FBXW11 contributes to stem-cell-like features and liver metastasis through regulating HIC1-mediated SIRT1 transcription in colorectal cancer

Abstract

Colorectal tumorigenesis is a heterogeneous disease driven by multiple genetic and epigenetic alterations. F-box and WD repeat domain containing 11 (FBXW11) is a member of the F-box protein family that regulates the ubiquitination of key factors associated with tumor growth and aggressiveness. Our study aimed to explore the role of FBXW11 in the development and metastasis of colorectal cancer (CRC). FBXW11 was overexpressed in colorectal tumor tissues and its overexpression was associated with a poor prognosis of CRC patients. The upregulation of FBXW11 not only promoted cell proliferation, invasion, and migration, but also contributed to maintaining stem-cell features in colorectal tumor cells. Further analysis revealed that FBXW11 targeted hypermethylated in cancer 1 (HIC1) and reduced its stability in CRC cells through ubiquitination. Moreover, the expression of sirtuin 1 (SIRT1), a deacetylase in tumor cells was upregulated by FBXW11 via regulating HIC1 expression. The mouse xenograft models of CRC confirmed that FBXW11 knockdown impeded colorectal tumor growth and liver metastasis in vivo. In summary, our study identified FBXW11 as an oncogenic factor that contributed to stem-cell-like properties and liver metastasis in CRC via regulating HIC1-mediated SIRT1 expression. These results provide a rationale for the development of FBXW11-targeting drugs for CRC patients.

Fig. 1. Expression of FBXW11 in CRC tissue samples and its correlation with the prognosis of CRC patients.

Colorectal tumor tissue and paired adjacent non-tumorous tissue samples were collected from 145 patients with CRC. A The mRNA level of FBXW11 in tissue specimens was measured by qRT-PCR. B The overall and disease-free survival of CRC patients with low or high FBXW11 expression was calculated using the Kaplan–Meier method. C The expressions of FBXW11 and ALDH1 in colorectal and adjacent non-tumorous tissue samples were detected by immunohistochemistry. Tissue samples with no staining (Normal, adjacent tissues), weak staining (Well), moderate staining (Moderate), and strong staining (Poor) were shown (×400 magnification). D The SI of each section was calculated by multiplying the immunohistochemical intensity score and the score of positively stained cells. An SI of ≤4 was defined as low expression, whereas an SI of ≥6 was considered high expression. The box plot shows the percentage of samples with low or high ALDH1 expression in tumors with low or high FBXW11 level. E Representative blots show the protein expressions of FBXW11 and ALDH1 in four pairs of tissue samples. Student’s t-test was used for statistical comparisons between the two groups.

Fig. 2. Effect of FBXW11 on the growth and migration of CRC cells.

Human CRC cell lines HCT116 and SW620 were transfected with recombinant lentiviral vectors carrying FBXW11 or shRNAs targeting FBXW11 (shFBXW11-1 and shFBXW11-2). The control groups were transfected with corresponding empty control vectors or scrambled shRNA sequence. A Transfection efficiency was evaluated by western blotting at 48 h post transfection. B The MTT assay was used to evaluate cell viability at 0, 24, 48, 72, and 96 h after transfection. C, F The colony formation assay was performed by culturing transfected CRC cells with culture medium supplemented with 10% FBS for 2 weeks. The number of colonies per well was counted. D, G The wound-healing assay was performed to evaluate the migration capacity of transfected CRC cells. The wound closure at 24 h post scratching was measured. E, H The Transwell assay was used to assess the invasion capacity of CRC cells. Cells were seeded in Matrigel-coated chambers filled with serum-free medium. The bottom chamber was added with the same volume of normal culture medium. After 24 h, the number of invaded cells per group was counted from six randomly selected fields (×400 magnification). ANOVA followed by Bonferroni’s post hoc test was used for statistical comparisons among multiple groups.

Fig. 3. Effect of FBXW11 on maintaining stem-cell-like properties in CRC cells.

HCT116 and SW620 cells were transfected with recombinant lentiviral vectors carrying FBXW11, shRNAs targeting FBXW11, or corresponding controls. A The sphere-forming ability of CRC cells following transfection was evaluated by cultivating one single cell in growth medium for 7 days. The number of spheres (diameter >50 μm) were counted and normalized to the group transfected with empty control lentiviral vector. B Transfected CRC cells were stained with anti-CD44-FITC and anti-CD133-APC antibodies, and then sorted by flow cytometry. The percentage of CD133⁺/CD44⁺ cells in each group was calculated. C The protein expressions of Nanog, ALDH1, and Oct4 were measured by western blotting. ANOVA followed by Bonferroni’s post hoc test was used for statistical comparisons among multiple groups.

