A novel mechanism of PPAR gamma induction via EGFR signalling constitutes rational for combination therapy in bladder cancer

Abstract

Background: Two signalling molecules that are attractive for targeted therapy are the epidermal growth factor receptor (EGFR) and the peroxisome proliferator-activated receptor gamma (PPARγ). We investigated possible crosstalk between these 2 pathways, particularly in light of the recent evidence implicating PPARγ for anticancer therapy.

Principal findings: As evaluated by MTT assays, gefitinib (EGFR inhibitor) and DIM-C (PPARγ agonist) inhibited growth of 9 bladder cancer cell lines in a dose-dependent manner but with variable sensitivity. In addition, combination of gefitinib and DIM-C demonstrated maximal inhibition of cell proliferation compared to each drug alone. These findings were confirmed in vivo, where combination therapy maximally inhibited tumor growth in contrast to each treatment alone when compared to control (p<0.04). Induction of PPARγ expression along with nuclear accumulation was observed in response to increasing concentrations of gefitinib via activation of the transcription factor CCAT/enhancer-binding protein-β (CEBP-β). In these cell lines, DIM-C significantly sensitized bladder cancer cell lines that were resistant to EGFR inhibition in a schedule-specific manner.

Conclusion: These results suggest that PPARγ agonist DIM-C can be an excellent alternative to bladder tumors resistant to EGFR inhibition and combination efficacy might be achieved in a schedule-specific manner.

Figure 1. Baseline expression of PPARγ and EGFR.

(A) Expression of PPARγ and EGFR relative to endogenous levels of β-actin and tubulin, respectively, and represented in units among 9 bladder cancer cell lines reflecting different stages of the disease (from Superficial to Invasive & no Metastasis, Invasive and Lymphatic Metastasis). (B) Dose-response of bladder cancer cell lines to PPARγ agonist (DIM-C) and EGFR inhibitor (gefitinib). The GI50 value was defined as the mean concentration of drug that generates 50% of growth inhibition as compared to controls.

Figure 2. Antiproliferative effects of combined therapy.

Growth was monitored by MTT assays. Cells were treated with gefitinib 5 µM and DIM-C 3 µM and compared to each drug alone. Red: gefitinib; Yellow: DIM-C; Blue: gefitinib+DIM-C.

Figure 3. Effect of combination therapy on EGFR downstream signaling.

(A) Phosphorylation pattern of p42/44 MAPK (Erk1/2) in cells treated with gefitinib 5 µM and DIM-C 3 µM for 5, 15 and 30 minutes. T0 is the untreated control. Whole-cell lysates were immunoblotted with phosho-p42/44 MAPK and p42/44 MAP. GAPDH was used as loading control on the Western blotting. (B) Comparison of PTEN expression in cell lines treated with gefitinib 5 µM and DIM-C 3 µM for 24 hs. T0 is the untreated control.

Figure 4. Effects of combination therapy in vivo.

Bladder tumor growth of combination treatment arm compared to control arm (P<0.02). Ten mice per group were treated with placebo; DIM-C was given 60 mg/Kg 3 times per week; Gefitinib was given 2 mg/day, 5 times per week. All drugs were administrated by oral gavage. Treatment was continued for 4 weeks.

Figure 5. Effects of combination therapy on p21 expression in vivo and in vitro.

(A) Immunohistochemistry (IHC) staining for p21 in tumor xenograft tissues. Mice were treated with placebo or combination therapy (Gefitinib 2 mg/day, 5 times per week and DIM-C 60 mg/Kg, 3 times per week). Graphic on the right side represents quantification of positive staining cells. (B) In vitro expression of p21 in Western blot of lysate cells treated with gefitinib 5 µM and DIM-C 3 µM for 24 hs. Graphic on the right side, represents quantification of p21 expression related to GAPDH.

Figure 6. Induction of PPARγ expression in response to different concentrations of gefitinib.

(A) Fold increase relative to control was determined after normalization with β-actin as external loading control. (B) Upregulation and nuclear accumulation of PPARγ following treatment with gefitinib (24 hs).

Figure 7. Immunohistochemistry (IHC) staining for PPARγ in tumor xenograft tissues Mice were treated with placebo or gefitinib (Gefitinib 2 mg/day, 5 times per week).

Graphic on the lower level represents quantification of positive staining cells. Black arrows indicate nuclear staining.

Figure 8. Schedule-specific efficacy of combination therapy.

Three relatively resistant cell lines to gefitinib (UM-UC3, UM-UC13, and KU-7) and one sensitive (UM-UC6). Growth (MTT assays) after 48 hs treatment. Gefitinib: 2 µM. DIM-C: 2 µM.

Figure 9. mRNA expression of CEBPβ after treatment with gefitinib.

(A) RT-PCR of cells treated with gefitinib (B) Western blot of CEBPβ expression in KU-7 and UM-UC-13 cell lines in response to different concentrations of gefitinib.