A FASN-TGF-β1-FASN regulatory loop contributes to high EMT/metastatic potential of cisplatin-resistant non-small cell lung cancer

Abstract

Cisplatin-resistant A549CisR and H157CisR cell lines were developed by treating parental A549 (A549P) and H157 (H157P) cells. These cisplatin-resistant cells showed slight growth retardation, but exhibited higher epithelial-mesenchymal transition (EMT) and increased metastatic potential compared to parental cells. We observed a highly up-regulated fatty acid synthase (FASN) level in A549CisR and H157CisR cells compared to parental cells and the up-regulation of FASN was also detected in A549P and H157P cells after short time treatment with cisplatin, suggesting that the high level of FASN in cisplatin-resistant cells may be from the accumulated cellular responses during cisplatin-resistance developmental process. We next investigated whether the inhibition of FASN by using a specific FASN inhibitor, cerulenin, can influence growth and EMT/metastatic potential of A549CisR and H157CisR cells. There was slight growth inhibition, but significantly reduced EMT/metastatic potential in cisplatin-resistant cells upon inhibitor treatment. The in vitro result was further investigated in orthotopic xenograft mouse models established with luciferase-tagged H157P and H157CisR cells. Mice were injected with cerulenin or vehicle after tumors were developed. No significant tumor regression was detected at the end of cerulenin treatment, but IHC staining showed higher expression of EMT/metastasis markers in H157CisR cell-derived tumors than H157P cell-derived tumors, and showed dramatic reduction of these markers in tumor tissues of cerulenin-treated mice, confirming the in vitro results. In mechanism dissection studies, we revealed the existence of the FASN-TGF-β1-FASN positive loop in A549CisR and H157CisR cells, but not in parental cells, which is believed to augment the FASN function in cisplatin-resistant cells.

Keywords: EMT; TGF-β1; cisplatin-resistance; fatty acid synthase (FASN); non-small cell lung cancer.

Figure 1. EMT and metastatic potential were increased in cisplatin-resistant NSCLC cells compared to parental cells

A. Cytotoxicity test of A549P/H157CisR and H157P/H157CisR cells against cisplatin treatment. Cisplatin-resistant cells were obtained by continuous treatment of cells with increasing dose of cisplatin for 6 months. Cisplatin cytotoxicities of A549P/H157CisR and H157P/H157CisR cells were analyzed in the presence of various concentrations of cisplatin in MTT assay. B. Morphology test. A549P/H157CisR and H157P/H157CisR cells (1 × 10³) were seeded and morphology was observed under microscope. C. Migration test. Cells (A549P/A549CisR and H157P/H157CisR, 1 × 10⁴) were placed in upper chamber of transwell plates (8 μm pore) and migrated cells to lower chamber were counted under microscope after crystal violet staining at the end of 24 hours of incubation. Quantitation shown on right. D. Western blot analysis showing an increase in EMT/metastasis markers in cisplatin-resistant cells compare to parental cells. Cell extracts were obtained from A549P/A549CisR and H157P/H157CisR cells and Western blot analyses were performed using antibodies against indicated molecules. *p<0.05, **p<0.01.

Figure 2. Increased FASN expressions in cisplatin-resistant NSCLC cells compared to parental cells

A. qPCR analysis of FASN in A549P/A549CisR and H157P/H157CisR cells. B. Western blot analysis of FASN in A549P/A549CisR and H157P/H157CisR cells. C. Western blot analysis of FASN in A549P and H157P cells upon cisplatin treatment. Cells were treated with 5 μM cisplatin for indicated time and cell extracts obtained were used in analyses. D. Oil Red staining. Equal number (1 × 10³) of A549P/A549CisR and H157P/H157CisR cells was plated in 24 well plates. After settlement, cells were stained with Oil Red O working solution for 15 min at room temperature, and washed with running tap water to remove unbound staining. The positively stained cells per phase were counted under a microscope. Quantitation shown on right. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Inhibition of FASN reduced EMT/metastasis potential of cisplatin-resistant cells

