Inhibition of PI3K/Akt/mTOR overcomes cisplatin resistance in the triple negative breast cancer cell line HCC38

Abstract

Background: Widely established targeted therapies directed at triple negative breast cancer (TNBC) are missing. Classical chemotherapy remains the systemic treatment option. Cisplatin has been tested in TNBC but bears the disadvantage of resistance development. The purpose of this study was to identify resistance mechanisms in cisplatin-resistant TNBC cell lines and select targeted therapies based on these findings.

Methods: The TNBC cell lines HCC38 and MDA-MB231 were subjected to intermittent cisplatin treatment resulting in the 3.5-fold cisplatin-resistant subclone HCC38CisR and the 2.1-fold more resistant MDA-MB231CisR. Activation of pro-survival pathways was explored by immunostaining of phospho-receptor tyrosine kinases. Targeted therapies (NVP-AEW541, lapatinib and NVP-BEZ235) against activated pathways were investigated regarding cancer cell growth and cisplatin sensitivity.

Results: In HCC38CisR and MDA-MB231CisR, phosphorylation of epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) was observed. In HCC38CisR, treatment with NVP-AEW541 increased potency of lapatinib almost seven-fold, but both compounds could not restore cisplatin sensitivity. However, the dual phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 acted synergistically with cisplatin in HCC38CisR and fully restored cisplatin sensitivity. Similarly, NVP-BEZ235 increased cisplatin potency in MDA-MB231CisR. Furthermore, NVP-AEW541 in combination with lapatinib restored cisplatin sensitivity in MDA-MB231CisR.

Conclusion: Simultaneous inhibition of EGFR and IGF1R in cisplatin-resistant TNBC cell lines was synergistic regarding inhibition of proliferation and induction of apoptosis. Co-treatment with NVP-BEZ235 or with a combination of NVP-AEW541 and lapatinib restored cisplatin sensitivity and may constitute a targeted treatment option for cisplatin-resistant TNBC.

Keywords: Cisplatin resistance; EGFR; HCC38; IGF1R; Lapatinib; MDA-MB231; NVP-AEW541; NVP-BEZ235; Triple negative breast cancer.

Fig. 1

Characterization of HCC38 and cisplatin-resistant HCC38CisR. (A) Weekly exposure of HCC38 with the IC₅₀ of cisplatin for 6 h resulted in the cisplatin resistant subclone HCC38CisR with a resistance factor of at least 3.5 (p < 0.001). IC₅₀ cisplatin HCC38: 2.7 μM; IC₅₀ cisplatin HCC38CisR: 9.4 μM. Shown are mean +/− SEM, n = 3. b Detail of phospho-RTK-array displays phosphorylation status of EGFR-family and IGF1R in HCC38 and HCC38CisR. c Immunostaining of expression and activation of signaling kinases. Shown is a representative experiment out of 3. HCC38 cells were treated with 2.5 μM cisplatin for 6 h followed by a recovery of 24 h or 1 week. Untreated HCC38 and HCC38CisR served as controls. d Densitometric analysis of the protein bands of HCC38 and HCC38CisR were performed using ImageJ software (NIH). Data are means ± SD, n = 3. All values have been normalized to HCC38 control. Statistical analysis was performed using one-way ANOVA test (* p < 0.05, ** p < 0.01, and *** p < 0.001). e Cell proliferation measured by flow cytometry-based cell counting. Doubling times were 23.6 h in HCC38 and 16.9 h in HCC38CisR and were significantly different (*** p < 0.001). Shown are mean +/− SEM, n = 3

