Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer

Abstract

Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we reported that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241-5253]. In general, various studies established inhibition of cell proliferation by SPRY in cancer. The mechanisms by which SPRY regulates cell proliferation in CRC are investigated. We demonstrate, for the first time, suppression of SPRY2 augmented EGF-dependent oncogenic signaling, however, surprisingly decreased cell proliferation in colon cancer cells. Our data suggest that cell cycle inhibitor p21(WAF1/CIP1) transcriptional activity being regulated by SPRY2. Indeed, suppression of SPRY2 significantly increased p21(WAF1/CIP1) mRNA and protein expression as well as p21(WAF1/CIP1) promoter activity. Conversely, overexpressing SPRY2 triggered a decrease in p21(WAF1/CIP1) promoter activity. Concurrent down-regulation of both SPRY1 and SPRY2 also increased p21(WAF1/CIP1) expression in colon cancer cells. Increased nuclear localization of p21(WAF1/CIP1) in SPRY2 downregulated colon cancer cells may explain the inhibition of cell proliferation in colon cancer cells. Underscoring the biological relevance of these findings in SPRY1 and SPRY2 mutant mouse, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression and nuclear localization of p21(WAF1/CIP1) and decreased cell proliferation. In CRC, the relationship of SPRY with p21 may provide unique strategies for cancer prevention and treatment. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Keywords: MEF; cancer; colon; mouse; p21; sprouty.

Figure 1

Silencing of SPRY2 augments EGF‐dependent signaling in Caco‐2 colon cancer cells. Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). Cells were serum starved overnight, treated with EGF (10 ng/ml) for the indicated times and cell lysates were western blotted. Results represent a representative experiment of three independent experiments.

Figure 2

Silencing of SPRY2 upregulates p21 expression and inhibits cell proliferation. (A) Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). Transfected cells (10 000/well) were then plated in a 96 well plate for 48 h and cell proliferation was assessed by MTT assay. Data represents means ± SD of three independent experiments, *P <  0.05, compared to Nsi cells. (B, C) Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). p21 mRNA and protein expression was assessed by RTPCR and western blotting, respectively. Data represents means ± SD of three independent experiments, *P < 0.05, compared to Nsi cells. (D) Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). Cells were serum starved overnight, treated with EGF (10 ng/ml) for the indicated times and cell lysates were western blotted for p21 expression. Results represent a representative experiment of three independent experiments. (E) SW480 and SW620 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). p21 protein expression was assessed by western blotting. Data represents means ± SD of three independent experiments, *P < 0.05, compared to Nsi cells.

Figure 3

Silencing of p21 reverts the effect of SPRY2 downregulation on EGF‐induced cell proliferation Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h) or a mixture of SPRY2 siRNA and p21 siRNA (50 nM each, 72 h). Transfected cells (10 000/well) were plated in a ninety six well plate, serum starved overnight, treated with EGF (10 ng/ml) or vehicle for 48 h and cell proliferation was assessed by MTT assay. Data represents means ± SD of three independent experiments, *P < 0.05 (one way ANOVA).

Figure 4

Silencing of SPRY2 delays cell cycle transition. Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or SPRY2 siRNA (50 nM, 72 h). Transfected cells were plated in six well plates for 24 h. Cells were synchronized by serum deprivation and then stimulated with EGF for indicated times. The percentage of cells in G0/G1, S and G2/M phases of cell cycle are shown within each histogram. Representative results from three independent experiments are shown.

Figure 5

Transcriptional regulation of p21 promoter by SPRY2. Caco‐2 cells were seeded in a 12 well plate for 24 h. Cells were then transiently transfected with (A) empty vector pHM6 or pHM6‐SPRY2 (1 μg) (B) Nsi RNA or siRNA‐SPRY2 (50 nM) for 72 h. Cells were then transfected with luciferase reporter construct pGL2‐Luc containing 2.4 kb of p21 native promoter or empty vector (pGL2) for 48 h. As an internal standard a Renilla luciferase vector (pRL‐TK) was included in all samples. After 48 h, cell lysates were prepared and the Firefly and Renilla luminescence were measured sequentially using the dual luciferase assay kit. Data represents means ± SD of three independent experiments, *P < 0.05, compared to control vector(s) or non‐silencing RNA treated cells.

