstrate that activation of EGFR is important for AREG and EREG are both members of the epidermal growth effective FGFR1-induced mammary tumorigenesis. Because there factor (EGF) family of ligands (reviewed by Schneider and Wolf, are currently no FGFR1-specific drugs that are used clinically, our 2009). These ligands are originally synthesized as transmembrane studies suggest that inhibition of EGFR might represent a useful proteins and are thought to be cleaved from the plasma membrane strategy for targeting breast cancers harboring FGFR1 by the ADAM17 (a disintegrin and metalloproteinase 17) sheddase amplifications. (Brown et al., 1998; Plowman et al., 1990; Sahin et al., 2004; Sternlicht et al., 2005; Sunnarborg et al., 2002; Toyoda et al., Results 1995a; Toyoda et al., 1995b). Furthermore, both ligands are known Activation of iFGFR1 in the mouse mammary gland results activators of the EGF receptor (EGFR); AREG exclusively binds to in increased expression of AREG and EREG and signals through EGFR (Shoyab et al., 1989), whereas EREG Owing to the prevalence of human breast cancer patients harboring can also bind to and signal through ERBB4 (Komurasaki et al., aberrant FGFR1 amplification, it is necessary to determine the 1997; Shelly et al., 1998). AREG is essential for normal ductal downstream molecular effects of FGFR1-induced signaling morphogenesis in the mammary gland (Ciarloni et al., 2007; pathways. In order to specifically study FGFR1 signaling, the Luetteke et al., 1999) and has also been linked to breast cancer iFGFR1 system previously engineered to mimic endogenous progression (Kenney et al., 1996a; Kondapaka et al., 1997; Ma et FGFR1 signaling was developed (Welm et al., 2002). Activation al., 1999; Silvy et al., 2001). Studies examining AREG expression of iFGFR1, by treatment with AP, within the mammary epithelial in human breast cancers have found AREG expression to cells of MMTV-iFGFR1 transgenic mice, results in the formation significantly correlate with regional lymph node metastases, large of hyperplastic lesions within 3 days, which progress to mammary tumor size and high-grade tumors (LeJeune et al., 1993; Ma et al., tumors upon prolonged AP treatment. To identify new mediators of 2001; Qi et al., 1994). Although EREG promotes proliferation of such FGFR1-induced tumorigenesis, previously published studies several normal and cancerous cell types (Komurasaki et al., 2002; described the results of microarray studies that were performed on Shirakata et al., 2000; Toyoda et al., 1997; Zhu et al., 2000), the role RNA isolated from the mammary glands of MMTV-iFGFR1 of EREG has not been extensively characterized in the mammary transgenic animals following AP stimulation (Schwertfeger et al., gland. However, recent studies have demonstrated that EREG is a 2006). Of the numerous genes induced by iFGFR1, two EGF family potent mediator of metastasis of breast cancer cells to the lung and ligands, Areg and Ereg, were significantly upregulated following that overexpression of EREG is an indicator of poor prognosis for iFGFR1 activation (Fig. 1A,B). Areg was significantly (P,0.05)

Journal of Cell Science inflammatory breast cancer patients (Bieche et al., 2004; induced within 8 hours of treatment, whereas Ereg was significantly Eltarhouny et al., 2008; Gupta et al., 2007). (P,0.05) induced following 16 hours of treatment. To validate the EGFR, a member of the ERBB receptor tyrosine kinase family, increased expression of AREG and EREG, immunohistochemical has been well studied in numerous systems (reviewed by Alroy and analysis of MMTV-iFGFR1 mammary gland sections was Yarden, 1997; Mitsudomi and Yatabe, 2010). In the mammary performed. As shown in Fig. 1C, both AREG and EREG proteins gland, it is known that EGFR is required for normal mammary were produced at elevated levels following iFGFR1 activation. gland ductal morphogenesis (Wiesen et al., 1999; Xie et al., 1997). These studies identify two new targets of FGFR1 activation in the However, overexpression or constitutive activation of EGFR in the mammary gland in vivo. mammary gland has been linked to mammary tumorigenesis (Brandt et al., 2000; Cho et al., 2008; Moscatello et al., 1995). Activation of iFGFR1 in HC11/R1 cells induces expression Additionally, overexpression of EGFR in the breast is associated of AREG and EREG with recurrence of earlier-stage breast cancers and decreased We next determined whether iFGFR1 was capable of inducing disease-free and overall survival in later-stage breast cancer Areg and Ereg in mouse mammary epithelial cells. For these patients (Fabian et al., 1994; Fox et al., 1994; Klijn et al., 1992; studies, HC11/R1 cells, an immortalized non-transformed Rimawi et al., 2010; Torregrosa et al., 1997). Therefore, together mammary epithelial cell line stably expressing iFGFR1, were with the fact that aberrant FGFR1 activation in the mammary gland used. Previous studies of HC11/R1 cells have demonstrated that leads to upregulation of the EGFR ligands AREG and EREG, these activation of this receptor, through treatment with AP, promotes data strongly support a model in which aberrant FGFR1 signaling cell survival, proliferation, migration, invasion and epithelial-to- requires EGFR activation for mammary tumor formation, a link mesenchymal transition (EMT) (Welm et al., 2002; Xian et al., that has not been previously established. Moreover, the EGFR 2009; Xian et al., 2007; Xian et al., 2005). Quantitative reverse kinase inhibitor erlotinib is currently an approved therapeutic for transcription PCR (qRT-PCR) was performed on RNA collected both non-small cell lung cancer patients and advanced pancreatic from HC11/R1 cells treated with AP for 0, 0.5, 1, 2 and 4 hours. cancer patients (reviewed by Gridelli et al., 2010; Squadroni and As shown in Fig. 2A,B, both Areg and Ereg transcript levels Fazio, 2010). However, erlotinib has not been fully tested as a increased following iFGFR1 activation in vitro. Areg transcript functional therapy for breast cancer patients. levels rapidly increased with AP treatment, peaking at 1 hour of Our current studies focus on identifying downstream mediators AP treatment, and then decreased with prolonged AP treatment. of iFGFR1-induced mammary tumorigenesis. We found that Ereg transcript levels rose more slowly to peak at 2 hours after 3108 Journal of Cell Science 124 (18)