A. Western blot analyses of FASN in A549CisR and H157CisR cells upon cerulenin (1 μg/ml, 48 hours) treatment. B. Growth assay. A549P and H157P and A549CisR and H157CisR (1 × 10³) were plated in 96 well plates, and the growth of cells in presence of cerulenin (1 μg/ml) or vehicle (control) was analyzed at each day in MTT assays. C. Morphology test. A549P, H157P, A549CisR and H157CisR cells were seeded. A549CisR and H157CisR cells were treated with cerulenin (1 μg/ml) or vehicle (control) for 48 hours and cell morphology was observed under a microscope. D. Migration test. A549P, H157P, A549CisR and H157CisR cells were seeded. A549CisR and H157CisR cells (1 × 10⁴) were treated with cerulenin (1 μg/ml) or vehicle (control) for 48 hours. Cells were then placed in upper chamber of transwell plates and migrated cells to the lower chamber at the end of 24 hours incubation were counted under a microscope after crystal violet staining. Quantitation shown on right. E. Western blot analyses of EMT/metastasis markers. Cell extracts were obtained from parental cells and cerulenin (or vehicle)-treated A549CisR and H157CisR cells and Western blot analyses were performed using antibodies against indicated molecules. F. Western blot analyses of EMT/metastasis markers in A549CisR-FASNsi/sc and H157CisRCisR-FASNsi/sc cell sets. Cell extracts were obtained and Western blot analyses were performed using antibodies against indicated molecules. G. Migration test. A549CisR-FASNsi/sc and H157CisRCisR-FASNsi/sc cells were placed in upper chamber of transwell plates and migrated cells to the lower chamber at the end of 24 hours incubation were counted under a microscope after crystal violet staining. Quantitation shown on right. *p<0.05, **p<0.01.

Figure 4. In vivo mice studies

A, B. IHC staining of tumor tissues using antibodies of Ki67 (A) and EMT/metastasis markers (B). Mice (H157CisR cell-derived xenograft) were sacrificed 3 days at the completion of cerulenin (or vehicle) treatment (H157P cell-derived xenografts did not receive treatment) and tumor tissues were obtained. The processed tumor tissues were cut into 5 μm, and stained with antibodies of FASN and EMT/metastasis markers. For Ki67 staining, tissues were subjected to antigen retrieval before staining. Magnification, x 100. Quantitation shown below. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. TGF-β1 is FASN downstream signaling that is important in mediating EMT/metastasis increase of cisplatin-resistant NSCLC cells

A. Western blot analysis showing TGF-β1 levels in A549P/A549CisR and H157P/H157CisR cells. B. Western blot analysis showing TGF-β1 increased expression in A549P and H157P cells treated with cisplatin at 5 μM for 48 hours or 72 hours. C. Western blot analysis showing TGF-β1 level decrease in A549CisR and H157CisR cells with cerulenin treatment (1 μg/ml, 48 hours). D. Western blot analysis showing TGF-β1 levels in A549CisR-FASNsi/sc and H157CisR-FASNsi/sc cells. E. TGF-β1 IHC staining of tumor tissues obtained in mice studies. F. qPCR (left) and Western blot (right) analyses showing expression of the EMT/metastasis markers in A549P/A549CisR and H157P/H157CisR cells after treatment with either vehicle or the TGF-β1 inhibitor, SB525334 (1 and 5 μg/ml) for 48 hours. G. Migration assay using A549CisR and H157CisR cells pre-treated with either vehicle or the TGF-β1 inhibitor, SB525334 (1 and 5 μg/ml, 48 hrs). *p<0.05, **p<0.01, ***p<0.001.

Figure 6. TGF-β1 induces FASN in cisplatin-resistant lung cancer cells

A, B. qPCR (A) and Western blot (B) analysis showing TGF-β1 and FASN mRNA (A) and protein (B) levels in A549CisR and H157CisR cells upon treatment with either vehicle or the TGF-β1 inhibitor SB525334 (SB) for 48 hours. C, D. qPCR (C) and Western blot (D) analysis showing FASN mRNA and protein levels in A549P/A549CisR and H157P/H157CisR cells after culturing with hrTGF-β1 (2 and 10 ng/ml) (24 hours of incubation for mRNA level study and 72 hours of incubation for protein analysis). *p<0.05, **p<0.01.