FGFR1 overexpression promotes resistance to PI3K inhibitor alpelisib in luminal breast cancer cells through receptor tyrosine kinase signaling-mediated activation of the estrogen receptor

Abstract

Aim: Resistance to PI3K inhibitor alpelisib is an emerging challenge in breast cancer treatment. FGFR1 is frequently amplified in breast cancer. We investigated FGFR1 overexpression-mediated alpelisib resistance and its mechanism. Methods: CCK-8, colony formation, and cell cycle assays assessed FGFR1 overexpression-induced alpelisib resistance in MCF-7 and T47D cells. FGFR1 siRNA knockdown validated FGFR1's role. Akt, Erk, and ER signaling were analyzed by Western blot. Synergistic effects of alpelisib with AZD4547 and fulvestrant were evaluated using the combination index. Results: FGFR1 overexpression conferred alpelisib resistance in MCF-7 and T47D cells, evidenced by increased viability, colony formation, and S-phase accumulation post alpelisib treatment. Knockdown of FGFR1 reverse alpelisib resistance in FGFR1 overexpressing MCF-7 and T47D cells. Resistance correlated with sustained activation of Akt and Erk1/2 pathways (p-Akt, p-Erk1/2, p-S6K, p-Rb) and attenuated suppression of ERα phosphorylation (S118/S167), highlighting RTK-ER crosstalk. Combining alpelisib with AZD4547 synergistically inhibited growth and suppressed both RTK signaling and ERα phosphorylation. While alpelisib-fulvestrant was effective, adding AZD4547 further enhanced inhibition, supporting triple therapy to overcome resistance. Conclusion: Our findings establish FGFR1 as a key mediator of alpelisib resistance in ER+ breast cancer. Combining FGFR1 inhibitors with alpelisib-based therapies offers a viable approach for FGFR1-overexpressing tumors.

Keywords: AZD4547; FGFR1; PI3K; alpelisib; estrogen receptor; fulvestrant; resistance.

Figure 1

FGFR1 overexpression promotes resistance to alpelisib in MCF-7 cells. (A) Protein levels of FGFR1 in MCF7/C and MCF7/FGFR1 cells detected by Western blotting; (B) Alpelisib-induced inhibition of MCF7/C and MCF7/FGFR1 cells assessed with the CCK-8 assay. Cells were treated with alpelisib at indicated concentrations for five days, and survival fractions were determined. IC₅₀ values were calculated using GraphPad Prism software; (C) IC₅₀ values based on three experiments were analyzed with Welch’s t-test. ^**P < 0.01; (D) Clonogenic assays of MCF7/C and MCF7/FGFR1 cells treated with alpelisib. Cells were seeded at 600 cells/well and treated with alpelisib for two weeks, followed by crystal violet staining and quantification; (E) Representative images from the clonogenic assay as described in (D); (F) Cell cycle analysis of MCF7/C and MCF7/FGFR1 cells treated with 0.3 μM alpelisib (Alp) for 24 h, followed by flow cytometry analysis in triplicate. Percentages of cells in the S phase are indicated for each sample; (G) Decrease in S-phase cells between alpelisib-treated MCF7/C and MCF7/FGFR1 cells as assessed in (F). The percentage of S-phase cells in drug-treated samples was normalized by dividing by the percentage of S-phase cells in the corresponding untreated cell line. ^**P < 0.01.

Figure 2

FGFR1 overexpression sustains Akt and Erk pathway activation in alpelisib-treated MCF-7/FGFR1 cells. (A) MCF7/C and MCF7/FGFR1 cells were treated with alpelisib (Alp) at the indicated concentrations for 24 h. Protein levels of p-Akt, Akt, p-Erk1/2, Erk1/2, p-S6K, S6K, p-Rb, Rb, and Cyclin D1 in each sample were detected by Western blotting, with GAPDH used as an internal control; (B) Densitometry analysis of phosphorylated/active signaling markers (p-Akt, p-Erk, p-S6K, p-Rb) and Cyclin D1 in MCF-7/C (M) and MCF-7/FGFR1 (MF) cells treated with alpelisib. Band intensities in (A) were quantified (ImageJ), normalized to loading controls (GAPDH) and total protein levels based on three repeats. ^*P < 0.05; ^**P < 0.01, based on comparisons of the marker between MCF-7/FGFR1 and MCF-7/C cells under the same treatment conditions.

Figure 3

FGFR1 knockdown restores alpelisib sensitivity in FGFR1-overexpressing MCF-7 cells. (A) Immunoblot analysis of FGFR1 protein levels in MCF-7/FGFR1 (MF) cells following transient transfection with FGFR1-targeting siRNA (MF/siFGFR1) or scramble control (MF/siC); (B) Dose-response curves of alpelisib treatment in MF cells with or without FGFR1 knockdown, demonstrating a significant reduction in IC₅₀ upon alpelisib treatment; (C) The statistical differences in IC₅₀ values between the two groups were analyzed using Welch’s t-test; (D and E) Clonogenic survival assays showing colony formation efficiency of MF cells treated with indicated concentrations of alpelisib (0.03-0.3 µM) following FGFR1 knockdown. Data represent triplicate measurements analyzed by two-way ANOVA. ^**P < 0.01.

