Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma

Abstract

Nicotinamide N-methyltransferase (NNMT) is an S-adenosyl-l-methionine (SAM)-dependent cytosolic enzyme, and a growing body of evidence suggest that it plays an essential role in cancer progression. Recently, NNMT has a role in methylation metabolism and tumorigenesis and was associated with a poor prognosis against numerous cancers. In addition, it has been reported that NNMT has been overexpressed in the stroma of advanced high-grade serous carcinoma and may contribute to decreased survival. This study aimed to identify novel biomarkers to predict resistance and investigate their clinicopathologic significance in paclitaxel-resistant advanced or recurrent ovarian clear cell carcinoma (OCCC). Fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE), immunohistochemical, and MASCOT analyses allowed us to identify the cytoplasmic metabolic enzyme NNMT. In cultured cell studies, NNMT protein expression was higher in paclitaxel-resistant OVMANA and OVTOKO cells than in paclitaxel-sensitive KK and ES-2 cells. Furthermore, although analysis of clinical tissue samples showed no association with poor prognosis in 7 individuals with low NNMT expression in the cytoplasm of OCCC cells, high expression of NNMT in the cytoplasm of OCCC cells may be associated with low sensitivity to paclitaxel in OCCC and may have prognostic implications. Therefore, targeting therapy to reduce cytoplasmic NNMT expression levels may increase the sensitivity of OCCC to paclitaxel.

Keywords: 2D-DIGE; Chemoresistance; Nicotinamide N-methyltransferase; Ovarian clear cell carcinoma; Paclitaxel.

Fig. 1

WST-8 assay of 4 ovarian clear cell cancer cell lines. Inhibition rate of the number of viable cells in the ovarian clear cell carcinoma (OCCC) cell lines, OVTOKO, OVMANA, ES-2, and KK assessed at indicated paclitaxel concentrations via the WST-8 assay. IC50 for OVTOKO and OVMANA was higher than IC50 for KK and ES-2. The number of independent experiments is 3

Fig. 2

The result of fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE). a Protein spots and quantity graph. Proteins extracted from OVTOKO, OVMANA, ES-2, and KK cells were separated using 2D-DIGE and the protein expression changes were analyzed using the PDQuest software. Image analysis using PDQuest software identified 5 protein spots with significantly higher or lower expression in OVTOKO and OVMANA than that in ES-2 and KK cells. b A representative gel image of 2D-DIGE is shown. The protein from ES-2 and OVMANA cells were labeled with Cy3 (green) and Cy5 (red), respectively. The red protein spot contained NNMT and its expression was higher in OVMANA than in ES-2 cells. The number of independent experiments is 3

Fig. 3

Mascot search result and western blot analysis. a The proteins identified using the MASCOT peptide mass fingerprinting server in one of the 5 protein spots. MASCOT analysis showed a top score of 159 for nicotinamide N-methyltransferase (NNMT). The number of independent experiments is 1. b Western blot analysis showed that NNMT was expressed in OVTOKO and OVMANA, but not in ES-2 and KK cells. The number of independent experiments is 3

Fig. 4

A list of the digested peptides and NNMT protein sequence. a A list of the digested peptides that covered the NNMT protein sequence. The number of independent experiments is 1. b The digested peptides covered 51% of the NNMT protein sequence. The number of independent experiments is 1

Fig. 5

The result of fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE). A representative gel image of 2D-DIGE is shown. a The protein from KK and OVMANA cells were labeled with Cy3 (green) and Cy5 (red), respectively. The red protein spot contained NNMT and its expression was higher in OVMANA than in KK cells. The number of independent experiments is 2. b The protein from ES-2 and OVTOKO cells were labeled with Cy3 (green) and Cy5 (red), respectively. The red protein spot contained NNMT and its expression was higher in OVTOKO than in ES-2 cells. The number of independent experiments is 2. c The protein from KK and OVTOKO cells were labeled with Cy3 (green) and Cy5 (red), respectively. The red protein spot contained NNMT and its expression was higher in OVTOKO than in KK cells. The number of independent experiments is 2

Fig. 6

Immunohistochemical staining of NNMT (cytoplasm) and survival analysis of NNMT. Immunohistochemical analysis of NNMT expression in primary OCCC tumors. a and b Representative NNMT immunohistochemical staining of primary ovarian clear cell carcinoma cells. High (a) or almost no (b) expression of NNMT was observed in the cytoplasm of primary OCCC cells. Magnification, × 400. c Kaplan–Meier curve showing the overall survival rates of patients with OCCC. There were no deaths in patients with OCCC, who showed lower NNMT levels in the cytoplasm. The number of samples in the low NNMT expression group was 7, and the number of samples in the high group was 32

Fig. 7

The result of NNMT siRNA and western blot analysis. We investigated changes in paclitaxel sensitivity after NNMT siRNA-dependent knockdown of OVTOKO and RMG1 using the WST-8 assay. The graphs were created using the mean of the three experiments and SEM is shown as error bars. a Paclitaxel IC50 in the NNMT siRNA-transfected OVTOKO cells is significantly lower than that of the control siRNA-transfected OVTOKO cells (< 0.7-fold, p = 0.00275, student t-test). The number of independent experiments is 3. b Paclitaxel IC50 in the NNMT siRNA-transfected RMG1 cells is significantly lower than that of the control siRNA-transfected RMG1 cells (< 0.44-fold, p = 0.00190, student t-test). The number of independent experiments is 3. c Paclitaxel IC50 in the NNMT siRNA-transfected ES-2 cells is not significantly lower than that of the control siRNA-transfected ES-2 cells (1.01-fold, p = 0.984, student t-test). The number of independent experiments is 3. d Western blot analysis showed reduced expression of NNMT protein in OVTOKO and RMG1 cells in the NNMT siRNA-treated group compared to the control siRNA-treated group. No difference was observed between the two groups in ES-2 cells because of the absence of NNMT expression. The number of independent experiments is 3

Fig. 8

The result of NNMT overexpression vector and western blot analysis. We investigated changes in paclitaxel sensitivity after NNMT overexpression in ES-2 cells using the WST-8 assay. a Paclitaxel IC50 in the NNMT-overexpressing viable ES-2 cells was not significantly higher than that of ES-2 transfected with empty vector (pCMV vector) (p = 0.360, student t-test). The number of independent experiments is 3. b Western blot analysis showed the increased expression of NNMT protein in ES-2 cells in the NNMT overexpression group compared to the empty vector-transfected group. The number of independent experiments is 3