CDP138 silencing inhibits TGF-β/Smad signaling to impair radioresistance and metastasis via GDF15 in lung cancer

Abstract

CDP138, a CDK5 binding partner, regulates cell proliferation and migration. However, the mechanisms by which CDP138 functions in these processes remain unclear. In this study, we show that CDP138 is frequently overexpressed and that high levels of CDP138 are correlated with lymph node metastasis in lung cancer. Furthermore, we provide evidence that CDP138-depleted lung cancer cells exhibit enhanced radiosensitivity as well as reduced migration and invasion. Mechanistically, we identify GDF15, a member of the TGF-β superfamily, as a key downstream effector of CDP138. CDP138 silencing attenuates TGF-β/Smad signaling activation at least in part through the downregulation of GDF15. More importantly, the observed phenotypes caused by CDP138 knockdown are partially dependent on GDF15 inhibition. Together, our findings demonstrate that CDP138 positively modulates the TGF-β/Smad signaling pathway via GDF15 to promote radioresistance and metastasis, suggesting CDP138 as a potential oncogenic biomarker and a promising therapeutic target in the treatment of lung cancer.

Figure 1

CDP138 is overexpressed in lung cancer cell lines and tissues. (a) Western blotting analysis of CDP138 expression in five human lung cancer cell lines and one normal human bronchial epithelial cell line. (b) Quantification of CDP138 protein expression in (a). (c) Representative IHC images of staining of CDP138 in tissue microarrays constructed from lung cancer and paired para-carcinoma tissues. (d) Summary of IHC staining for CDP138 expression in tissue microarrays. Differences between variables were assessed using the χ² test

Figure 2

CDP138 silencing results in impaired proliferation and enhanced radiosensitivity in lung cancer cells. (a) Western blotting analyses revealed that CDP138 is efficiently knocked down in H1299 and HCC827 cells. (b) Knocking down CDP138 suppresses cell growth. H1299 and HCC827 cells were seeded, and cell numbers were evaluated every day. (c) Colony formation is significantly reduced in CDP138-depleted cells. **P<0.01 and ***P<0.001 compared with controls cells. (d) Radiation sensitivity of H1299 and HCC827 cells lacking CDP138. H1299 cells transfected with indicated siRNAs and stable HCC827 cells were irradiated at indicated doses. Percentages of surviving colonies were examined two weeks later. (e,f) Left panel: H1299 cells (e) or HCC827 cells (f) were exposed to 2 Gy radiation and harvested at indicated time points. Immunostaining was performed to determine γ-H2AX foci formation. Right panel: Quantification of γ-H2AX foci in H1299 cells (e) or HCC827 cells (f). *P<0.05, **P<0.001 compared with controls cells

Figure 3

Knocking down CDP138 inhibits the migration and invasion of lung cancer cells. (a) Cell migration is decreased in CDP138 knockdown cells as determined using wound healing assay. (b,c) Left panel: Transwell assays showing that CDP138-depleted H1299 cells (b) or HCC827 cells (c) have lower migratory and invasive capacity than those of control cells. Right panel: Quantification of migration and invasion in H1299 cells (b) or HCC827 cells (c). *P<0.05 and **P<0.01 compared with controls cells. (d) HBE cells were transfected with the indicated plasmids. After 24 h, cells were harvested and analyzed via Western blotting with indicated antibodies. (e) Left panel: cell migration and invasion is measured using transwell assays. Right panel: quantification of migration and invasion in HBE cells. *P<0.05 and **P<0.01 compared with control cells

Figure 4

CDP138 knockdown attenuates the TGF-β/Smad signaling pathway at least in part via the downregulation of GDF15. (a) Heat map produced from mRNA microarray analysis. H1299 cells were transfected with indicated siRNAs for 48 h, and mRNA was isolated and evaluated using microarray analysis. (b) List of differentially expressed genes related to cell proliferation and metastasis (fold change>2). (c) H1299 cells were transfected with indicated siRNAs for 48 h and then harvested. Levels of indicated mRNAs were determined using quantitative real-time PCR. (d) GDF15 protein level was dramatically reduced in CDP138-depleted cells. (e) Knockdown of CDP138 decreases p-Smad2 protein expression. H1299 cells transfected with indicated siRNAs were treated with TGF-β (10 ng/ml) for 24 h. Cells were harvested and subjected to western blotting with indicated antibodies. (f) GDF15 overexpression partially rescues the reduction in p-Smad2 level. H1299 cells were transfected with indicated siRNAs for 24 h, and GDF15 expression plasmid was introduced. Cells were harvested after TGF-β treatment (10 ng/ml), and the lysates were subjected to western blotting with indicated antibodies

Figure 5

GDF15 overexpression partially rescues the impaired radioresistance and migration in lung cancer cells. (a) H1299 cells were transfected with the indicated siRNAs. After 24 h, the cells were then transfected with GDF15 expression plasmids. After 24 h, the cells were harvested and analyzed via western blotting. (b) GDF15 partially rescues the defects in cell growth. H1299 cells transfected with indicated siRNAs and plasmids were seeded, and cell numbers were evaluated every day. (c) Upper panel: H1299 cells were seeded and grown for two weeks. Number of colonies was counted. Lower panel: quantification of colony formation. (d) Upper panel: cell migration is determined using transwell assays. Lower panel: quantification of migration in H1299 cells. (e) Upper panel: H1299 cells transfected with indicated siRNAs and plasmids were exposed to 2 Gy radiation and harvested at the indicated time points. Immunostaining was performed to determine γ-H2AX foci formation. Lower panel: quantification of γ-H2AX foci in H1299 cells. *P<0.05 and **P<0.01. n.s. indicates no statistically significant difference (P>0.05)

Figure 6

GDF15 is required for radioresistance and migration in CDP138-depleted H1299 cells. (a) H1299 cells were transfected with the indicated siRNAs. After 48 h, the cells were harvested and analyzed via Western blotting. (b) H1299 cells transfected with the indicated siRNAs were seeded at low density, and cell numbers were evaluated every day. (c) Upper panel: H1299 cells were transfected with indicated siRNAs and grown for two weeks. Number of colonies was counted. Lower panel: quantification of colony formation. (d) Upper panel: cell migration is determined using transwell assays. Lower panel: quantification of migration in H1299 cells. (e) Upper panel: H1299 cells transfected with indicated siRNAs were exposed to 2 Gy radiation and harvested at the indicated time points. Immunostaining was performed to examine γ-H2AX foci formation. Lower panel: quantification of γ-H2AX foci in H1299 cells. *P<0.05 and **P<0.01. n.s. indicates no statistically significant difference (P>0.05)