Figure 4

The combination of FGFR1-targeting AZD4547 with alpelisib induces significant synergistic inhibitory effects. (A) and (B) Combination index (CI) of MCF7/C (A) and MCF7/FGFR1 (B) cells treated with alpelisib (Alp) and AZD4547 in various combinations. Cells were treated with the drugs at indicated concentrations for 5 days, followed by CCK-8 assays. CI values were calculated using CompuSyn software according to the Chou-Talalay method. Only CI values corresponding to a fractional effect (FA) greater than 0.5 were presented; (C) and (D) Clonogenic assays of MCF7/C and MCF7/FGFR1 cells treated with alpelisib (Alp) or AZD4547 (AZD), alone or in combination. Cells were treated with 0.3 μM alpelisib, 3 μM AZD4547, or the combination for 2 weeks, followed by crystal violet staining; (C) Representative images of the assays; (D) Quantification of colony numbers based on triplicate experiments, displayed as a bar graph; (E) Cell cycle analysis of MCF7/C and MCF7/FGFR1 cells treated with alpelisib (Alp) or AZD4547 (AZD), alone or in combination. Cells were treated with 1 μM alpelisib, 10 μM AZD4547, or the combination for 24 h, followed by flow cytometry analysis in triplicate. ^**P < 0.01 indicates significant differences in S-phase cell populations between the indicated groups.

Figure 5

Combination of alpelisib with AZD4547 induces enhanced inhibition of the PI3K/Akt/mTOR/S6K and MAPK/Erk1/2 pathways. MCF7/C and MCF7/FGFR1 cells were treated with 2 μM alpelisib (Alp), 6 μM AZD4547 (AZD), or the combination for 24 h. Cells were then collected for Western blot analysis to assess the protein levels of p-Akt, Akt, p-Erk1/2, Erk1/2, p-S6K, S6K, p-Rb, Rb, Cyclin D1, with GAPDH as an internal control.

Figure 6

ERα phosphorylation/activation in FGFR1-driven alpelisib resistance and sensitization by combination therapy. (A) Sustained ERα phosphorylation in alpelisib (Alp)-treated MCF7/FGFR1 cells. Cells of both sublines were treated with alpelisib at the indicated concentrations for 24 h. Protein levels of p-ERα/S118, p-ERα/S167, ERα, and the internal control GAPDH were detected by Western blotting; (B) Combination of alpelisib with AZD4547 enhances inhibition of ERα phosphorylation/activation. MCF7/C and MCF7/FGFR1 cells were treated with 2 μM alpelisib (Alp), 6 μM AZD4547 (AZD), or the combination for 24 h, followed by Western blot analysis of p-ERα/S118, p-ERα/S167, ERα, and GAPDH in the indicated groups.

Figure 7

The combination of alpelisib with fulvestrant induces a synergistic effect that is further enhanced by the addition of AZD4547. (A) Fulvestrant-induced inhibition of MCF7/C and MCF7/FGFR1 cells. Cells were treated with fulvestrant at the indicated concentrations for 5 days, followed by CCK-8 assays. IC₅₀ values were analyzed with GraphPad; (B and C) Synergistic effect of alpelisib with fulvestrant in MCF7/C (B) and MCF7/FGFR1 (C) cells. Cells were treated with alpelisib (Alp) and fulvestrant at the indicated concentrations for 5 days, followed by CCK-8 assays. CI values were calculated using CompuSyn software according to the Chou-Talalay method. Only CI values corresponding to a fractional effect (FA) greater than 0.5 were presented; (D and E) Addition of AZD4547 (AZD) further enhances the alpelisib-fulvestrant (Alp-Ful) combination in four different treatment regimens. MCF7/C (D) and MCF7/FGFR1 (E) cells were treated with AZD4547 and four different combinations of alpelisib and fulvestrant at the indicated concentrations for 5 days, followed by CCK-8 assays. CI values were calculated as above.

Figure 8

FGFR1 overexpression confers alpelisib resistance in T47D cells. (A) Immunoblot analysis of FGFR1 overexpression in T47D/FGFR1 and control T47D/C cells; (B) Dose-response curves of T47D/C and T47D/FGFR1 cells measured with CCK-8 assays; (C) Quantification of IC₅₀ values derived from the dose-response assays in panel B. Statistical differences in IC₅₀ values between the two groups were analyzed using Welch’s t-test. ^**P < 0.01; (D and E) Clonogenic survival assays of T47D/C (control) and T47D/FGFR1 cells treated with indicated concentrations of alpelisib, performed using the same methodology as in Figure 1D and E. ^**P < 0.01.

Figure 9

FGFR1 knockdown reverses alpelisib resistance in T47D/FGFR1 cells. (A) Immunoblot analysis of FGFR1 protein levels in T47D/FGFR1 (TF) cells following transfection with FGFR1-targeting siRNA (TF/siFGFR1) or scramble control (TF/siC); (B) CCK-8 measurement of the dose-response curves of TF/siC and TF/siFGFR1 cells followed by IC₅₀ calculation; (C) Comparison of the IC₅₀ values between the two groups analyzed with Welch’s t-test; (D and E) Clonogenic assays of TF/siC and TF/siFGFR1 cells. Twenty-four h after siRNA transfection, the cells were reseeded and treated with alpelisib at indicated concentrations. Quantified colony formation efficiencies were analyzed with a two-way ANOVA test. ^**P < 0.01.

Figure 10

FGFR1 inhibition enhances alpelisib efficacy and synergizes with alpelisib + fluvastatin in T47D cells. (A and B) Synergistic interaction between alpelisib (ALP) and AZD4547 (AZD) in T47D/C and T47D/FGFR1 cells. Combination index (CI) values were calculated using the Chou-Talalay method (as described in Figure 4), with CI < 1 indicating synergism; (C and D) Effect of the alpelisib and AZD4547 combination on colony formation in T47D/C and T47D/FGFR1 cells. The combination treatment led to enhanced inhibition of colony formation; (E and F) Synergy analysis of AZD4547 in combination with alpelisib and fluvastatin. The triple combination (ALP + Fluvastatin + AZD) significantly suppressed cell proliferation, particularly in T47D/FGFR1 cells.