ANKFN1 plays both protumorigenic and metastatic roles in hepatocellular carcinoma

Abstract

Ankyrin repeat and fibronectin type III domain containing 1 (ANKFN1) is reported to be involved in human height and developmental abnormalities, but the expression profile and molecular function of ANKFN1 in hepatocellular carcinoma (HCC) remain unknown. This study aimed to evaluate the clinical significance and biological function of ANKFN1 in HCC and investigate whether ANKFN1 can be used for differential diagnosis in HCC. Here, we showed that ANKFN1 was upregulated in 126 tumor tissues compared with adjacent nontumorous tissues in HCC patients. The upregulation of ANKFN1 in HCC was associated with cirrhosis, alpha-fetoprotein (AFP) levels and poor prognosis. Moreover, silencing ANKFN1 expression suppressed HCC cell proliferation, migration, invasion, and metastasis in vitro and subcutaneous tumorigenesis in vivo. However, ANKFN1 overexpression promoted HCC proliferation and metastasis in an orthotopic liver transplantation model and attenuated the above biological effects in HCC cells. ANKFN1 significantly affected HCC cell proliferation by inducing G1/S transition and cell apoptosis. Mechanistically, we demonstrated that ANKFN1 promoted cell proliferation, migration, and invasion via activation of the cyclin D1/Cdk4/Cdk6 pathway by stimulating the MEK1/2-ERK1/2 pathway. Moreover, ANKFN1-induced cell proliferation, migration, and invasion were partially reversed by ERK1/2 inhibitors. Taken together, our results indicate that ANKFN1 promotes HCC cell proliferation and metastasis by activating the MEK1/2-ERK1/2 signaling pathway. Our work also suggests that ANKFN1 is a potential therapeutic target for HCC.

Fig. 1. ANKFN1 is frequently overexpressed and associated with a poor prognosis in HCC patients.

A ANKFN1 was found to be overexpressed in HCC tissues versus normal tissues according to analysis of TCGA HCC data using the R language package “limma”. B qRT–PCR analysis of ANKFN1 mRNA expression levels in HCC tissue compared with paracarcinoma tissue. C qRT–PCR analysis of ANKFN1 mRNA expression levels in HCC cells (SMMC-7721, HLE, Hep G2, HuH7, BEL-7404) compared with normal liver cells (LO2). D Representative images of ANKFN1 IHC staining in HCC tissues of high and low level. Original magnification: ×100. E HCC tissues stratified by the IHC staining index. F Kaplan–Meier analysis of the OS of 126 HCC patients stratified by ANKFN1 expression. ANKFN1 ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma, TCGA The Cancer Genome Atlas, qRT‐PCR quantitative real‐time polymerase chain reaction, IHC immunohistochemistry. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. Knockdown of ANKFN1 suppressed HCC cell growth through induction of G1-S cell cycle arrest and cell apoptosis.

A qRT‐PCR assay analyses of ANKFN1 expression levels in SMMC-7721 and HLE cells infected with sh-ANKFN1-RNA. B Immunofluorescence assay analyses of ANKFN1 expression levels in SMMC-7721 and HLE cells infected with sh-ANKFN1-RNA. Scale bars, 50 μm, 20 μm. C The effect of ANKFN1 knockdown on SMMC-7721 and HLE cell proliferation was assessed by the CCK-8 assay. D The effect of ANKFN1 knockdown on SMMC-7721 and HLE cell proliferation was assessed by the BrdU assay. E and F Flow cytometry analysis of cell apoptosis and cycle distribution in SMMC-7721 and HLE cells with or without ANKFN1 knockdown. ANKFN1 ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma, qRT‐PCR quantitative real‐time polymerase chain reaction, IHC immunohistochemistry. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Knockdown of ANKFN1 inhibits HCC cell migration and invasion in vitro.

A The results of a wound-healing assay showed the effects of ANKFN1 on the cell migration ability of sh‐ANKFN1 #2‐ and sh‐ANKFN1 #3‐infected SMMC-7721 cells. B The results of a wound-healing assay showed the effects of ANKFN1 on the cell migration ability of sh‐ANKFN1 #2‐ and sh‐ANKFN1 #3‐infected HLE cells. C The effect of ANKFN1 knockdown on SMMC-7721 cell migration and invasion was assessed by a transwell assay. D The effect of ANKFN1 knockdown on HLE cell migration and invasion was assessed by a transwell assay. ANKFN1 ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. The downregulation of ANKFN1 inhibits HCC growth and promotes apoptosis in nude mice.