Fig. 4. Effect of FBXW11 on the stability of HIC1.

A, B HCT116 and SW620 cells were transfected with recombinant lentiviral vectors carrying FBXW11, shRNAs targeting FBXW11, or corresponding controls. The A mRNA and B protein levels of HIC1 in transfected CRC cells were determined by qRT-PCR and western blotting, respectively. C HEK-293T cells were transfected with FLAG-tagged FBXW11 or HA-tagged HIC1, or both for 48 h. Then cells were stained antibodies against FLAG tag or HA tag. The immunoprecipitated proteins were analyzed by western blotting. D The ubiquitination level of HIC1 in transfected CRC cells was examined by treating them with 20 μM of MG132 for 3 h. Cells were then mixed with conjugated beads followed by immunoprecipitation with anti-poly-ubiquitin antibody. E Transfected HCT116 and SW620 cells were treated with 10 µg/mL of CHX. The protein expression of HIC1 was analyzed by western blotting at 0, 2, 4, 8, and 12 h after treatment. F The qualification curves of HIC1 expression in both cells lines at different time points were plotted. Student’s t-test and ANOVA followed by Bonferroni’s post hoc test were used for statistical comparisons between two groups or among multiple groups, respectively.

Fig. 5. FBXW11 mediates the expression of HIC1 via SIRT1.

A, B HCT116 and SW620 cells were transfected with recombinant lentiviral vectors carrying FBXW11, shRNAs targeting FBXW11, or corresponding controls. The A mRNA and B protein expressions of SIRT1 in transfected CRC cells were determined by qRT-PCR and western blotting, respectively. C, D HCT116 and SW620 cells were co-transfected with recombinant lentiviral vectors carrying FBXW11 (or empty control vectors) and vectors carrying HIC1 (or empty control vectors). The C mRNA and D protein expressions of SIRT1 were measured by qRT-PCR and western blotting, respectively. The protein expressions of HIC1 and FBXW11 were also detected. E The expressions of FBXW11, HIC1, and SIRT1 in human colorectal tumor tissue and adjacent non-tumorous tissue specimens were detected by immunohistochemistry (×400 magnification). Each set of slides (each column) was from the same subject. F The percentages of cells positively stained with FBXW11, HIC1, or SIRT1 were calculated. Their relationships were analyzed by Pearson’s correlation coefficient. ANOVA followed by Bonferroni’s post hoc test was used for statistical comparisons among multiple groups.

Fig. 6. FBXW11 mediates stem-cell-like properties of CRC cells via SIRT1.

HCT116 and SW620 cells were co-transfected with recombinant lentiviral vectors carrying FBXW11 (or empty control vectors) and shRNA targeting SIRT1 (or scrambled shRNA sequence). A Cell viability at different time points after transfection was evaluated by the MTT assay. B, E The wound-healing assay was used to assess the migration capacity of CRC cells following transfection. The wound closure at 24 h after scratching was measured. C, F The invasion capacity of transfected CEC cells was determined by the Transwell assay. The number of invaded cells per field at 24 h post plating was calculated. D, G The sphere-forming ability of CRC cells following transfection was evaluated by the sphere formation assay. The number of spheres with a diameter of over 50 μm was counted. H The protein expressions of Nanog, ALDH1, and Oct4 were assessed by western blotting. ANOVA followed by Bonferroni’s post hoc test was used for statistical comparisons among multiple groups.

Fig. 7. Effect of FBXW11 knockdown on colorectal tumor growth and liver metastasis.

A–C A mouse CRC xenograft model was established by injecting male BALB/c nude mice with transfected CRC cells into the subcutaneous region of the flank (n = 6 per group). Tumor growth was monitored for 35 days. A Tumor xenografts were collected and weighed at the end of the experiment. B Tumor volume was recorded every 5 days and a growth curve was plotted. C Tumor tissues were sectioned and immunostained FBXW11, SIRT1, HIC1, and Ki-67 (200× magnification). D–F A splenic liver metastasis model was established by injecting male BALB/c nude mice with transfected CRC cells into the spleen (n = 6 per group). At 3 weeks post implantation, mice were killed and livers were collected. D Black arrows indicate metastatic foci in the liver. E The number of metastases was counted and the liver was weighed. F Liver tissues were sectioned and stained for H&E. Black arrows indicate metastases. Student’s t-test was used for statistical comparisons between two groups.