Fig. 1. AREG and EREG are induced following FGFR1 activation in vivo. (A,B) MMTV-iFGFR1 animals were administered by intraperitoneal injections of 1 mg per kg of body weight AP for the times indicated. Following AP treatment, the animals were killed, and their fourth inguinal mammary glands were removed for either isolation of RNA or paraffin embedding and subsequent tissue analysis. An analysis of the data from previously published microarray experiments (Schwertfeger et al., 2006) demonstrates that Areg (A) and Ereg (B) were both significantly upregulated at the indicated timepoints. Error bars represent s.e.m. *P,0.05. (C) AREG and EREG detection by immunohistochemistry on mammary gland sections from either WT or MMTV-iFGFR1 mice treated with AP for 48 hours. Images are representative of at least three mice per treatment group. Scale bars: 50 mm. Journal of Cell Science AP treatment and then, like Areg, decreased with continued AP Pond et al., 2010) and was used to verify that FGF treatment leads treatment. These data are consistent with the in vivo analysis of to increased expression of AREG and EREG. As shown in Fig. 2, Areg and Ereg transcripts, in that an increase in Areg mRNA is qRT-PCR performed on RNA isolated from MCF7 cells treated detectable earlier than an increase in Ereg mRNA, and with 50 ng/ml basic FGF (bFGF) for 4, 6 and 24 hours displayed a demonstrate that Areg and Ereg are induced in mouse significantly (P,0.001) increased fold change in both AREG and mammary epithelial cells following iFGFR1 activation. EREG mRNA as compared with that in the no-treatment control To verify that Areg and Ereg transcripts are indeed translated samples (Fig. 2F,G). As in the mouse, human AREG and EREG are into mature AREG and EREG proteins in vitro, AREG and shed from the cell membrane. Thus, conditioned medium was EREG protein levels were quantified. Because it is known that collected to detect AREG protein levels through ELISA (Fig. 2E). EGF family ligands are shed from their membrane-bound Compared with the no-treatment control, 50 ng/ml bFGF treatment precursors into the extracellular matrix (ECM) (Sahin et al., of MCF7 cells for 4, 6 and 24 hours significantly (P,0.001) 2004; Sunnarborg et al., 2002), soluble AREG and EREG protein induced soluble human AREG protein. These results demonstrate concentration was measured by ELISA from the conditioned that activation of the endogenous FGF pathway by treating cells medium of HC11/R1 cells treated overnight with either AP or its with ligand also increases AREG and EREG expression. solvent, ethanol. Compared with the ethanol controls, HC11/R1 Furthermore, as MCF7 cells are a human-derived breast cancer cells treated with AP had significantly (P,0.05) increased levels cell line, these studies suggest that activation of the FGF pathway of soluble AREG and EREG (Fig. 2C,D). These data suggest that signals an increase in both AREG and EREG expression in human iFGFR1 activation induces both gene and protein expression of cells, as well as mouse cells. AREG and EREG. AREG and EREG both individually promote cellular AREG and EREG expression are upregulated following migration and proliferation of mammary epithelial cells bFGF treatment in MCF7 cells in vitro Further studies were performed to confirm that AREG and EREG On the basis of the fact that both AREG and EREG are are regulated by FGF signaling in another breast cancer model. The upregulated following iFGFR1 activation and that iFGFR1 MCF7 human breast cancer cell line has been previously used to activation induces numerous tumorigenic phenotypes including study mechanisms of FGF-induced tumorigenesis (Liu et al., 2002; migration and proliferation, we tested whether AREG and/or EGFR promotes FGFR1-driven tumors 3109 Journal of Cell Science

Fig. 2. FGFR1 activation in mammary epithelial cells in vitro induces expression of AREG and EREG. (A,B) Mouse mammary epithelial HC11/R1 cells were treated with 30 nM AP for the indicated times. Following AP treatment, qRT-PCR analysis was performed on RNA isolated at each timepoint for both the Areg transcript (A) and the Ereg transcript (B), normalized to mouse cyclophilin B. Experiments were performed in biological triplicates. Error bars represent s.e.m. *P,0.05; **P,0.01; ***P,0.005; ****P,0.001. (C,D) To detect soluble AREG (C) and EREG (D) ligands, ELISAs were performed on conditioned HC11/R1 medium following treatment of cells with either 30 nM AP or its solvent, ethanol. Experiments were performed in biological triplicates. Error bars represent s.e.m. *P,0.05 (E) The conditioned medium of MCF7 human breast cancer cells treated with 50 ng/ml bFGF for 4, 6 or 24 hours were collected and soluble AREG was detected through an ELISA. Experiments were performed in biological triplicates. Error bars represent s.e.m. *P,0.05; ****P,0.001. (F,G) MCF7 cells were treated with 50 ng/ml bFGF for 4, 6 or 24 hours. Following bFGF treatment, qRT-PCR analysis was performed on RNA isolated at each timepoint for both AREG transcript (F) and EREG transcript (G), normalized to human cyclophilin B. NT, no treatment. Experiments were performed in biological triplicates. Error bars represent s.e.m. ****P,0.001.

