Loss of FBXW7 and accumulation of MCL1 and PLK1 promote paclitaxel resistance in breast cancer

Abstract

FBXW7 is a component of SCF (complex of SKP1, CUL1 and F-box-protein)-type ubiquitin ligases that targets several oncoproteins for ubiquitination and degradation by the proteasome. FBXW7 regulates cellular apoptosis by targeting MCL1 for ubiquitination. Recently, we identified PLK1 as a new substrate of FBXW7 modulating the intra-S-phase DNA-damage checkpoint. Taxanes are frequently used in breast cancer treatments, but the acquisition of resistance makes these treatments ineffective. We investigated the role of FBXW7 and their substrates MCL1 and PLK1 in regulating the apoptotic response to paclitaxel treatment in breast cancer cells and their expression in breast cancer tissues. Paclitaxel-sensitive MDA-MB-468 and a paclitaxel-resistant MDA-MB-468R subclone were used to study the role of FBXW7 and substrates in paclitaxel-induced apoptosis. Forced expression of FBXW7 or downregulation of MCL1 or PLK1 restored sensitivity to paclitaxel in MDA-MB-468R cells. By contrary, FBXW7-silenced MDA-MB-468 cells became resistant to paclitaxel. The expression of FBXW7 and substrates were studied in 296 invasive carcinomas by immunohistochemistry and disease-free survival was analyzed in a subset of patients treated with paclitaxel. In breast cancer tissues, loss of FBXW7 correlated with adverse prognosis markers and loss of FBXW7 and MCL1 or PLK1 accumulation were associated with diminished disease-free survival in paclitaxel-treated patients. We conclude that FBXW7 regulates the response to paclitaxel by targeting MCL1 and PLK1 in breast cancer cells and thus targeting these substrates may be a valuable adjunct for paclitaxel treatment. Also, FBXW7, MCL1 and PLK1 may be relevant predictive markers of tumor progression and response to paclitaxel treatment.

Keywords: FBXW7; MCL1; PLK1; Pathology Section; apoptosis; paclitaxel.

Figure 1. Expression of FBXW7 and its substrates in breast cancer cell lines

A. Western blot analyses of FBXW7, AURKA, c-Myc, Cyclin E, PLK1 and MCL1 in MCF7, T47D, MDA-MB-231 and MDA-MB-468 cells in basal conditions are shown, using β-actin as loading control. Quantifications of indicated proteins are shown as histograms and data are presented as mean ± SEM. B. Western blot analyses of MCF7 and MDA-MB-468 cells silenced with FBXW7 siRNA and with a nontargeting control are shown; β-actin was used to ensure equal loading. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM comparing siRNA control versus siRNA FBXW7 in MDA-MB-468 cells and siRNA control versus siRNA FBXW7 in MCF7 cells. *p < 0.05, **p < 0.01 and ***p < 0.001 from Student's t-test. C. FBXW7, AURKA, c-Myc, Cyclin E, PLK1 and MCL1 were detected by Western blot in MDA-MB-468 and MDA-MB-468R cells in basal conditions. β-actin is shown as loading control. Densitometric analyses of indicated proteins are shown as histograms. Data are presented as mean ± SEM comparing MDA-MB-468 and MDA-MB-468R cells in basal conditions. *p < 0.05, **p < 0.01 and ***p < 0.001 from Student's t-test. IC50 curves for paclitaxel in MDA-MB-468 (solid line) and MDA-MB-468R (dotted line) cell lines are shown. Data are presented as mean ± SEM.

Figure 2. Paclitaxel-induced apoptosis and cell cycle analysis of MDA-MB-468 and MDA-MB-468R cells

Cells were treated with 0.025 μM paclitaxel during 24 and 48 h. A. Intact and cleaved PARP, cleaved caspase-9 (CASP-9), cleaved caspase-3 (CASP-3), MCL1, Cyclin B1, PTTG1, phospho-histone H3 (p-His H3), PLK1 and BubR1 proteins were detected by western blot. β-actin is shown as loading control. Densitometric analysis of cleaved PARP, cleaved CASP-9 and cleaved CASP-3 are shown as histograms. Data from triplicate experiments are presented as mean ± SEM comparing paclitaxel-treated MDA-MB-468 versus paclitaxel-treated MDA-MB-468R at 24 and 48 h. *p < 0.05 and ***p < 0.001 from Student's t-test. B. Cell cycle analysis of propidium iodide-stained cells by flow cytometry. Quantification of each phase and sub-G1 cells are shown as histograms. C. Ploidy analysis by FISH. The number of signals per cells was determined for chromosomes 1, 11 and 17 in at least 100 cells. Representative photographs are shown. Histograms represent the percentage of cells with normal or higher ploidy for each condition.

