Six1 promotes proliferation of pancreatic cancer cells via upregulation of cyclin D1 expression

Abstract

Six1 is one of the transcription factors that act as master regulators of development and are frequently dysregulated in cancers. However, the role of Six1 in pancreatic cancer is not clear. Here we show that the relative expression of Six1 mRNA is increased in pancreatic cancer and correlated with advanced tumor stage. In vitro functional assays demonstrate that forced overexpression of Six1 significantly enhances the growth rate and proliferation ability of pancreatic cancer cells. Knockdown of endogenous Six1 decreases the proliferation of these cells dramatically. Furthermore, Six1 promotes the growth of pancreatic cancer cells in a xenograft assay. We also show that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Overexpression of Six1 upregulates cyclin D1 mRNA and protein, and significantly enhances the activity of the cyclin D1 promoter in PANC-1 cells. We demonstrate that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. These data suggest that Six1 is overexpressed in pancreatic cancer and may contribute to the increased cell proliferation through upregulation of cyclin D1.

Figure 1. Expression of Six1 in pancreatic cancer.

(A) The relative mRNA expression level of Six1 was determined by quantitative Real-time RT-PCR in 51 pancreatic ductal adenocarcinomas and 13 adjacent non-tumor pancreatic tissue samples. Results were normalized to the expression level of GAPDH mRNA in each sample. *P<0.05. (B) ONCOMINE database was used to analyze previously published microarray data. Levels of Six1 mRNA are increased in pancreatic cancer when compared with adjacent non-tumor pancreatic tissue. All results, including p-values, were calculated using ONCOMINE data. N, adjacent non-tumor pancreatic tissue; T, pancreatic ductal adenocarcinomas; *P<0.05.

Figure 2. Six1 increases pancreatic cancer cell proliferation in vitro and in vivo.

Cell numbers (A), percentage of BrdU positive cells (B) and number of colonies (C) of PANC-1 cells stably transfected with either Six1 or control plasmids. Solid line, control; dashed line, Six1. D, Number of PANC-1 cells transfected with Six1 siRNA and scramble control. Solid line, scramble control siRNA; dashed line, Six1 siRNA. E, Tumor size of subcutaneous xenografts measured within 5-day interval. The tumor volume was calculated using the formula: (Length × Width²)/2. F, Representative subcutaneous tumor xenografts generated in mice 5 weeks after inoculation (upper panel), and the weight of the tumors (lower panel). G, The expression of Six1 in both PANC-1 and MIA PaCa-2 cells was determined by western blot analyses. GAPDH was used as an internal control. All experiments were performed in triplicate; bars, s.e.m.; *P<0.05.

Figure 3. Six1 transcriptionally activates the gene encoding cyclin D1 in pancreatic cancer cells.

A and B, The effect of Six1 on the expression of cyclin D1 protein (A) and mRNA (B) was examined in PANC-1 cells by western blot analyses. GAPDH was used as an internal control. C, cyclin D1 promoter activity was shown to be responsive to increased amounts of a Six1 expression vector, using a luciferase reporter. D, the physical interaction of Six1 to the cyclin D1 promoter in chromatin of PANC-1 cells was shown using a ChIP-qPCR assay. The recruitment of Pol II to the GAPDH proximal promoter was used as a positive control, and the negative control is normal IgG, which controls for the non-specific immunoselection of chromatin by immunoglobulins. Soluble chromatin was prepared and immunoprecipitated with the indicated antibodies. The final DNA extractions were amplified using three pairs of primers as indicated. It showed that Six1 could bind to the −1189 to −985 region of the cyclin D1 promoter but not to the irrelevant −348 to −190 region. All experiments were performed in triplicate; NS, not significant; *P<0.05.

Figure 4. Six1 correlates with cyclin D1 in human pancreatic cancer.

A, Six1 levels correlate with cyclin D1 in the xenograft tumors from PANC-1 cells stably transfected with either Six1 or control plasmids as determined by western blot and then quantified by Fluorchem computer analysis (r = 0.903, P = 0.0003). B, the correlation between the two in 27 advanced stage (III & IV) of human pancreatic cancer samples as determined by qRT-PCR (r = 0.541, P = 0.020). C, the correlation between the two in three other independent pancreatic cancer microarray datasets (GSE15471: r: 0.477, P: 0.0021; GSE32676: r: 0.372, P: 0.036; and GSE28735: r: 0.767, P<0.0001). Correlation between the two was quantified by Spearman’s rank correlation. *P<0.05.

Figure 5. Six1 regulates pancreatic cancer cell growth and cell cycle progression through cyclin D1.

A, Knockdown of cyclin D1 decreases proliferation of PANC-1 cells transduced with Six1. B, C, and D, overexpression of cyclin D1 rescued the inhibitory effect of Six1 silencing on the growth of PANC-1 cells, which was examined by BrdU incorporation assay (B) and flow cytometry analyses (C). The phosphorylation levels Rb (phospho-Rb) was also examined by western blot analyses. The expression of proteins was quantified using the expression of GAPDH as calibrator and normalized against the control group value (D). All the experiments were performed in triplicate; bars, s.e.m.; *P<0.05.