EREG affects these processes. To study the effects of AREG and of wound closure was calculated from the start of the treatment EREG on migration, recombinant mouse AREG (rmAreg) or (0 hours) up to 18 hours after recombinant protein treatment. recombinant mouse EREG (rmEreg) was added to freshly Compared with controls treated only with DMSO, addition of scratch-wounded serum-starved HC11 mouse mammary either rmAreg or rmEreg significantly (P,0.001) increased epithelial cells, which do not express iFGFR1. The percentage HC11 migration (Fig. 3A,B). Because AREG and EREG both 3110 Journal of Cell Science 124 (18)

Fig. 3. Treatment of HC11 cells with either AREG or EREG in vitro stimulates cellular migration, cellular proliferation and ERK activation. (A) Freshly wounded mouse mammary epithelial HC11 cells were treated with either DMSO, 20 ng/ml rmAreg+DMSO, 1 mM erlotinib, 20 ng/ml rmAreg+1 mM erlotinib, 0.5 mM erlotinib or 20 ng/ml rmAreg+0.5 mM erlotinib for 18 hours. Five representative images were taken per well at 0 and 18 hours, and the area of the open space of the wound was determined using Leica LAS Software. Migration was measured as the percentage of wound closure. Each treatment was performed in biological triplicates. Error bars represent s.e.m. ****P,0.001. (B) The migration assay was performed as in A using 10 ng/ml rmEreg instead of rmAreg. Each treatment was performed in biological triplicates. Error bars represent s.e.m. ****P,0.001. (C) HC11 cells were treated with DMSO, 100 ng/ml rmAreg, 1 mM erlotinib, or 100 ng/ml rmAreg+1 mM erlotinib for 48 hours. Proliferation was measured with an MTT assay and normalized to the levels at treatment day 0. Each treatment was performed in biological triplicates. Error bars represent s.e.m. **P,0.01; ***P,0.005. (D) HC11 cells were treated as in C, but with 10 ng/ml rmEreg in place of rmAreg. Proliferation was measured with an MTT assay at 48 hours and normalized to the levels at day 0 of treatment. Each

Journal of Cell Science treatment was performed in biological triplicates. Error bars represent s.e.m. ***P,0.005. (E) HC11 cells were treated with either 20 ng/ml rmAreg or 10 ng/ml rmEreg for the indicated times. Following treatment, whole cell lysates were collected and analyzed by immunoblot for phosphorylated ERK (pERK) and total ERK. The experiment shown is representative of three individual experiments. (F) HC11 cells were treated with combinations of 20 ng/ml rmAreg or 10 ng/ml rmEreg and DMSO, 1 mM erlotinib or 0.5 mM erlotinib as designated for 10 minutes. Whole cell lysates were collected and analyzed by immunoblot for phosphorylated ERK and total ERK. The experiment shown is representative of three individual experiments.

activate EGFR (Shoyab et al., 1989; Komurasaki et al., 1997; migration of the HC11 cells as compared with the DMSO- Shelly et al., 1998), we examined the effects of blocking treated controls (Fig. 3A,B), suggesting that the reduction in signaling through EGFR on the migratory potential of the migration is not due to erlotinib-induced off-target effects but is rmAreg- or rmEreg-treated cells. For these studies, we used the due to an inhibition of EGFR signaling. These data suggest that EGFR inhibitor erlotinib, which exerts its effects by blocking treatment of cells with either AREG or EREG alone is enough to EGFR kinase function. Cells treated with either rmAreg or promote increased mammary epithelial cell migration, a hallmark rmEreg together with erlotinib had significantly (P,0.001) of tumor progression. Furthermore, this migration is promoted at decreased migration compared with the rmAreg- or rmEreg- least in part through EGFR, as blocking EGFR activity by treated cells, similar to the DMSO-treated control levels addition of erlotinib significantly impairs the ability of these (Fig. 3A,B). Moreover, erlotinib alone did not affect the epithelial cells to migrate into the open space of the wound. EGFR promotes FGFR1-driven tumors 3111

Previous studies have also demonstrated that activation of evaluated. As shown in Fig. 4A, AP treatment stimulated iFGFR1 induces proliferation of mammary epithelial cells (Welm phosphorylation of residues Y845, Y1068 and Y1173, as et al., 2002; Xian et al., 2009; Xian et al., 2005). Because EGFR compared with that upon ethanol treatment, suggesting that activation has also been linked to increased proliferation, we activation of iFGFR1 does subsequently cause EGFR activation. hypothesized that the upregulation of EGF ligands, such as Because treatment of cells with recombinant AREG and EREG AREG and EREG, might promote proliferation. Therefore, initial protein stimulates migration and proliferation of HC11 cells, and studies were performed to determine the ability of AREG and because activating iFGFR1 results in EGFR phosphorylation, we EREG to stimulate mammary epithelial cell proliferation. For next tested whether iFGFR1-induced cellular processes were these studies, HC11 cells were treated with rmAreg or rmEreg and dependent upon EGFR activity. Freshly wounded and serum- either DMSO or erlotinib for 48 hours before performing an MTT starved HC11/R1 cells were treated overnight with either AP and assay. Treatment of HC11 cells with either rmAreg (Fig. 3C) or DMSO, or ethanol and DMSO. The percentage of wound closure rmEreg (Fig. 3D) significantly (P,0.005) promoted proliferation was calculated from the start time of treatment (0 hours) to compared with DMSO-treated controls, and this proliferation was 18 hours post treatment. As expected based on previous studies blocked with erlotinib treatment. Taken together, these data (Xian et al., 2005), AP and DMSO treatment significantly demonstrate that treatment of mammary epithelial cells with stimulated migration of the HC11/R1 cells compared to ethanol recombinant AREG or EREG promotes EGFR-dependent and DMSO-treated controls (Fig. 4B). To determine whether migration and stimulates EGFR-dependent mammary epithelial EGFR activation is required for the increased iFGFR1-induced cell proliferation. migration of HC11/R1 cells, we also added erlotinib to AP- or ethanol-treated cells. As shown in Fig. 4B, addition of erlotinib to AREG and EREG stimulate ERK activation in vitro, which is AP-treated cells partially, but significantly decreased migration to inhibited by erlotinib levels close to the ethanol and DMSO-treated control. Erlotinib The results described above, in which AREG and EREG treatment of the ethanol control cells did not inhibit migration as treatment stimulates migration and proliferation of HC11 compared with that in the ethanol and DMSO-treated control cells, mammary epithelial cells, and that this is blocked by addition demonstrating that the decreased migration of the AP- and of the EGFR kinase inhibitor erlotinib, suggest that EGFR erlotinib-treated HC11/R1 cells is not due to non-specific off- activation is required for AREG and EREG to exert their target effects of erlotinib. These results suggest that iFGFR1 signaling effects. Because a known target of EGFR activation is activation signals through EGFR to promote migration in vitro. ERK, we next examined the activation profile of ERK following To determine whether iFGFR1-mediated cellular proliferation rmAreg or rmEreg treatment. HC11 cells were treated with either could also be blocked by erlotinib treatment, an MTT assay was rmAreg or rmEreg for various times up to 1 hour, and performed. As shown in Fig. 4C, AP and DMSO treatment of immunoblot analysis was performed to examine levels of HC11/R1 cells induced greater cellular proliferation than ethanol phosphorylated and total ERK. Both rmAreg and rmEreg and DMSO-treated controls. This proliferation was partially, but treatments led to increased ERK activation by 5 minutes, significantly (P,0.05), inhibited by erlotinib treatment, which was largely diminished within 15 minutes (Fig. 3E). suggesting that iFGFR1 activation, at least in part, signals