Figure 3. FBXW7 downregulation induces mitotic arrest and resistance to paclitaxel in MDA-MB-468 and MCF7 cells

MDA-MB-468 A. and MCF7 B. cells were silenced with FBXW7 siRNA or with nontargeting control and treated with 0.025 μM paclitaxel during 24 h. Expression levels of the indicated proteins were assessed by western blot, using β-actin as loading control. Densitometric analyses of cleaved PARP, cleaved caspase-9 (CASP-9) and cleaved caspase-3 (CASP-3) are shown as histograms. Data from triplicates are presented as mean ± SEM comparing paclitaxel-treated siRNA control versus paclitaxel-treated siRNA FBXW7. *p < 0.05 from Student's t-test. C. MDA-MB-468 cell cycle analysis of propidium iodide-stained cells by flow cytometry. Quantification of each phase and sub-G1 cells are shown as histograms and data from triplicates are presented as mean ± SEM.

Figure 4. FBXW7 overexpression restores sensitivity to paclitaxel in resistant MDA-MB-468R cells

Cells were transiently transfected with pCMVHA-FBXW7 or with empty vector and treated with 0.025 μM paclitaxel during 24 h. A. FBXW7, HA, AURKA, c-Myc, Cyclin E, PLK1 and MCL1 proteins were detected by western blot. β-actin is shown as loading control. B. Western blot analysis of cleaved PARP, cleaved caspase-9 (CASP-9) and cleaved caspase-3 (CASP-3) is shown; β-actin was used to ensure equal loading. Densitometric analyses of cleaved PARP, cleaved CASP-9 and cleaved CASP-3 are shown as histograms. Data from triplicates are presented as mean ± SEM comparing paclitaxel-treated empty vector versus paclitaxel-treated HA-FBXW7. *p < 0.05 from Student's t-test. C. Cell cycle analysis of propidium iodide-stained cells by flow cytometry. Quantification of each phase and sub-G1 cells are shown as histograms.

Figure 5. Silencing PLK1 or MCL1 restores sensitivity to paclitaxel in resistant MDA-MB-468R cells

Cells were silenced A. with siRNA MCL1 or B. with siRNA PLK1 or with nontargeting controls and treated with 0.025 μM paclitaxel during 24 h. Apoptosis induction was measured by appearance of cleaved PARP, cleaved caspase-9 (CASP-9) and cleaved caspase-3 (CASP-3) by western blot. MCL1, PLK1, Cyclin B1 and p-His H3 were also assessed by western blot, using β-actin as a loading control. Densitometric analysis of cleaved PARP, cleaved caspase-9 (CASP-9) and cleaved caspase-3 (CASP-3) are shown as histograms. Data from triplicates are presented as mean ± SEM comparing paclitaxel-treated siRNA control versus paclitaxel-treated siRNA MCL1 or siRNA PLK1. *p < 0.05 and **p < 0.01 from Student's t-test.

Figure 6. Immunohistochemical expression of FBXW7 and substrates in breast carcinomas

Representative micrographs of FBXW7, Cyclin E, AURKA, MCL1 and PLK1 in breast carcinomas, tumor grade 1 to 3 (top to bottom), are shown. Scale bars, 100 μm.

Figure 7. Expression levels of FBXW7, MCL1 and PLK1 and association with disease-free survival (DFS) in paclitaxel-treated patients

Kaplan-Meier analysis of disease-free survival in patients treated with FEC (5-Fluorouracil, epirubicin and cyclophosphamide; left) and FECP (5-Fluorouracil, epirubicin, cyclophosphamide and paclitaxel; right) showing either high (dotted line) or low (solid line) expression of FBXW7, MCL1 and PLK1. Tick marks represent censored patients. The p values were obtained from the long-rank test of Mantel and Cox.