FoxM1 mediates resistance to herceptin and paclitaxel

Abstract

Inherent and acquired therapeutic resistance in breast cancer remains a major clinical challenge. In human breast cancer samples, overexpression of the oncogenic transcription factor FoxM1 has been suggested to be a marker of poor prognosis. In this study, we report that FoxM1 overexpression confers resistance to the human epidermal growth factor receptor 2 monoclonal antibody Herceptin and microtubule-stabilizing drug paclitaxel, both as single agents and in combination. FoxM1 altered microtubule dynamics to protect tumor cells from paclitaxel-induced apoptosis. Mechanistic investigations revealed that the tubulin-destabilizing protein Stathmin, whose expression also confers resistance to paclitaxel, is a direct transcriptional target of FoxM1. Significantly, attenuating FoxM1 expression by small interfering RNA or an alternate reading frame (ARF)-derived peptide inhibitor increased therapeutic sensitivity. Our findings indicate that targeting FoxM1 could relieve therapeutic resistance in breast cancer.

Figure 1. Overexpression of FoxM1 renders multiple HER-2 amplified cell lines resistant to the effects of Herceptin treatment

(A) The response of SKBR3, MDA-MB-453, and BT474 stable cell lines to Herceptin was tested by colony forming assay. Cell lines were treated continuously with either 0 or 10ug/ml Herceptin for 14 days, media was changed every 3 days. Cells were plated in triplicate and the experiment was repeated three times. Representative wells for SKBR3 cells are shown. Graphs provide average quantification as a percentage of the untreated wells. (B) Stable cell lines expressing either pBabe or FoxM1 were treated with 10ug/ml of Herceptin for 48 hours, stained with propidium iodide, and subjected to FACS analysis. Percentage change in G1 phase is shown. Inset shows relative protein expression in FoxM1 versus pBabe stable cell lines. (C) SKBR3-pBabe and FoxM1 lines were either untreated or treated for 72 hours with Herceptin followed by a pulse of BrdU for 2 hours. Percentage of BrdU positive compared to DAPI positive cells are shown for each group, 500 cells in each experiment were counted. Average values are shown above error bars and representative pictures are shown below the graph.

Figure 2. SKBR3-FoxM1 cell lines fail to accumulate p27 after treatment with Herceptin

(A) SKBR3-pBabe and FoxM1 expressing cell lines were treated with increasing doses of Herceptin for 48 hours. Representative western blots of FoxM1 and p27 levels are shown. Cdk2 was used as a loading control. (B) SKBR3 stable cell lines were treated with 10ug/ml of Herceptin for 24, 48, and 72 hours. FoxM1 and p27 levels are shown by western blot. (C) SKBR3-pBabe cell lines were treated with 10ug/ml of IgG for indicated periods of time. FoxM1 and p27 were assayed by western blot and Cdk2 was used as a loading control.

Figure 3. FoxM1 expression is higher in resistant lines and can be targeted to reintroduce sensitivity

(A) Western blot showing FoxM1 protein levels in SKBR3, BT474, and MDA-MB-453 parental and resistant lines. Lines were continuously cultured in 5ug/ml of Herceptin for six months. Cells were placed in drug free media for 7 days prior to treatment and lysates were collected 72 hours after treatment with 10ug/ml of Herceptin. Quantification of FoxM1 bands by Image J is shown, using untreated parental lines for normalization. (B) Semi-quantitative RT-PCR using cDNA from either parental or resistant SKBR3 cells was used to analyze target gene expression. Representative gel pictures are shown and quantification values normalized to GAPDH are shown above. (C) Parental and resistant SKBR3 and MDA-MB-453 cells were transfected with control or FoxM1 specific siRNA. 48 hours later, 3 × 10³ cells were plated in each well or a 24-well plate and left untreated or treated with 10ug/ml Herceptin. Media was changed every 3–4 days. After 14 days, colonies were stained with crystal violet and quantified by photoshop.

Figure 4. FoxM1 expression induces resistance to Taxol alone by increasing Stathmin expression and activity

(A) Top Panel: SKBR3-pBabe and FoxM1 expressing lines were treated continuously with 0.1 uM of Taxol for 7 days and viable cell numbers were determined by luminescent measurement of ATP. Bottom Panel: SKBR3 parental cells were treated with control siRNA or siRNA specific to FoxM1 for 72 hours then treated with indicated doses of Taxol for 24 hours. CellTiter Glo, a luminescence assay was used to measure cell viability. (B) Polymerized and soluble tubulin fractions from untreated and treated SKBR3-pBabe and FoxM1 cell lines were generated by centrifugation. Western blot was used to assay α-tubulin and β-tubulin ratios in polymerized and soluble fractions. Relative percentages are shown above western blot. (C) RNA from SKBR3 pBabe and FoxM1 lines were collected and RT-PCR was used to measure stathmin. Values were normalized against cyclophilin. Inset shows relative protein expression by western blot. (D) Chromatin immunoprecipitation assay (ChIP) was performed in SKBR3 cells using an antibody specific to FoxM1 or IgG as a control. PCR was used to amplify the region surrounding the putative FoxM1 binding site at −5793 upstream of the transcriptional start site and the region surrounding −1371 as a non-specific control. Representative PCR results are shown.

Figure 5. FoxM1 protects cells against treatment with Herceptin and Taxol in combination

(A) Cell lines were pretreated with 10ug/ml of Herceptin for 3 days then treated with 0.1uM of taxol for 7 days. Relative cell number was determined by cell titer glo measurement every other day for seven days. (B) SKBR3 parental cells were treated with control or FoxM1 siRNA for 72 hours followed by 10ug/ml of Herceptin for 3 days. Equal numbers of cells were treated for 24 hours with increasing amounts of taxol and ATP was measured by luminescence. (C) 3–5 ×10³ SKBR3, MDA-MB-453, or BT474 cells were seeded in each well in triplicate. Cells were either left untreated or pre-treated in 10ug/ml Herceptin for 72 hours then pulsed in 0.1µM Taxol for 4 hours. Wells that received Herceptin were continuously cultured in 10ug/ml for the duration of the experiment. Media was changed every 3 days. Cells were stained after a total of 17 days in crystal violet and representative wells are shown in the left panel. Graph shows quantification of triplicates from three separate experiments. Representative wells of SKBR3-pBabe and FoxM1 cells are shown in upper left.

Figure 6. Targeting FoxM1 with ARF-peptide is sufficient to overcome inherent Herceptin resistance and to sensitize pBabe or FoxM1 overexpressing cells to treatment

(A) ARF-peptide or mutant peptide (2µM) was used to treat either parental or resistant SKBR3 and MDA-MB-453 cells. Cells were pretreated with peptide for 3 days then treated with both 10ug/ml Herceptin and peptide. Media was changed daily and cells were stained with crystal violet after 17 total days. Graph shows quantification of colony forming assay by Photoshop. (B) SKBR3-pBabe and FoxM1 cell lines were treated with either mutant or ARF-peptide for three days. Wells receiving both Herceptin and Taxol were pre-treated for 72 hours with Herceptin before receiving indicated doses of Taxol. Quantification of triplicates is shown in the graph with representative images below.