Journal of Cell Science These results suggest that AREG or EREG treatment induces through EGFR to promote cellular proliferation in vitro. rapid and transient ERK activation. Moreover, erlotinib treatment of the ethanol control cells did To confirm that the detected phosphorylated ERK is induced not significantly block proliferation as compared with the downstream of EGFR, HC11 cells were treated with rmAreg or ethanol- and DMSO-treated control cells, again demonstrating rmEreg in the presence or absence of erlotinib for 10 minutes. that the reduction in proliferation of the AP- and erlotinib-treated Compared with DMSO-treated controls, rmAreg- or rmEreg-only HC11/R1 cells is not due to off-target effects of erlotinib. treated cells showed increased phosphorylation of ERK (Fig. 3F), To be certain that erlotinib does not inhibit iFGFR1-driven as expected on the basis of our above results. Notably, treatment of migration and proliferation by blocking iFGFR1 activity in a cells with erlotinib at either 1 mMor0.5mM, in addition to rmAreg non-specific manner, HC11/R1 cells were treated with AP in the or rmEreg, blocked ERK phosphorylation (Fig. 3F), suggesting that presence of erlotinib and phosphorylation of ERK, a well- blocking EGFR activation, and thus signaling through EGFR, established downstream target of FGFR1 signaling, was blocks activation of ERK in mammary epithelial cells. These data determined. For these studies, serum-starved cells were treated imply that EGFR must be activated in order for AREG and EREG for 0, 5, 10, 15, 30 and 60 minutes with AP in the presence of to exert their effects and that blocking EGFR signaling effectively either erlotinib or DMSO. As shown in Fig. 4D, erlotinib did not inhibits the effects of AREG and EREG stimulation. block iFGFR1-induced activation of ERK during this timecourse. These results demonstrate that erlotinib is not blocking migration Activation of iFGFR1 in mammary epithelial cells induces and proliferation by non-specifically inhibiting iFGFR1 activity. migration, proliferation and ERK activation in vitro, which On the basis of these results, we predicted that the are inhibited by erlotinib accumulation of the ligands AREG and EREG over time leads As shown thus far, iFGFR1 activation upregulates expression of to activation of EGFR and its downstream signaling pathways, the EGFR ligands AREG and EREG, and the EGFR-specific and that this autocrine stimulation of EGFR contributes to inhibitor erlotinib blocks Areg- and Ereg-induced cellular FGFR1-induced migration and proliferation. To address this processes and signaling. Therefore, we next assessed whether prediction, immunoblot analysis of signaling molecules EGFR is activated following iFGFR1 activation in HC11/R1 downstream of EGFR was performed on HC11/R1 cells treated cells. Serum-starved HC11/R1 cells were treated with AP or with AP or ethanol in the presence or absence of erlotinib or ethanol for 18 hours before the phosphorylation status of three DMSO for 7 or 18 hours. As expected, phosphorylation of Akt crucial tyrosine residues within the EGFR kinase domain was and ERK was higher in the 18-hour AP-treated samples as 3112 Journal of Cell Science 124 (18) Journal of Cell Science Fig. 4. Erlotinib inhibits FGFR1-induced migration, proliferation and ERK activation in vitro. (A) HC11/R1 cells were treated with either 30 nM AP or ethanol for 18 hours. Whole cell lysates were collected and analyzed by immunoblot for total EGFR and EGFR phosphorylated at Y845, Y1068, and Y1173 (pY845, pY1068 and pY1173). Experiments shown are representative of three individual experiments. (B) Freshly wounded mouse mammary epithelial HC11/R1 cells were treated with ethanol+DMSO, 30 nM AP+DMSO, ethanol+1 mM erlotinib, or 30 nM AP+1 mM erlotinib for 18 hours. Five representative images were taken per well at 0 and 18 hours, and the area of the open space of the wound was determined using Leica LAS Software. Migration was measured as percent wound closure. Each treatment was performed in biological triplicates. Error bars represent s.e.m. ***P,0.005; ****P,0.001. (C) HC11/R1 cells were treated as in B for 72 hours. Proliferation was then measured with an MTT assay and normalized to the levels at day 0 of treatment. Each treatment was performed in biological triplicates. Error bars represent s.e.m. *P,0.05; ****P,0.001. (D) HC11/R1 cells were treated with 30 nM AP in the presence of either DMSO or 2 mM erlotinib for the times indicated. Whole cell lysates were collected and analyzed by immunoblot for phosphorylated ERK (pERK) and total ERK. The experiment shown is representative of three individual experiments. (E) HC11/R1 cells were treated with 30 nM AP or ethanol and either DMSO or 1 mM erlotinib for 7 [for measuring the cyclin D1 (Cyc D1) and b-tubulin (b-tub) levels] or 18 (for Akt and ERK) hours. Whole cell lysates were collected and analyzed by immunoblot for phosphorylated Akt (pAkt), total Akt, Cyc D1, b-tubulin, pERK and total ERK. Experiments shown are representative of three individual experiments.

