Oncotarget, 2016, 7(50): 83424-83436 

FGFR signaling maintains a drug persistent cell population following epithelial-mesenchymal transition

Michael K. Wendt


摘要:

An emerging characteristic of drug resistance in cancer is the induction of epithelial-mesenchymal transition (EMT). However, the mechanisms of EMT-mediated drug resistance remain poorly defined. Therefore, we conducted long-term treatments of human epidermal growth factor receptor-2 (Her2)-transformed breast cancer cells with either the EGFR/Her2 kinase inhibitor, Lapatinib or TGF-β, a known physiological inducer of EMT. Both of these treatment regimes resulted in robust EMT phenotypes, but upon withdrawal a subpopulation of TGF-β induced cells readily underwent mesenchymal-epithelial transition, where as Lapatinib-induced cells failed to reestablish an epithelial population. The mesenchymal population that remained following TGF-β stimulation and withdrawal was quickly selected for during subsequent Lapatinib treatment, manifesting in inherent drug resistance. The Nanostring cancer progression gene panel revealed a dramatic upregulation of fibroblast growth factor receptor 1 (FGFR1) and its cognate ligand FGF2 in both acquired and inherent resistance. Mechanistically, FGF:Erk1/2 signaling functions to stabilize the EMT transcription factor Twist and thus maintain the mesenchymal and drug resistant phenotype. Finally, Lapatinib resistant cells could be readily eliminated using recently characterized covalent inhibitors of FGFR. Overall our data demonstrate that next-generation targeting of FGFR can be used in combination with Her2-targeted therapies to overcome resistance in this breast cancer subtype.Acquisition and/or inherent resistance to Her2 targeted therapies can take place through a number of mechanisms that may be unique to the other underlying mutations that are specific to particular tumor models [20]. Therefore, in an effort to elucidate more global mediators of resistance to the clinically used EGFR/Her2 kinase inhibitor, Lapatinib, we utilized a model in which directed overexpression of Her2 mediates transformation of otherwise normal mammary epithelial cells [21]. As shown in Figure 1A Her2 was overexpressed in non-transformed human mammary epithelial cells (HMLE) (Figure 1A). Ectopic expression of Her2 allowed for culture of the HMLE cells in standard serum containing media (not shown), reduced expression of CD24, facilitated aberrant growth under 3D culture conditions and led to tumor formation in mice (Figure 1B and 1C, Supplementary Figure S1A, S1B and S1C). Importantly, Her2-driven cell growth in 3D culture and tumor growth in vivo could be significantly, but not completely, inhibited by treatment with Lapatinib (Figure 1B and 1D). Long-term culture (4 weeks) of Her2-transformed HMLE cells with regular addition of Lapatinib yielded a proliferative cell population that displayed a highly mesenchymal morphology (Figure 1E). A similar yet distinct cell morphology could also be elicited in these cells upon long-term culture with TGF-β1 (Figure 1E). Treatment of parental HMLE-Her2 cells with the covalent ErbB inhibitor Afatinib lead to a similar mesenchymal morphology but a proliferative population could not be established (Supplementary Figure S1B). Both TGF-β and Lapatinib-induced EMT events lead to the dramatic upregulation of CD44. However, upon withdrawal of these differential stimuli only those cells induced to undergo EMT by TGF-β reestablished an epithelial population where as a Lapatinib-induced EMT event was stably maintained following withdrawal of the drug (Figure 1E and 1F). The stable versus transient EMT events induced by Lapatinib and TGF-β respectively could further be visualized by immunoblot and immunofluorescence for the mesenchymal marker vimentin and the epithelial marker E-cadherin (Figure 1G, 1h and Supplementary Figure S1C). Overall, these data clearly establish the stable verses transient nature of EMT induced by EGFR/Her2 inhibition versus that induced by TGF-β. Furthermore, they demonstrate how TGF-β-induced EMT and mesenchymal-epithelial transition (MET) results in the formation of a heterogeneous cell population consisting of both epithelial and mesenchymal cells.Human mammary epithelial cells (HMLE) were obtained from Sendurai A. Mani (MD Anderson Cancer Center, Houston TX) and NMuMG and BT474 cells were purchased from the ATCC (Manassas, VA, USA). The HMLE cells were cultured in DMEM:F12 supplemented with insulin (10 μg/ml), EGF (10 ng/ml), and hydrocortisone (250 μg/ml), this media was mixed 1:1 with Mammary Epithelial Cell Growth Medium (MEGM) purchased from Lonza (Allendale, NJ, USA). Bioluminescent HMLE cells were engineered to stably express firefly luciferase via lentiviral transduction under the selection of Blasticidin. Her2 and Twist expressing HMLE cells and Twist expressing NMuMG cells were constructed via stable transduction using pBabe viral particles and selected for using puromycin. NMuMG, Her2 transformed HMLE and BT474 cells were cultured in DMEM supplemented with 10% FBS, 1% Pen/Strep, and 10 μg/mL of insulin. Plasmids encoding eGFP, FGFR1-α-IIIc (NM_023110.2) or FGFR1-β-IIIc (NM_023105.2) were purchased from Cyagen Biosciences (Santa Clara, CA, USA). These constructs were used to construct lentiviral particles, and stable transduction was selected for under Hygromycin selection. TGF-β1 and basic FGF (FGF2) were purchased from R&D systems (Minneapolis, MN). BGJ-398, Lapatinib, Afatinib, Trametinib, VX11e, AZD-6244, and Idelalisib were purchased from Selleckchem (Houston, TX), solubilized in DMSO and used at the indicated concentrations. FIIN-2 and FIIN-4 were synthesized as previously described and similarly solubilized in DMSO [15, 18].Members of the Wendt Laboratory are thanked for critical reading of the manuscript. We kindly acknowledge Drs. Nathanael Gray and Li Tan for providing the FIIN2 and FIIN4 compounds. We also acknowledge the expertise of the personnel within the Purdue Center for Cancer Research Biological Evaluation Core and Biosciences Imaging core (P30 CA023168).CONFLICTS OF INTERESTThe authors declare no conflicts of interest.GRANT SUPPORTThis research was supported in part by the National Institutes of Health (R00 CA166140) and the METAvivor foundation to M.K.W.
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