Fibroblast growth factor receptor splice variants are stable markers of oncogenic transforming growth factor β1 signaling in metastatic breast cancers
© Wendt et al.; licensee BioMed Central Ltd. 2014
Received: 27 September 2013
Accepted: 28 February 2014
Published: 11 March 2014
Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle.
An in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor β1 (TGF-β1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398.
Metastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-β1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-β was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth.
The results of our study strongly suggest that FGFR1-β is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.
The reported results from several recent studies suggest that metastatic breast cancer (BC) cells undergo epithelial–mesenchymal transition (EMT) during invasion and dissemination and that the reverse process of mesenchymal–epithelial transition (MET) occurs at some point during metastatic tumor outgrowth [1–3]. In fact, the ability of BCs to transition between an epithelial and mesenchymal state seems to be a key feature of the metastatic process and has recently been more accurately termed epithelial–mesenchymal plasticity. Indeed, many of the well-established changes in gene expression that take place during EMT return to baseline during MET. However, stable phenotypic markers capable of distinguishing metastatic BC cells that have undergone an EMT:MET cycle from their indolent counterparts that have not undergone an EMT program remain to be identified.
Recently, we demonstrated that transforming growth factor β (TGF-β)–induced EMT empowers BCs with the ability to invade in response to paracrine epidermal growth factor (EGF) stimulation, thereby facilitating the egress of BC cells from the primary tumor . Intriguingly, we also observed that cells proficient in undergoing metastatic outgrowth downregulate EGF receptor (EGFR) during the MET process . These findings may serve to explain the contrasting clinical data that establish EGFR as a predictor of poor prognosis for BC patients , yet administration of monotherapies directed against EGFR or in conjunction with other chemotherapies has failed to provide a clinical benefit for BC patients [7–9].
Increased expression of fibroblast growth factor receptor (FGFR) types 1 and 3 have recently been identified as two of six receptor tyrosine kinases associated with poor disease-free survival and/or decreased overall survival in BC patients . FGFR1, FGFR2 and FGFR3 all exist as several different isoforms generated via alternative splicing . Two of the best-described variants are generated via inclusion of unique versions of the third (III) extracellular immunoglobulin (III-Ig) domain, and hence they are termed FGFR-IIIb and FGFR-IIIc. The III-Ig domain governs the specificity of FGFR binding to the 18 different FGF ligands. FGF2 (basic FGF), for instance, has an extremely high affinity for the IIIc isoform . Another FGFR splicing event results in either the inclusion (FGFR-α) or exclusion (FGFR-β) of the first Ig domain and/or the linker region between IgI and IgII, an area termed the acid box. Importantly, IgI and the linker region regulate the affinity of FGFR for its particular ligand . Furthermore, increased expression of the β versus α isoform of FGFR1 has been correlated with reduced relapse-free survival in a cohort of BC patients . The use of antisense morpholino oligonucleotides to convert FGFR splicing from the β to the α isoform can induce apoptosis in glioblastomas . Currently, the upstream mechanisms that regulate FGFRα/FGFRβ splicing remain poorly defined, but TGF-β and its induction of EMT can cause upregulation of FGFR1-IIIc and downregulation of FGFR2-IIIb . However, little is known about the mechanism by which either of these events drives the metastatic progression of BCs.
In The present study, we sought to identify factors that are stably altered during EMT:MET cycles and thus might act as drivers of metastatic tumor outgrowth. To this end, we utilized microarray expression analyses of BC cells that had been treated and withdrawn from exogenous TGF-β to uncover a stable upregulation of the FGFR1-β isoform, an event that was also readily detected in samples obtained from BC patients and in metastatic human BC cell lines. Along these lines, genetic depletion of total FGFR1 and/or ectopic overexpression of FGFR1-α in the D2.A1 model of pulmonary metastatic outgrowth potently inhibited pulmonary tumor formation. Collectively, these studies establish FGFR1-β as a critical player whose expression is stably altered in metastatic BCs that have experienced oncogenic TGF-β signaling and undergone EMT:MET cycles. Our findings highlight the need to further elucidate the pro- and antitumorigenic nature of FGFR to appropriately administer small-molecule inhibitors for the treatment of metastatic BC.