compared with ethanol-treated samples (Fig. 4E). Moreover, the epithelial cells (Welm et al., 2002). On the basis of our above expression of the cell cycle regulator cyclin D1 was upregulated results in which AREG and EREG promote cellular proliferation following 7 hours of AP treatment as compared with 7 hours of in vitro in an erlotinib-dependent manner, we wanted to determine ethanol treatment. Importantly, erlotinib inhibited both Akt and whether erlotinib could also inhibit aberrant cellular proliferation ERK phosphorylation and cyclin D1 expression (Fig. 4E), in vivo. For these studies, MMTV-iFGFR1 transgenic mice were suggesting that EGFR activation in HC11/R1 cells results in treated with erlotinib daily through oral gavage for 3 days in subsequent activation of several downstream signaling pathways. conjunction with intraperitoneal injections of AP on the second day of erlotinib treatment. At 2 days after iFGFR1 activation, the Activation of iFGFR1 in vivo induces cellular proliferation mice were injected with BrdU 2 hours before killing. The fourth of mouse mammary epithelial cells, which is inhibited inguinal mammary glands were harvested and tissue sections were by erlotinib stained for further analysis. Using hematoxylin and eosin (H&E) Activating iFGFR1 in vivo is known to stimulate aberrant staining, it was found that, compared with wild-type controls, epithelial cell budding due to increased proliferation of the mammary glands from mice in which iFGFR1 was activated EGFR promotes FGFR1-driven tumors 3113

exhibited more aberrant epithelial budding around the ductal breast cancers (Gelsi-Boyer et al., 2005; Kwek et al., 2009; lumens (Fig. 5A–D). This aberrant budding significantly (P,0.05) Letessier et al., 2006; Ugolini et al., 1999). Although it is still not decreased when the transgenic animals were treated with erlotinib clear whether FGFR1 is the driving oncoprotein in this region, in conjunction with iFGFR1 activation, suggesting that activation studies that focused on identification of smaller segments within of EGFR is, at least in part, required for the anomalous mammary the amplicon have found that the region containing FGFR1 has epithelial cell budding (Fig. 5A–D). been linked to poor prognosis (Gelsi-Boyer et al., 2005). To ensure that the observed phenotype is due to aberrant Furthermore, recent studies have demonstrated that although cellular proliferation and not just a rearrangement of the cellular FGFR1 might not be sufficient to drive tumor formation on its structure of the ductal tree, histological sections were stained own, it can act in concert with genes in other co-amplified with an anti-BrdU antibody. As shown in Fig. 5E–H, iFGFR1 regions, such as MYC on 11q13, to promote tumorigenesis (Kwek activation in transgenic animals greatly increased cellular et al., 2009). In agreement with this hypothesis, studies using proliferation of the ductal epithelial cells as compared with mouse models have demonstrated that FGFR1 activation, in wild-type controls. Erlotinib treatment of iFGFR1 transgenic conjunction with another oncogenic signal, such as WNT1, can mice significantly (P,0.001) reduced the number of BrdU- dramatically decrease tumor latency (Pond et al., 2010). Finally, positive epithelial cells. These data suggest that iFGFR1-induced recent studies have implicated FGFR1 in breast cancer, cellular proliferation in vivo requires activation of EGFR, as particularly in the resistance of breast cancer cells to inhibiting the EGFR kinase domain with erlotinib treatment endocrine- and chemotherapy-based treatments (Chin et al., blocks the ability of the epithelial cells to proliferate. 2006; Turner et al., 2010). Therefore, FGFR1 might represent a novel therapeutic target in breast cancer patients, particularly in Discussion patients that do not respond well to standard therapies. The gene encoding FGFR1 is located on human chromosome On the basis of the potential contributions of FGFR1 to breast 8p11-12, which is a common site of amplification in human tumorigenesis, we have utilized both in vitro and in vivo models Journal of Cell Science

Fig. 5. Erlotinib inhibits FGFR1-induced mammary epithelial cell budding and proliferation in vivo. (A–C) Representative images of H&E-stained sections of paraffin-embedded mammary glands from wild-type (control) mice (A), and MMTV-iFGFR1 transgenic mice treated with 1 mg per kg of body weight per day AP through intraperitoneal injections and either DMSO (B) or 25 mg per kg of body weight per day erlotinib (C) through oral gavage. Scale bars: 50 mm. (D) Quantification of the number of budding ductal structures. Every ductal structure distal to the lymph node was counted in H&E-stained sections of the 3 classes of mice, and was then grouped into budding or not budding structures. At least three sections were analyzed per mouse. Error bars represent s.e.m. *P,0.05. (E) Cellular proliferation was measured by immunofluorescence through BrdU staining. All epithelial cells were counted in ten ductal structures per section through DAPI staining, and the percentage of BrdU-positive epithelial cells was determined. A minimum of 1500 cells was counted per treatment group. Error bars represent s.e.m. ****P,0.001. (F–H) Representative images of BrdU-stained (red) sections of paraffin-embedded mammary glands from wild-type (control) mice (F), and MMTV- iFGFR1 transgenic mice treated with 1 mg per kg of body weight per day AP through intraperitoneal injections and either DMSO (G) or 25 mg per kg of body weight per day erlotinib (H) through oral gavage. The sections were also stained with DAPI (blue) to visualize all cells. Scale bars: 50 mm. 3114 Journal of Cell Science 124 (18)

