SKA1 regulates actin cytoskeleton remodelling via activating Cdc42 and influences the migration of pancreatic ductal adenocarcinoma cells

Figures

• 

  Figure 1. SKA1 is overexpressed in PDAC and associated with differentiation level and poor prognosis. A, Representative immunohistochemistry staining of different degrees of differentiation in 145 PDAC patients’ tissues for SKA1 is shown (left), and statistical histogram shows that the expression level SKA1 was increased along with poorer differentiated PDAC (right). B and C, Expression of SKA1 in one normal pancreatic duct epithelial cell line (hTERT‐HPNE), five well‐differentiated cell lines (Capan‐2, HPAF‐II, CFPAC‐1, SW1990 and Capan‐1), two moderate differentiated cell lines (AsPC‐1 and BxPC‐3) and two poor differentiated cell lines (MIA PaCa‐2 and PANC‐1) was measured by Immunoblotting and qRT‐PCR, respectively. GAPDH was used as the loading control. D, No significance of SKA1 expression level was observed among different stages in TCGA database. E‐G, Kaplan‐Meier survival curves of overall survival according to SKA1 expression in 145 PDAC patients of the testing cohort were performed using the log‐rank test. High SKA1 expression predicts poor overall survival in PDAC patients (P = .019), especially for patients with relative early TNM stage (P = .011), but not for late TNM stage (P…

• 

  Figure 2. SKA1 promotes PDAC proliferation in vitro and in vivo. A, Immunoblotting was performed in PANC‐1 and BxPC‐3 cells transfected with control shRNA (sh‐ctr) and SKA1 knock‐down shRNAs (sh‐SKA1), in Capan‐1 and SW1990 cells transfected with empty vector (vector) and lentivirus‐mediated flag‐tagged overexpression SKA1(SKA1). B, MTT assay showed the SKA1 facilitates PDAC cell growth ability, the significances were identified based on the comparison of counterpart. *P < .05. C and D, Cell cycle analysis by flow cytometry presented a significantly increased percentage of sh‐SKA1 cells in the G2/M phase, and related proteins were detected by Immunoblotting. E, The subcutaneous tumorigenic ability of tumour cells was measured (n = 5 per group). Expression of SKA1 promoted tumour growth and increased tumour weight in nude mice (P = .03). F, Percentage of positive Ki67 staining cells in tumour tissues was counted by immunohistochemical analysis. Data are presented as the mean ± SEM from three independent cell function experiments

• 

  Figure 3. SKA1 accelerates tumour invasion and metastasis via EMT. A and B, Transwell migration and Matrigel invasion assay. sh‐SKA1 infectants exhibited significantly reduced migration and invasion capacity than the sh‐ctr infectants, except for SW1990 cell line. C, Wound‐healing assay measured the effect of SKA1 on PDAC cell motility. Left: Representative images of scratched and recovering of wounded areas taken at different time points. Right: Semi‐quantitative analysis of gap size by ImageJ. The significances were calculated based on the comparison of the control group. *P < .05; **P < .01; ***P < .001. D, Representative haematoxylin‐eosin staining and summarized data on number of metastasis of lung in nude mice at 12 wk after tail vein injection of PANC‐1 or Capan‐1 cells with SKA1 knock‐down or overexpression (n = 10 per group), red arrow, metastatic nodule (P = .033, P = .013, respectively)

• 

  Figure 4. SKA1 facilitates EMT in PDAC cells. A, A significant reduction of vimentin and N‐cadherin but increase E‐cadherin was detected in PANC‐1‐shSKA1 cells compared to PANC‐1‐sh‐ctr cells by immunofluorescence staining, and vice versa in Capan‐1 cells. B, The protein expression levels of EMT markers and transcription factors (E‐cadherin, N‐cadherin, ZO‐1, Vimentin, Snail, Slug, Twist, TCF8/ZEB1) in modified PANC‐1 and Capan‐1 cells were assayed by Western blotting. GAPDH was used as an internal control

• 

  Figure 5. Proteomic analysis of SKA1‐induced biological process. A, Volcano plot showing the quantitative protein expression from sh‐SKA1 and sh‐ctr in PANC‐1 cells. Proteins differentially expressed with fold change over 1.5 were marked in blue and red. B, Subcellular distribution of the differentially expressed proteins. C, The top enriched biological processes, cellular components and molecular functions based on the gene ontology (GO) analysis with the sequence data of differentially expressed proteins. D, Bubble chart that illustrate the top 20 signalling pathways enriched based on the KEGG pathway analysis. E, Network of gene ontology and related differential proteins

• 

  Figure 6. SKA1 modulates the formation of cytoskeletal actin filament and shared partial co‐localization with F‐actin in PDAC cells. A, Combined rhodamine‐phalloidin staining for F‐actin (red) and SKA1 (green) immunofluorescence, nuclei were counterstained with DAPI (blue). Upper two panels: stable knock‐down of SKA1 reduced actin protrusions and stress fibres in PANC‐1 cells. Lower two panels: stable overexpression of SKA1 altered the cellular morphology and lead to longer invadopodia formation compared with vector group in Capan‐1 cells. Additionally, SKA1 and phalloidin‐stained F‐actin shared partial co‐localization to the cytoplasm. Co‐localization correlation of intensity distributions curves between two channels was drawn by ImageJ and measured by Pearson's correlation coefficient analysis (right). B, With cytochalasin B treatment, F‐actin filaments stained with phalloidin were not seen in both PANC‐1 and Capan‐1 cells’ infectants

• 

  Figure 7. SKA1 remodels actin cytoskeleton via activating Cdc42. A, Pearson's correlation test was used to analyse the correlation between the expression level of SKA1 and Cdc42 according to TCGA database (R = .51, P < .01). B, Immunoblotting performed in PANC‐1 and Capan‐1 cells’ infectants to verify the proteins that regulate actin cytoskeleton. C, Cdc42‐GTP pull‐down and Western blot analyses of the activation state of Cdc42 expression and total expression of Cdc42 in the indicated cells. D and E, The application of ZCL278 resulted in a dose‐dependent decrease in Cdc42 activity, followed by inhibition of endogenous Arp2/3, N‐WASP. F, Cells were fixed and stained with rhodamine‐phalloidin to label filamentous actin following ZCL278 treatments. Results showed that ZCL278 inhibits microspike formation and stress fibres in PANC‐1 and Capan‐1 cells. White asterisks indicate the subcellular locations that normally show stress fibre distribution. White arrows point to the seemingly disruption Golgi organization. G, RhoA, RhoB, RhoC and Rac1/2/3 protein expression were detected, and only a total of Rac1/2/3 increase accompanied by SKA1 overexpression was observed. H, FAK, Talin and ɑ‐actinin…