Cell lines and reagents
Murine D2.A1 and human MCF-10A derivatives (MCF-10A, T1K and Ca1h) were ethically obtained from Dr Fred Miller (Wayne State University, Detroit, MI, USA) [18, 19], and murine 4T1, human MCF-7 and human MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). In addition, the following human BC cell lines were originally purchased from the ATCC and ethically obtained from Dr John J Pink (Case Western Reserve University, Cleveland, OH, USA): MDA-MB-361, ZR-75-1, T47D, T47D-C42W and BT549 . The aforementioned human and murine BC cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as described previously . Bioluminescent 4T1 and D2.A1 cells were engineered to stably express firefly luciferase under the selection of Zeocin (InvivoGen, San Diego, CA, USA) as described previously [2, 5, 21]. Bioluminescent normal mammary epithelial (NME) cells were constructed and cultured as previously described . Dual-bioluminescent E-cad reporter cells were generated by stably transfecting 4T1 cells with pcDNA3.1/Hygro mammalian expression vector (Invitrogen, Carlsbad, CA, USA) encoding Renilla luciferase under control of the cytomegalovirus (CMV) promoter and pGL4.20[luc2/Puro] vector (Promega, Madison, WI, USA) that encodes firefly luciferase under control of the human Cdh1 promoter . Cellular depletion of FGFR1 expression was achieved by glycoprotein of vesicular stomatitis virus lentiviral transduction of TRC pLKO.1 short hairpin RNA (shRNA) vectors (Thermo Scientific, Pittsburgh, PA, USA) (Additional file 1: Table S1) as described previously [2, 21]. Ectopic expression of FGFR1-α-IIIc was accomplished as described previously and selected for using neomycin .
In vivobioluminescence imaging of tumor growth and metastasis
Parental (that is, scrambled shRNA) and FGFR1-manipulated D2.A1 cells were injected into the lateral tail veins of 5-week-old female BALB/C mice (The Jackson Laboratory, Bar Harbor, ME, USA). Where indicated, mice bearing D2.A1 pulmonary tumors were treated daily with BGJ-398 (ChemieTek, Indianapolis, IN, USA) or PF-573271 (PF271; Pfizer Pharmaceuticals, New York, NY, USA) at 50 mg/kg by oral gavage. Alternatively, Cdh1 reporter 4T1 cells (1 × 104 cells) were engrafted onto the mammary fat pads of 4-week-old BALB/c mice. Circulating 4T1 tumor cells were isolated from the inferior vena cava at the time of necropsy using 3% sodium citrate. Following lysis of red blood cells, circulating tumor cells were selected for with 5 μg/ml Zeocin (the selectable marker for firefly luciferase). Luciferase-expressing NME cells (1 to 2 × 106 cells) were engrafted onto the mammary fat pads of 5-week-old female nu/nu mice. All bioluminescent images were captured using a Xenogen IVIS 200 preclinical imaging system (Caliper Life Sciences/PerkinElmer, Hopkinton, MA, USA) within the Small Animal Imaging Resource Center at the Case Comprehensive Cancer Center as previously described [5, 21, 23].