to better understand the mechanisms by which FGFR1 promotes activation stimulates phosphorylation of EGFR and that blocking mammary tumor formation. Previous studies have demonstrated EGFR activity inhibits iFGFR1-induced migration and that activation of FGFR1 in mammary epithelial cells in vitro proliferation. Moreover, investigation of downstream molecules results in increased proliferation, survival, migration, invasion demonstrated that AP treatment of HC11/R1 cells stimulated and EMT (Welm et al., 2002; Xian et al., 2007; Xian et al., 2005). activation of Akt and ERK and expression of cyclin D1 in an Furthermore, activation of FGFR1 in mammary epithelial cells in EGFR-dependent manner. However, analysis of other signaling vivo leads to the formation of alveolar hyperplasias, ultimately pathways revealed that both MAPK14 and phospholipase Cc resulting in the formation of tumors with both adenocarcinoma were regulated solely by iFGFR1 and not by EGFR (data not and squamous characteristics (Schwertfeger et al., 2006; Welm et shown). Although erlotinib treatment significantly inhibited al., 2002). We have further utilized these cell culture and iFGFR1-driven proliferation and migration, this inhibition was transgenic mouse models to delineate the mechanisms by which not complete, suggesting that EGFR-independent pathways also FGFR1 promotes mammary tumor formation. Obtaining a better contribute to these phenotypes. Therefore, it is possible that using understanding of these mechanisms will ultimately lead to the a combination of erlotinib and additional inhibitors that development of therapeutic strategies to target tumors in breast specifically target these pathways will lead to a more complete cancer patients with high levels of FGFR1 and increased inhibition of iFGFR1-driven phenotypes. Overall, the results resistance to conventional therapies. presented here support the hypothesis that iFGFR1 leads to Previous gene expression studies, using microarray analysis, activation of EGFR, which then contributes to the regulation of led to the identification of numerous potential genes involved in some signaling pathways that promote iFGFR1-induced promoting FGFR1-driven tumorigenesis (Schwertfeger et al., tumorigenesis. 2006). Our recent studies have focused primarily on secreted Because the concentration of AREG in the medium was factors regulated by FGFR1 because of the potential ability of consistently higher than that of EREG, we predicted that AREG secreted factors to regulate both epithelial and stromal cells. would be the dominant ligand responsible for EGFR activation. During the analysis of these previous studies, we found a However, knockdown of Areg in the HC11/R1 cells using shRNA significant induction of two EGF ligands, AREG and EREG, strategies did not reveal any differences in iFGFR1-induced following iFGFR1 activation. This finding was further verified in migration or proliferation (data not shown). Therefore, it is our iFGFR1 in vitro system. Importantly, AREG and EREG possible that EREG compensates for the loss of AREG and that expression was increased following iFGFR1 activation at both targeting single ligands might not be an effective therapeutic the transcript and protein levels. This observation was also strategy when multiple EGFR ligands are present. Current studies confirmed in the MCF7 human breast cancer cell line, which has are focusing on understanding the regulation of the sheddase been previously used to study FGF signaling in breast cancer ADAM17 by FGFR1 and the ability of this protease to release of cells. We chose to further evaluate the ability of these ligands to both AREG and EREG into the medium. Targeting the regulation act through the EGFR pathway to promote FGFR1-driven of AREG and EREG release into the medium might represent a mammary tumorigenesis. better strategy than targeting the ligands themselves. Both AREG and EREG have been implicated in breast cancer Using a mouse model of mammary tumorigenesis, we have

Journal of Cell Science (Bieche et al., 2004; Eltarhouny et al., 2008; Gupta et al., 2007; demonstrated that treatment of mice with erlotinib inhibits Kenney et al., 1996b; Kondapaka et al., 1997; Ma et al., 1999; epithelial proliferation and subsequent formation of early-stage Silvy et al., 2001). High levels of AREG expression have been hyperplastic lesions following iFGFR1-activation in vivo. identified in cancerous lesions but not in neighboring non- Although our cell culture studies focused on the autocrine cancerous tissue (LeJeune et al., 1993; Panico et al., 1996; Qi et effects of iFGFR1-induced activation of EGFR, it is possible that al., 1994). This expression has also been associated with large paracrine stimulation of EGFR is also involved in regulating the high-grade tumors and metastases found in lymph nodes, tumorigenic phenotype in vivo. For example, transplantation suggesting that high levels of AREG expression are a marker studies using different combinations of AREG and EGFR-null for aggressive invasive breast cancers (LeJeune et al., 1993; Ma and -positive epithelial and stromal cell types found that, for et al., 2001; Qi et al., 1994). EREG expression has also been proper mammary gland development, AREG expression is found to correlate with breast cancer metastasis to the lung, required in the mammary epithelial cells, whereas EGFR is suggesting that it too promotes invasive breast cancer progression required in the stromal, and not the epithelial, compartment (Eltarhouny et al., 2008; Gupta et al., 2007). AREG is known to (Sternlicht et al., 2005; Wiesen et al., 1999). These data indicate exclusively bind and activate EGFR (Shoyab et al., 1989), that AREG can act in a paracrine manner through stromal EGFR whereas EREG acts through both EGFR and ERBB4 to stimulate expression of growth factor(s) that can act to drive (Komurasaki et al., 1997; Shelly et al., 1998). In these studies, epithelial cell proliferation, which is necessary for proper ductal we found that AREG and EREG were both able to induce network formation within the mammary gland. Further studies proliferation, migration and activation of the ERK pathway in are required to better understand the effects of AREG and EREG HC11 cells. Furthermore, AREG and EREG both act primarily on cells within the stroma and how these interactions might through EGFR, as demonstrated by the inhibition of these promote tumorigenesis. phenotypes by the EGFR-specific inhibitor erlotinib. Taken together, our results reveal the complex interactions Because AREG and EREG, which are both EGFR ligands, are between various signaling pathways that can regulate both induced by iFGFR1 activation and are capable of promoting tumorigenesis and suggest that targeted therapies need to take both migration and proliferation of the HC11 cells, we into account more than one pathway. To date, FGFR1 inhibitors hypothesized that activation of EGFR could be a mechanism have not yet been tested in patients, although there are reports of by which iFGFR1 promotes these phenotypes. In agreement with FGFR1 inhibitors in preparation (Zhou et al., 2010). However, this hypothesis, the present study demonstrates that iFGFR1 EGFR inhibitors have been successfully utilized in patients with EGFR promotes FGFR1-driven tumors 3115