Figure 6

Increased nuclear localization of p21 in SPRY2 downregulated cells. Representative confocal images showing expression of p21 in Nsi RNA or SPRY2 siRNA (50 nM, 72 h) treated Caco‐2 cells. Cells were fixed with cold methanol, permeabilized, and blocked with normal goat serum. Sequentially, the cells were incubated with rabbit anti‐p21 and Alexa Fluor 488‐conjugated goat anti‐rabbit immunoglobulin. The cells were then stained with DAPI to label the nuclei. Images were obtained using a Leica TCS SP8 confocal laser scanning microscope. Cytoplasmic (white arrow head) and nuclear (red arrow head) localization of p21 is shown. All images were collected using identical microscope settings and magnification. The panels are representative of at least three independent experiments in which similar patterns were obtained for the indicated cells.

Figure 7

Concurrent SPRY1 and SPRY2 suppression in Caco‐2 cells upregulates p21 expression. Caco‐2 cells were transiently transfected with non‐silencing control (Nsi) or a mixture of SPRY1 and SPRY2 siRNA (50 nM each, 72 h). (A) SPRY1, SPRY2, and p21 mRNA, and (B) p21 protein expression was assessed by RTPCR and western blotting, respectively. Nsi arbitrary value 1 represents mRNA expression levels of SPRY1, SPRY2 and p21 in non‐targeted siRNA transfected cells. Data represents means ± SD of three independent experiments, *P < 0.05, compared to non‐silencing RNA treated cells.

Figure 8

Spry1 and Spry2 deletion in mouse embryonic fibroblasts upregulates nuclear p21 expression and inhibits EGF‐dependent proliferation (A) Tamoxifen dependent recombination and a complete deletion of Spry1 and Spry2 in MEFs increases p21 expression. Spry1^f/f; Spry2^f/f and Spry1^−/−; Spry2^−/− MEFs were cultured for 48–120 h. Total RNA was extracted and mRNA transcripts of SPRY1, SPRY2, and p21 were assessed by RTPCR. Data represents means ± SD of three independent experiments, ^#,*P < 0.05, compared to control Spry1^f/f; Spry2^f/f MEFs (B) Deletion of Spry1 and Spry2 in MEFs reduces EGF‐dependent cell proliferation. Spry1^f/f; Spry2^f/f and Spry1^−/−; Spry2^−/− MEFs were plated and treated with vehicle or EGF (10 ng/ml) for 48 h. Cell proliferation was assessed by MTT assay. Groups compared; vehicle and EGF treated Spry1^f/f; Spry2^f/f cells, vehicle and EGF treated Spry1^−/−; Spry2^−/− cells and EGF treated Spry1^−/−; Spry2^−/− cells compared to EGF treated Spry1^f/f; Spry2^f/f cells. Data represents means ± SD of three independent experiments, *P < 0.05 (one way ANOVA). (C) Representative confocal images showing the expression of p21 in Spry1^f/f; Spry2^f/f and Spry1^−/−; Spry2^−/− MEFs. Cells were fixed with cold methanol, permeabilized, and blocked with normal goat serum. Sequentially, the cells were incubated with mouse anti‐p21 and Alexa Fluor 488‐conjugated goat anti‐mouse immunoglobulin. The cells were then stained with DAPI to label the nuclei. Images were obtained using a Leica TCS SP8 confocal laser scanning microscope. Cytoplasmic (white arrow head) and nuclear (red arrow head) localization of p21 is shown. All images were collected using identical microscope settings and magnification. The panels are representative of at least three independent experiments in which similar patterns were obtained for the indicated cells.