Gene expression profiling
NME cells were cultured in the presence of TGF-β1 (5 ng/ml) for 4 weeks, at the end of which TGF-β1 supplementation was discontinued and the cells were allowed to recover for an additional 4 weeks. Total RNA was prepared from unstimulated cells of similar passage (pre-TGF) and the post-TGF NME cells. Microarray analyses were performed in triplicate using the GeneChip Mouse Gene ST 1.0 Array (Affymetrix, Santa Clara, CA, USA). Genes regulated more than twofold are given in Additional file 2: Table S2. The complete data set has been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database [GEO:GSE54491] .
mRNA transcript analyses
For real-time PCR analysis, normal murine mammary gland (NMuMG) and NME cells were stimulated with TGF-β1 (5 ng/ml) for varying lengths of time, and then total RNA was isolated using RNeasy Plus Mini Kit (QIAGEN, Valencia, CA, USA). Afterward, total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), and semiquantitative real-time PCR was conducted using iQ SYBR Green Supermix (Bio-Rad Laboratories) as described previously . Identification of FGFR splice variants was accomplished by visualizing PCR products separated by gel electrophoresis. The oligonucleotide primer pairs used are provided in Additional file 1: Table S1.
Immunoblotting and immunohistochemical analyses
For immunoblot assays, lysates generated from two- and three-dimensional cultures were prepared as described previously . The antibodies we used are described in Additional file 3: Table S3. For immunohistochemistry, tissues were fixed in 10% formalin, and histological sections were prepared by the Tissue Procurement and Histology Core at the Case Comprehensive Cancer Center. Sections were deparaffinized and stained with the indicated antibodies given in Additional file 3: Table S3.
Three-dimensional organotypic growth assays
Cells were diluted in complete media supplemented with 5% Cultrex reagent (Trevigen, Gaithersburg, MD, USA) and seeded onto solidified Cultrex cushions (50 μl/well) contained in 96-well plates (1 × 104 cells/cm2). Longitudinal bioluminescence growth assays were performed as described previously [2, 5]. Pharmacological inhibitors against FGFR (BGJ-398) or focal adhesion kinase (FAK) (PF271) were added to cultures at the indicated concentrations and times.
All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Case Western Reserve University School of Medicine. For human BC specimens, primary tumors and matched normal tissues were collected and processed under protocols approved by the Institutional Review Board of the Cleveland Clinic. All patients provided their written informed consent allowing the study investigators to have access to their tumor specimens and clinical data.
Statistical values were defined using an unpaired Student’s t-test, where a P-value less than 0.05 was considered significant. Statistically significant differences in the overall survival of mice bearing control and FGFR-manipulated D2.A1 pulmonary tumors were analyzed using a logrank test. P values for all experiments are indicated.
E-cadherin is dynamically regulated during spontaneous breast cancer metastasis
TGF-β treatment is sufficient to drive orthotopic mammary tumor metastasis
FGFR is stably upregulated following TGF-β treatment and withdrawal
Given the transient nature of exogenous TGF-β treatment prior to NME cell engraftment, we next sought to assess the regulation of MET in this model. Consistent with our IHC analysis (Figure 2E), ex vivo subculture of post-EMT NME primary tumors displayed a highly mesenchymal phenotype in two- and three-dimensional cultures and downregulation of E-cad (Additional file 4: Figures S1A and S1B). TGF-β-treated NME tumor cells that had undergone metastasis were subcultured from the lungs of mice. In comparison to their parental NME counterparts, these cells underwent dramatically enhanced primary tumor formation, postsurgical recurrence and spontaneous pulmonary metastasis upon secondary mammary fat pad engraftment (Additional file 4: Figure S1C). These resulting metastases were subcultured and termed the NME lung metastatic (NME-LM2) cell line (Additional file 5: Figure S2A). Further in vitro analyses of the NME-LM cell lines revealed a return of E-cad expression to levels that approximated those detected in their untreated NME parental cells (Additional file 5: Figures S2B and S2C). Taken together, these results indicate that depletion of E-cad by exogenous TGF-β treatment was stably maintained during formation of the primary tumor but readily returned to baseline expression levels during formation of macroscopic pulmonary metastases.