certain types of cancers, including lung cancers (Gridelli et al., for 4, 6 or 24 hours. Conditioned medium was collected and used to quantify the soluble ligand concentration of AREG and/or EREG using ELISA assays (R&D 2010; Squadroni and Fazio, 2010). Therefore, inhibition of the Systems). Experiments were performed in biological triplicates. Statistical analysis EGFR pathway might be an alternative targeting strategy in was performed using the unpaired Student’s t-test to compare two means breast cancer patients with high levels of FGFR1. Further studies (GraphPad QuickCalcs). are required to determine the link between FGFR1 expression and EGFR activation in human breast cancer tissue samples. Migration assays For AREG and EREG overexpression, HC11 cells were grown to confluence and In summary, we have demonstrated that the EGFR pathway is an then starved in SF-RPMI. The next day, a P10 pipette tip was used to make a important downstream regulator of FGFR1-induced mammary scratch down the center of each well. Cells were then treated with either 20 ng/ml tumor formation. Growth factor receptors and their downstream rmAreg or 10 ng/ml rmEreg (both R&D Systems) in the presence or absence of signaling pathways in breast cancer have been the focus of numerous either 1 mM or 0.5 mM erlotinib, or an equivalent amount of DMSO. To test iFGFR1-induced migration, HC11/R1 cells were grown to confluence and then studies. However, these studies have generally focused on single starved in SF-RPMI overnight. Serum starved cells were treated with 30 nM AP or pathways at a time. It is becoming clear that these pathways probably an equivalent amount of ethanol overnight. Additionally, the HC11/R1 cells were interact and that understanding the key points at which these treated with 1 mM erlotinib or an equivalent amount of DMSO. All treatments pathways communicate is important for generating novel therapies were performed in biological triplicates. Five representative pictures of each scratch were taken at the start time of treatment and 18 hours later. Areas of the that most efficiently inhibit tumor formation and progression. open space in each picture were determined using the Leica LAS software, and the percentage of wound closure was calculated for each treatment. Statistical analysis Materials and Methods was performed using the unpaired Student’s t-test to compare two means Cell culture and treatment (GraphPad QuickCalcs). Generation of HC11 cells stably expressing the iFGFR1 construct (HC11/R1 cells) was described previously (Xian et al., 2005), and the cells were obtained from Jeff MTT assays Rosen (Baylor College of Medicine, Houston, TX). Cells were maintained in HC11 or HC11/R1 cells were plated in complete medium on day 0 into 24-well HC11/R1 complete medium [serum free (SF)-RPMI (Invitrogen) supplemented tissue culture plates at 20,000 cells per well. Cells were grown overnight and then with of 10% fetal bovine serum (FBS) (Invitrogen), 1% penicillin-streptomycin starved in SF-RPMI. The next day, cells were stimulated with 100 ng/ml rmAreg (Invitrogen), 5 mg/ml (Sigma-Aldrich), 10 ng/ml EGF (Invitrogen) and (HC11 cells), 10 ng/ml rmEreg (HC11 cells) or 30 nM AP (HC11/R1 cells) in the 0.7 mg/ml puromycin (Sigma-Aldrich) (final percentages or concentrations)]. presence or absence of 1 mM erlotinib or an equivalent amount of DMSO. After 48 HC11 cells were maintained in HC11 complete medium (HC11/R1 complete (HC11 cells) or 72 (HC11/R1 cells) hours, Thiazolyl Blue Tetrazolium Bromide medium without puromycin). Before treatment with 30 nM AP (Ariad (Sigma-Aldrich) was added to each well at a final concentration of 0.3 mg/ml for Pharmaceuticals, Cambridge, MA) or its solvent ethanol, confluent cells were 2 hours. The medium was then removed from the wells, and the cells were rinsed twice with 16PBS (Cellgro, Manassas, VA) and incubated in SF-RPMI solubilized in 95% DMSO and 5% 16PBS. The solubilized dye was detected on a overnight. 2 mM erlotinib (Boynton Pharmacy, University of Minnesota, plate reader at an absorbance at 570 nm with background fluorescence at 650 nm Minneapolis, MN, USA), 1 mM erlotinib, 0.5 mM erlotinib, or DMSO (Sigma- subtracted from each value. Experiments were performed in biological triplicates. Aldrich) as a solvent control, was added to the SF media as indicated. MCF7 cells Statistical analysis was performed using the unpaired Student’s t-tests to compare were previously obtained from the American Type Culture Collection (ATCC, two means (GraphPad QuickCalcs). Manassas, VA) and maintained in MCF7 complete medium [SF-Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% FBS, 1% penicillin-streptomycin and 5 mg/ml insulin (final percentages or concentrations)]. Immunoblot analysis Before treatment with bFGF (Invitrogen), confluent MCF7 cells were rinsed twice HC11 or HC11/R1 cells were grown to confluence and then starved in SF-RPMI with 16PBS and starved overnight in SF-DMEM. For bFGF treatment, cells were overnight. The next day, each well of HC11 cells was treated with 20 ng/ml incubated for the indicated time with 50 ng/ml bFGF. All cells were grown and rmAreg or 10 ng/ml rmEreg for the indicated time (Fig. 3E) or overnight (Fig. 3F) maintained at 37 C under a 5% CO atmosphere. in the presence or absence of either 1 mM or 0.5 mM erlotinib, or an equivalent Journal of Cell Science ˚ 2 amount of DMSO. HC11/R1 cells were treated with either 30 nM AP or ethanol in the presence of either 1 mM erlotinib or an equivalent amount of DMSO for 7 or RNA isolation and quantitative RT-PCR analysis 18 hours (Fig. 4A,E). Alternatively, HC11/R1 cells were treated with 30 nM AP RNA was isolated from HC11/R1 and MCF7 cells and from the fourth inguinal with 2 mM erlotinib or an equivalent amount of DMSO for the times indicated mammary glands of MMTV-iFGFR1 transgenic mice using TRIzol (Invitrogen) as (Fig. 4D). Following all treatments, lysates were collected in RIPA buffer, and the described previously (Reed et al., 2009; Schwertfeger et al., 2006). For qRT-PCR total protein concentration was determined using a Bradford assay. At total of analysis, cDNA was generated using the Quantitect reverse transcription kit (Qiagen) 30 was analyzed by SDS-PAGE and immunoblotting for and used in quantitative SYBR green qRT-PCR reactions as described previously m (Yuen et al., 2002) using the Bio-Rad iQ5 system. Relative quantification of gene phosphorylated Akt (1:2000, 9271, Cell Signaling), cyclin D1 (1:2000, 2926, expression was calculated and normalized to averaged cyclophilin B expression Cell Signaling), b-tubulin (1:1000, 2146, Cell Signaling), EGFR (1:100,000, levels using the 2–DDCt method (Livak and Schmittgen, 2001). The following mouse ab52894, Abcam), EGFR phosphorylated at Y845 (1:1000, 2231, Cell Signaling), primer sequences were used: AREG, 59-GGGGACTACGACTACTCAGAG-39 and EGFR phosphorylated at Y1068 (1:1000, 3777, Cell Signaling), EGFR 59-TCTTGGGCTTAATCACCTGTTC-39;EREG,59-TCCGAGGATAACTGT- phosphorylated at Y1173 (1:1000, ab5652, Abcam), or phosphorylated ERK ACCGC-39 and 59-CTCTCATGTCCACCAGGTAGAT-39; and cyclophilin B, 59-TG- (1:500, 9101, Cell Signaling). Additionally, 5 mg protein was analyzed by SDS- AGCACTGGGGAGAAAGG-39 and 59-TTGCCATCCAGCCACTCAG-39.For PAGE and immunoblotting for total Akt (1:1000, 9272, Cell Signaling) or ERK MCF7 cells, the following human primers were used: AREG, 59-GTGGTGCT- (1:2000, sc-94, Santa Cruz Biotechnology). Experiments were performed at least GTCGCTCTTGATA-39 and 59-ACTCACAGGGGAAATCTCACT-399; EREG, 59- three separate times. CTGCCTGGGTTTCCATCTTCT-39 and 59-GCCATTCATGTCAGAGCTACA- CT-39; and cyclophilin B, 59-GAAAGAGCATCTACGGTGAGC-39 and 59-GTC- Animals TTGACTGTCGTGATGAAGAA-39. Experiments were performed in biological Generation of MMTV-iFGFR1 transgenic mice has been described previously triplicates. Statistical analysis was performed using the unpaired Student’s t-test to (Welm et al., 2002) and the mice were obtained from Jeff Rosen. Animal care and compare two means (GraphPad QuickCalcs). procedures were approved by the Institutional Animal Care and Use Committee of the University of Minnesota and were in accordance with the procedures detailed Immunohistochemistry in the Guide for Care and Use of Laboratory Animals. The following antibodies were used: anti-AREG (AF989) and anti-EREG (AF1068) antibodies (R&D Systems). For both antibodies, sodium citrate Treatment of mice antigen retrieval was used as described previously (Grimm and Rosen, 2003) Six-week-old MMTV-iFGFR1 female mice were administered with 25 mg per kg and primary antibodies were used at a concentration of 15 mg/ml. of body weight per day erlotinib or an equivalent amount of the DMSO solvent Immunohistochemistry analysis was performed on mammary gland sections through oral gavage once daily for 3 days. On the second day of oral gavage from a minimum of three mice per treatment and genotype. treatment, the mice were also given intraperitoneal injections of 1 mg per kg of body weight AP to activate iFGFR1. At 24 hours after the third oral gavage, the ELISA mice were given intraperitoneal injections of 0.3 mg per kg of body weight BrdU Serum-starved HC11/R1 cells were treated with either 30 nM AP or its solvent (GE Healthcare). Mice were killed 2 hours after the BrdU injections, and their ethanol overnight. Serum-starved MCF7 cells were treated with 50 ng/ml bFGF fourth inguinal mammary glands were harvested for further analysis. 3116 Journal of Cell Science 124 (18)