A Representative images of the subcutaneous tumors formed in nude mice between the scramble and ANKFN1-knockdown groups. B Statistical comparison of the difference in tumor volume between the scramble and ANKFN1-knockdown groups. C Statistical comparison of the difference in tumor weight between the scramble and ANKFN1-knockdown groups. D Bioluminescence images of the subcutaneous tumors formed in nude mice between the scramble and ANKFN1-knockdown groups. E IHC analysis of HE and Ki-67 expression levels in xenograft tumor tissues in the scramble and ANKFN1-knockdown groups. Scale bars, 200 μm. F TUNEL assay for cell apoptosis in tumor tissues developed from SMMC-7721 cells with or without ANKFN1 knockdown. Scale bars, 20 μm. G Western blot analysis of the expression of the MAPK signaling pathway proteins p-ERK1/2 and p-JNK1/2 and c-Myc, Cdk6, Cdk4, PCNA, and RhoA in SMMC-7721 and HLE cells with or without ANKFN1 knockdown. ANKFN1 ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Overexpression of ANKFN1 promotes HCC cell growth, migration, and invasion in vitro.

A qRT‐PCR assay analyses of ANKFN1 overexpression levels in SMMC-7721 and HLE cells infected with or without LV-ANKFN1. B Immunofluorescence assay analyses of ANKFN1 overexpression levels in SMMC-7721 and HLE cells infected with or without LV-ANKFN1. C The effect of ANKFN1 overexpression on SMMC-7721 and HLE cell proliferation was assessed by the CCK-8 assay. D The effect of ANKFN1 overexpression on SMMC-7721 and HLE cell proliferation was assessed by the BrdU assay. E The effect of ANKFN1 overexpression on the apoptosis of SMMC-7721 and HLE cells was analyzed by flow cytometry. F The cell cycle distribution of ANKFN1-overexpressing SMMC-7721 and HLE cells was analyzed by flow cytometry. G The results of a wound-healing assay showed the effects of ANKFN1 on the cell migration ability of LV-Ctrl- and LV-ANKFN1-infected SMMC-7721 and HLE cells. H The effect of ANKFN1 overexpression on SMMC-7721 and HLE cell migration and invasion was assessed by a transwell assay. ANKFN1 ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6. Overexpression of ANKFN1 promotes HCC migration and metastasis in vivo.

A Liver tissues from animals with tumor xenografts inoculated with SMMC-7721 cell lines stably expressing ANKFN1. B HE staining of orthotopic liver transplantation tumors. C IHC analysis of HE and Ki-67 expression levels in xenograft tumor tissues in the scramble and ANKFN1-overexpression groups. Scale bars, 200 μm. D Statistical graph of the number and area of liver metastases in nude mice. E TUNEL assay for cell apoptosis in tumor tissues developed from SMMC-7721 cells with or without ANKFN1 overexpression. Scale bars, 20 μm. F Western blot analysis of the expression of the MAPK signaling pathway proteins p-ERK1/2 and p-JNK1/2 and c-Myc, Cdk6, Cdk4, PCNA, and RhoA in SMMC-7721 and HLE cells with or without ANKFN1 overexpression. ANKFN1, ankyrin repeat and fibronectin type III domain containing 1, HCC hepatocellular carcinoma; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7. ANKFN1 downregulation is mainly mediated by the downregulation of p-ERK in HCC cells.

A and B qRT–PCR analyses of the expression of ANKFN1 in LV-ANKFN1-SMMC-7721 cells after induction with the ERK inhibitor FR180204 at 48 h and 72 h. C The effect of FR180204 on SMMC-7721 cells infected with LV-ANKFN1 cell proliferation was assessed by the CCK-8 assay at 24 h, 48 h, and 72 h. D The effect of FR180204 on SMMC-7721 cells infected with LV-ANKFN1 cell proliferation was assessed by the BrdU assay at 72 h. E and F Flow cytometry analysis of the cell cycle distribution in SMMC-7721 cells infected with LV-ANKFN1 and induced with the ERK inhibitor FR180204 at 24 h, 48 h, and 72 h. G Western blot analysis of p-ERK, c-Myc, cyclin D1, Cdk4, Cdk6, and PCNA levels in SMMC-7721 cells infected with LV-ANKFN1 and induced with the ERK inhibitor FR180204 at 24 h, 48 h, and 72 h. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 8. Diagram of the mechanism of the ANKFN1 gene in HCC.

ANKFN1 expression promoted proliferation and metastasis and inhibited apoptosis in HCC in vitro and in vivo by activating the MEK/ERK/c-Myc/cyclin D1/Cdk4/Cdk6 signaling pathway. ANKFN1 can regulate HCC migration and invasion directly or indirectly via the RhoA/ROCK/JNK pathway.