Given that TGF-β treatment and metastasis are capable of selecting for a spontaneously metastatic cell model that has renewed E-cad expression, we next sought to identify stable changes in gene expression that could characterize cells that reside in either a pre- or post-TGF-β exposure state. To do so, we conducted microarray analyses of NME cells that had undergone 4 weeks of exogenous TGF-β1 treatment, which was followed by an additional 4-week withdrawal of exogenous TGF-β1 (Additional file 2: Table S2). Indeed, following this experimental protocol, the expression patterns of traditional EMT markers, such as E-cad, N-cadherin, vimentin, fibronectin, α-smooth muscle actin, Twist and Snail all returned to baseline levels (Additional file 2: Table S2). Surprisingly, 98 genes were stably modulated more than threefold following this TGF-β treatment and withdrawal protocol. Gene set enrichment analysis revealed that the highest degree of overlap between genes upregulated in our gene list was that of genes downregulated in mouse embryonic fibroblasts after 10 hours of TGF-β treatment [GEO:GSE15871] . Furthermore, 15 upregulated and 28 downregulated genes in our data set were shared with a data set generated upon knockout of the TGF-β family member bone morphogenic protein 2 . The transcription factor Snai2, which functions to inhibit the expression of E-cad , was downregulated tenfold in cells that had undergone this cycle of TGF-β treatment and withdrawal. Taken together, these findings suggest that certain aspects of TGF-β signaling can be stably altered, leading to a unique transcriptional profile in cells following prolonged ligand exposure and withdrawal.
FGFR1-β-IIIc is selected for during metastatic progression
Genetic depletion of FGFR1 prevents pulmonary tumor outgrowth
Pharmacologic inhibition of FGFR kinase activity delays pulmonary outgrowth
EMT:MET cycles represent essential physiological processes that occur during critical points in the development, maintenance and repair of wounded epithelial tissues . Through these processes, normal epithelial cells have the capacity to take on certain mesenchymal characteristics and then accurately return to their initial epithelial state. However, as has been observed in numerous other studies and quantified in our present study as shown in Figure 1, the EMT:MET process is pathologically engaged by carcinoma cells during the metastatic cascade. Therefore, in the present study, we sought to address the hypothesis that, following initiation of metastatic outgrowth, tumor cells inaccurately complete the MET program and enter into a secondary epithelial state that is similar to, but critically unique to, the epithelial characteristics of the primary tumor cells from which they are derived. Using microarray analyses, we defined numerous factors that are differentially expressed between nonmetastatic mammary tumor cells and those that have undergone a metastasis-inducing treatment with TGF-β. Overall, these findings will contribute to our understanding of the unique growth properties of metastatic lesions as compared to their corresponding primary tumors.
Using the spontaneously metastatic 4T1 model in combination with a stable E-cad-luciferase reporter system, we quantified the dynamic regulation of E-cad expression during the various steps of the metastatic cascade (Figure 1). Because these cells were derived from a spontaneous tumor, however, they almost certainly underwent one or more EMT:MET cycles during their original and natural development. Therefore, we utilized our EGFR transformation model to specifically demonstrate that TGF-β treatment is sufficient for the acquisition of metastatic properties (Figure 2). Indeed, immunohistochemical analyses and subculture of the primary tumors demonstrated that several changes in gene expression initiated in vitro were maintained in the primary tumor. However, ex vivo subculture of the resulting metastases clearly indicates that these cells have renewed expression of E-cad and enter into a secondary epithelial state. Just as with the 4T1 model, this new epithelial state easily gives way to a secondary spontaneous EMT and metastatic cycle, as secondary engraftment of these cells onto the mammary fat pad leads to robust pulmonary metastasis without TGF-β treatment prior to their inoculation into mice. Subculture of these metastases yielded the NME-LM2 cell line and established an isogenic progression series of cell lines that possess increasing metastatic potential, ranging from normal (NMuMG) to low-grade and nonmetastatic (NME) to high-grade and metastatic (NME-LM).