Mammary gland histology and measurement of epithelial budding Gridelli, C., Maione, P., Bareschino, M. A., Schettino, C., Sacco, P. C., Ambrosio, Glands were fixed in fresh 4% paraformaldehyde (PFA) for 2 hours on ice and R., Barbato, V., Falanga, M. and Rossi, A. (2010). Erlotinib in the treatment of non- then stored in 70% ethanol. For histological and immunofluorescence analysis, small cell lung cancer: current status and future developments. Anticancer Res. 30, glands were embedded in paraffin and sectioned (5 mm thickness). H&E-stained 1301-1310. sections were used to calculate the percentage of budding structures. Each Grimm, S. L. and Rosen, J. M. (2003). The role of C/EBPbeta in mammary gland epithelial structure distal to the lymph node was counted and grouped into either development and breast cancer. J. Mammary Gland Biol. Neoplasia 8, 191-204. no budding, moderate budding (1–4 buds) or severe budding (.4 buds). The Grose, R. and Dickson, C. (2005). Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev. 16, 179-186. percentage of budding was calculated by comparing all budding structures to all Gupta, G. P., Nguyen, D. X., Chiang, A. C., Bos, P. D., Kim, J. Y., Nadal, C., Gomis, structures for each category of mice. At least three sections were analyzed per R. R., Manova-Todorova, K. and Massague, J. (2007). Mediators of vascular mouse. For immunofluorescence analysis, 5-mm sections were stained with anti- remodelling co-opted for sequential steps in lung metastasis. Nature 446, 765-770. BrdU antibody (1:300, ab6326, Abcam,) according to standard protocols. Sections Kenney, N. J., Smith, G. H., Maroulakou, I. G., Green, J. H., Muller, W. J., were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) to Callahan,R.,Salomon,D.S.andDickson,R.B.(1996a). Detection of visualize the nuclei. A total of 10 epithelial ductal structures distal to the lymph amphiregulin and Cripto-1 in mammary tumors from transgenic mice. Mol. node were imaged per mammary gland section. On the basis of the DAPI staining, Carcinog. 15, 44-56. all epithelial cells within each structure were counted and all BrdU-positive Kenney, N. J., Smith, G. H., Rosenberg, K., Cutler, M. L. and Dickson, R. B. epithelial cells of the same structure were counted to determine the percentage of (1996b). Induction of ductal morphogenesis and lobular hyperplasia by amphiregulin BrdU-positive cells. A minimum of 1500 epithelial cells were counted per in the mouse mammary gland. Cell Growth Differ. 7, 1769-1781. treatment group, and all studies were performed in a blinded manner. Statistical Klijn, J. G., Berns, P. M., Schmitz, P. I. and Foekens, J. A. (1992). 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