Fig. 2

Combination of lapatinib and NVP-AEW541 is hyper-additive but not reversing cisplatin resistance in HCC38CisR. a Coincubation with 1.5 μM NVP-AEW541 significantly decreased IC₅₀ of lapatinib in HCC38CisR, whereas this treatment had no effect in HCC38. b Coincubation with 2 μM lapatinib significantly decreased IC₅₀ of NVP-AEW541 in HCC38CisR but had no effect in HCC38. c In HCC38CisR (but not in HCC38), the combination of NVP-AEW541 and lapatinib significantly induced apoptosis in a hyper-additive manner (***p < 0.001). NVP-AEW541 and lapatinib were used at 2 μM. Cells were treated for 48 h and the amount of apoptotic nuclei in the control was subtracted from treated samples. d Effect of NVP-AEW541 or lapatinib (2 μM, respectively) on cell cycle in HCC38CisR. Combination of 2 μM NVP-AEW541 and 2 μM lapatinib significantly (***p < 0.001) increased cell population in G₁ (77.7 ± 1.2% vs. 67.3 ± 1.4%) while reducing cell population in G₂/M phase (14.2 ± 1.5% vs. 25.7 ± 1.6%). Incubation time was 48 h. e Western blot analysis of p-EGFR, p-IGF1R, and p-Akt upon treatment of HCC38CisR with an IC₅₀ of lapatinib or NVP-AEW541 or both compounds for 6 h. f Densitometric analysis of the protein bands for p-AKT, p-EGFR, and p-IGF1R of HCC38CisR were performed using ImageJ software (NIH). Data are means ± SD, n = 3. All values have been normalized to untreated HCC38 CisR. Statistical analysis was performed using one-way ANOVA test (* p < 0.05). g Effect of 1 μM lapatinib and 1.5 μM NVP-AEW541 on cisplatin sensitivity either alone or in combination. Lapatinib and/or NVP-AEW541 were added 48 h prior to cisplatin treatment. IC₅₀ of cisplatin did not significantly differ. All data shown are mean +/− SEM, n = 3, except (e) showing a representative experiment out of 3

Fig. 3

NVP-BEZ235 treatment fully restores cisplatin sensitivity in HCC38CisR. a 20 nM NVP-BEZ235 added 48 h prior to cisplatin treatment significantly reduced IC₅₀ of cisplatin in HCC38CisR (p < 0.001) but not in HCC38. b 1 μM KU0063794 or 5 μM LY294002 or their combination significantly reduced IC₅₀ of cisplatin in HCC38CisR (p < 0.001). c Western blot analysis of PARP and cleaved PARP in HCC38CisR used as an indicator of active Caspase 3. For combination of NVP-BEZ235 and cisplatin, 20 nM NVP-BEZ235 was incubated 48 h prior to addition of 3 μM cisplatin for 6 h. d Induction of apoptosis by NVP-BEZ235 and cisplatin. 20 nM NVP-BEZ235 was incubated 24 h prior to addition of 5 μM cisplatin for 6 h followed by 24 h of recovery. Combination of NVP-BEZ235 with cisplatin increased apoptotic nuclei (35.3 ± 3.7%) compared to cisplatin alone (11.4 ± 2.3%) and NVP-BEZ235 alone (4.6 ± 2.0%) (***p < 0.001). e Western blot analysis of p-EGFR, p-IGF1R, and p-Akt in HCC38CisR upon 48 h treatment with 20 nM or 280 nM NVP-BEZ235. f Densitometric analysis of the protein bands of p-EGFR, p-IGF1R, and p-Akt in HCC38 and HCC38CisR were performed using ImageJ software (NIH). Data are means ± SD, n = 3. All values have been normalized to HCC38 control. Statistical analysis was performed using one-way ANOVA test (* p < 0.05, ** p < 0.01, and *** p < 0.001). g Effect of 20 nM or 280 nM NVP-BEZ235 on cell cycle in HCC38CisR. 280 nM NVP-BEZ235 gave a slight but significant (*p < 0.5) reduction of cells in G₁ phase (67.3 ± 1.6% vs. 60.0 ± 0.9% in control) accompanied by an increase in cells in G₂/M phase (23.6 ± 1.4% vs. 28.3 ± 0.5% in control). All data shown are mean +/− SEM, n = 3, except (C/E) showing a representative experiment out of 3

Fig. 4

Combination of lapatinib and NVP-AEW541 – but not NVP-BEZ235 inhibits cell migration in HCC38CisR measured by scratch assay. a Microscopic images were obtained before (0 h) and 24 h after applying a pipet tip-induced scratch in a nearly confluent cell monolayer. Data shown are one typical experiment out of three independent experiments. b Average migration, estimated as space occupied after 24 h, showed that only the combination of 1.5 μM NVP-AEW541 and 1 μM lapatinib significantly reduced cell migration (**p < 0.01). c Cell proliferation assays applied under the conditions of (b) (24 h incubation). Only 20 nM NVP-BEZ235 significantly reduced cellular proliferation (n = 3, *p < 0.05)