Using our NME progression series, various human BC cell lines and patient-derived tissue samples, we verified the stable upregulation of FGFR1-β-IIIc that we initially identified in our in vitro TGF-β treatment and recovery microarray analyses. As such, we further investigated the role of FGFR in facilitating late-stage metastatic tumor outgrowth. Our data clearly indicate that cellular transformation is required in conjunction with TGF-β treatment to facilitate sustained upregulation of FGFR1 (Figure 3). In this case, the means of cellular transformation is overexpression of EGFR, and we previously demonstrated that EGFR is downregulated following the in vitro TGF-β treatment and recovery protocol used in our present study . Therefore, it is interesting to note that maintained upregulation of FGFR may serve to explain the disparity between the power of EGFR as a predictive marker of poor prognosis in BC  and the failure of EGFR-targeted therapies in the treatment of metastatic BC [7–9]. Studies aimed at further elucidating the role of FGFR in the inherent resistance of BCs to EGFR-targeted therapies are currently underway in our laboratory. Along these lines, we previously demonstrated that metastatic D2.A1 cells have diminished expression of EGFR compared to their nonmetastatic and isogenic D2.OR counterparts . Interestingly, D2.A1 cells predominantly express FGFR1-β-IIIc, which is consistent with their metastatic phenotype. Importantly, depletion of total FGFR1 and ectopic expression of FGFR1-α-IIIc similarly inhibited pulmonary tumor outgrowth in the present study (Figure 6). Thus, our findings expand upon the results of previous studies that have linked FGFR1-β expression to the development of BC  and work in glioblastomas whose apoptosis was readily induced by administration of morpholino oligonucleotides to reestablish inclusion of the α exon .
Using flanking primer sets, we were able to identify human mammary tumor samples that not only upregulated FGFR1 expression but also aberrantly excluded the α exon as compared to their matched normal samples (patient 1 and patient 7) (Figure 4). Developing this assay as a diagnostic screening test to detect individual FGFR isoform expression could prove to be highly beneficial in prospectively identifying those patients who would most likely benefit from FGFR inhibitor therapy. Indeed, administration of BGJ-398 completely inhibited the activity of FGFR under physiologic conditions and potently delayed pulmonary tumor outgrowth of the D2.A1 cells. These data are consistent with the fact that the D2.A1 cells primarily express FGFR1-β. However, given our genetic studies identifying the antitumorigenic nature of FGFR1-α, administration of FGFR inhibitor therapies to patients whose tumors express this isoform (patients 2 and 13) (Figure 4) could potentially prove to be detrimental. In fact, this pro- and antitumorigenic dichotomy between FGFR isoforms likely contributes to the limited efficacy of BGJ-398 in inhibiting the outgrowth of the D2.A1 cells, as these cells do endogenously express detectable levels of the FGFR1-α isoform. In contrast to this cell culture model, the breast tumor samples of two patients analyzed in our present study (patients 1 and 13) yielded very clear upregulation of one isoform or the other, again supporting the use of this FGFR diagnostic approach as a predictive biomarker for initiation of FGFR inhibitor therapy.
Our studies demonstrate the dynamics of EMT and MET as BC progresses from carcinoma in situ to full-blown metastatic disease. FGFR and several other factors identified herein represent a signature of oncogenic TGF-β signaling that does not return to baseline during recovery from ligand exposure. This failure to accurately execute the MET process sets the stage for FGFR to act as a potent driver of pulmonary metastatic outgrowth, even if it may not have been an initiator of primary tumor formation. Overall, our findings have important implications related to the means by which science and medicine undertake targeting of FGFR for the treatment of metastatic BC.
Circulating tumor cell
Epidermal growth factor receptor
Estrogen receptor α
Fibroblast growth factor receptor
Transforming growth factor β.
Members of the Schiemann Laboratory are thanked for critical reading of the manuscript. This research was support in part by grants from the National Cancer Institute, National Institutes of Health (NIH) (CA129359 and CA177069 to WPS and CA166140 to MKW). We also acknowledge the expertise of the personnel within the Case Comprehensive Cancer Center Small Animal Imaging Core and the Gene Expression and Genotyping Core Facilities (NIH project P30 CA043703).
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