FBXW7 Reduces the Cancer Stem Cell-Like Properties of Hepatocellular Carcinoma by Regulating the Ubiquitination and Degradation of ACTL6A

Abstract

Cancer stem cells (CSCs) comprise a subset of tumor cells that can initiate tumorigenesis and promote tumor advance. A previous study showed that the expression of FBXW7 in hepatocellular carcinoma (HCC) clinical samples was lower than that in the adjacent nontumor tissues and was negatively correlated with the invasion and migration of HCC cells. However, the biological characteristics and the underlying molecular mechanisms of FBXW7 in HCC stemness are yet to be elucidated. In present study, we found that FBXW7 participates in the self-renewal, tumorigenicity, sorafenib therapy, and stem cell-like properties of HCC cells in vivo and in vitro. The upregulation of FBXW7 inhibited the stemness and reduced the tumorigenicity and drug resistance of HCC cells. Mechanistically, proteins binding to FBXW7 were identified by coimmunoprecipitation and protein colocalization assays. We confirmed ACTL6A as a novel downstream target for FBXW7. The in vivo ubiquitination assay showed that FBXW7 repressed HCC malignancy by regulating the oncogenic activity of ACTL6A in a ubiquitin-dependent manner. Furthermore, we found that ACTL6A overexpression inversed the self-renewal abilities and tumorigenic abilities depressed by overexpressing FBXW7. The current findings suggested that FBXW7 reduces the stemness of HCC cells by targeting and degrading ACTL6A and provides a novel target for the diagnosis and treatment of HCC.

Figure 1

FBXW7 overexpression inhibits the stem cell-like properties of HCC cells. (a) FBXW7 expression was detected in spheres and their adherent HCC cells by RT-qPCR and Western blot. (b) RT-qPCR and Western blot analysis of mRNA and protein level of FBXW7 in the cell lines infected with LV-FBXW7 and LV-Vector. (c, d) CD133 surface marker expression was analyzed in the two cell lines by flow cytometry and IF staining. (e) Representative images and quantitative analysis of spheres. Scale bar: 100 mm. (f, g) Pluripotent transcription factors, including Nanog, SOX2, OCT4, and c-Myc, were analyzed by q-PCR (f) and Western blot (g). (h) Flow cytometry showed the effect of FBXW7 on the cell cycle in MHCC-97H and Huh7 cells. (i) Western blot analysis of cyclin E1 and p21 after FBXW7 alternation in MHCC-97H and Huh7 cells. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 2

FBXW7 downregulation promotes the stem cell-like properties of HCC cells. (a) RT-qPCR and Western blot analysis of mRNA and protein level of FBXW7 in the cell lines infected with LV-shFBXW7#1, #2, and shNC. (c–h) The experimental method is the same as Figure 1. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 3

FBXW7 inhibits the tumorigenesis in vivo and interferes with sorafenib resistance of HCC cells. (a) Images of xenografts in nude mice 6 weeks after subcutaneous injection of indicated cells. (b, c) Xenograft tumors growth curve and weight. Tumor volume was calculated by (length × width²)/2. (d) IHC staining of FBXW7 and pluripotency-associated markers in respective xenograft tumor tissues. (e) FBXW7 overexpression increased the sensitivity to sorafenib, while FBXW7 knockdown reduced the sensitivity to sorafenib. (f) The influence of FBXW7 or sorafenib on the sphere formation in MHCC-97H or Huh7 cells. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 4

E3 ubiquitin ligase FBXW7 interacts with ACTL6A. (a) Cell extracts from MHCC-97 cells with Flag-FBXW7 expression were immunopurified on an anti-Flag affinity column and eluted with Flag peptides. The eluates were analyzed by mass spectrometry. (b) Predictive analysis of E3 ubiquitin ligases that might target ACTL6A was performed using UbiBrowser database. (c) Co-IP and immunoblotting detected the interaction between FBXW7 and ACTL6A. (d) IF staining of FBXW7 and ACTL6A in the above cells. (e) Western blot was used to detect the effect of FBXW7 on the ACTL6A protein level. (f, g) Comparison of the expression of pluripotency-associated markers in HCC cells of different groups by Western blot. (h, i) Self-renewal abilities were measured by sphere formation assays in MHCC-97H and Huh7 cells transfected with LV-ACTL6A or shACTL6A#2. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01.

Figure 5

FBXW7 negatively regulates ACTL6A stability via the ubiquitin-proteasome pathway. (a) RT-qPCR analysis of ACTL6A transcript level in MHCC-97H and Huh7 cells. (b) The above cells were incubated with MG132 (10 μM) for 12 h, and total protein was extracted and subjected to Western blot. (c–f) MHCC-97H and Huh7 cells were treated with CHX (50 μg/mL), harvested, and immunoblotted for ACTL6A and GAPDH. Quantification of the ACTL6A protein levels relative to GAPDH expression. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01.

Figure 6

Ubiquitination of ACTL6A in MHCC-97H and Huh7 cells was regulated by FBXW7 overexpression or knockdown in the presence of MG132 (10 μM, 12 h) detected by IP and immunoblotting. (a, c) MHCC-97H cells. (b, d) Huh7 cells.

Figure 7

FBXW7 inhibits HCC cell stemness and malignancy by downregulating the levels of ACTL6A. (a) Flow cytometry analysis of the positivity rate of CD133 in different groups. (b, c) Representative images and quantification of the size and generation rate of tumor spheres. Scale bars, 100 μm. (d) Western blot was used to detect the expression of pluripotent transcription factors in HCC cells with FBXW7 or ACTL6A. (e) Representative images and quantification of cell cycle distribution detected by flow cytometry. (f) Expression of cell cycle-related proteins detected by Western blot. Graph represents mean ± SD; ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 8

FBXW7 regulates ACTL6A expression levels to inhibit HCC growth in vivo and resistance of sorafenib in vitro. (a–c) The indicated engineered MHCC-97H or control cells were injected into subcutaneous and representative images of tumors (a), tumor growth curve (b), and tumor weight (c). (d) The expression of ACTL6A, CD133, Nanog, and OCT in xenograft tumor tissues by IHC staining. (e) Overexpression of ACTL6A in MHCC-97H decreased the sensitivity to sorafenib, which was rescued by FBXW7 upregulation. (f) Effect of ACTL6A overexpression combined with sorafenib on tumor spheroid formation in MHCC-97H cells. Graph represents mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.