Fig 2: A schematic model illustrating the transcriptional reduction of SOX2 by ANGPTL1 through the induction of FOXO3a in CRC, and how this affects cancer stemness and metastasis

Fig 3: The clinical significance of ANGPTL1 expression in colorectal cancer (CRC)(A) The expression of ANGPTL1 negatively correlates with CRC, metastatic stage, and recurrence. ONCOMINE datasets: Hong_Colon, Bitner_Colon, and Jorissen_Colon. (B) IHC staining of ANGPTL1 in colon and CRC TMA tissues with scores of 0–3 was represented. Positive control: Carbon; scale bar: 100 µm. (C) The box plot representation of scores based on IHC staining of ANGPTL1 in 59 paired specimens of CRC and adjacent normal colon tissues. P-value was analyzed by Chi-square analysis followed by Fisher’s exact test. (D) The overall survival of 58 patients in TMA with low and high ANGPTL1 expression was analyzed by Kaplan–Meier analysis (P=0.0372). ANGPTL1 expression was grouped based on the median of the IHC score of specimens. (E) ANGPTL1 expression inversely associated with metastatic tissues compared with paired specimens of CRC tissues in TMA (n=10). *P<0.05 was obtained by paired two-tailed Student’s t-tests.

Fig 4: ANGPTL1 suppressed cell migration and invasion through FOXO3a induction in HCT116 cells(A) The protein expression of FOXO family was assessed by Western blotting. The expression of a-tubulin used as an internal loading control. (B) The mRNA expression of FOXO3 was analyzed using RT-PCR analysis. GAPDH was used as reference gene; *, P<0.05 (C) The p-FOXO3a and FOXO3a expression in ANGPTL1-overexpressed cells was performed. The expression of a-tubulin is as an internal loading control. (D) The expression of FOXO3a in the nucleus and cytosol was assessed by Western blotting. The expression of Lamin A/C used as nuclear internal loading control and a-tubulin as a cytosol internal loading control. (E) The knockdown efficiencies of FOXO3a were analyzed. The expression of a-tubulin is as an internal loading control. (F–H) Both migration and invasion abilities were examined and quantified. Results are shown as the mean ± S.D. of three independent experiments. *, P<0.05, compared with indicated cells; scale bar = 100 µm.

Fig 5: Effects of ANGPTL1 overexpression on liver metastasis and tumor growth in CRC(A) The liver metastasis of CRC was performed with an intrasplenic injection mouse model for four weeks (n = 7/group). Black arrows indicated the metastatic tumor nodules in the liver. (B) The tumor nodules were counted and analyzed by Student’s t test. (C,D) The liver and body weights were weighted; *, P<0.05. (E,F) The xenograft subcutaneous model was performed by implanted the HCT116 cells with stable expression of vector or ANGPTL1 into the dorsal flanks of nude mice. The tumor volume and tumor weight were measured. Results are represented as the mean ± S.D. *, P<0.05, compared with vector groups (N = 8/group).