FNDC3B promotes cell migration and tumor metastasis in hepatocellular carcinoma

Abstract

Recurrence and metastasis are common in hepatocellular carcinoma (HCC) and correlate with poor prognosis. We investigated the role of fibronectin type III domain containing 3B (FNDC3B) in HCC metastasis. Overexpression of FNDC3B in HCC cell lines enhanced cell migration and invasion. On the other hand, knockdown of FNDC3B using short-hairpin RNA reduced tumor nodule formation in both intra- and extra-hepatic metastasis. High levels of FNDC3B were observed in metastatic HCCs and correlated with poor patient survival and shorter recurrence time. Mutagenesis and LC-MS/MS analyses showed that FNDC3B promotes cell migration by cooperating with annexin A2 (ANXA2). Furthermore, FNDC3B and ANXA2 expression correlated negatively with patient survival. Our results indicate that FNDC3B behaves like an oncogene by promoting cell migration. This suggests FNDC3B could serve as a biomarker and therapeutic target for HCC metastasis.

Keywords: FNDC3B; hepatocellular carcinoma; metastasis.

Figure 1. FNDC3B enhanced cell migration and invasion

(A) Migration assay for overexpressed or knockdown cells utilized a transwell system. (B) Cell invasion assays (similar to the migration assays) with collagen pre-coated inserts (10 μg/cm²). **and*** represent p < 0.005 and p < 0.0005, respectively.

Figure 2. Knockdown of endogenous FNDC3B expression suppressed tumor metastasis in nude mice model

Metastatic nodules in nude mice by (A) orthotopical intrahepatic or (B) tail-vein injection of Mahlavu cells. The white arrows indicate metastatic tumors. The metastatic number in liver and lung was calculated in nude mice. *represents p < 0.05. (C) Immunohistochemical staining of FNDC3B in liver tumor model section. Scale bar = 400 μm.

Figure 3. FNDC3B overexpression correlates with HCC metastasis and patient survival

(A) FNDC3B levels in tissue arrays detected by FNDC3B antibodies in metastatic and primary HCC. (B) Differential immunohistochemical (IHC) score of FNDC3B between metastatic and primary HCC. (C) Overall survival and (D) recurrence-free survival of HCC patients categorized accordingly to the expression of FNDC3B. ↑ and ↓ stand for high (T/N ≥ 1.5) and low (T/N < 1.5) expression groups, respectively.

Figure 4. FNIII domains 1–4 were essential for FNDC3B to enhance cell migration and invasion

(A) The construction map of variant FNIII domain deletion mutants of FNDC3B. (B) Cellular localization of full-length FNDC3B and variant domain deletion mutants assessed by immunofluorescence. Calnexin and Flag-FNDC3B visualized with TRITC-conjugated calnexin (red) and anti-Flag antibody (green), respectively. (C) Migration assays for FNIII domain deletion mutants performed using the transwell system. ***represent p < 0.0005.

Figure 5. ANAX2 interacting FNIII domains 1–4 of FNDC3B were essential for FNDC3B enhanced cell migration

(A) Co-immunoprecipitation of Flag-FNDC3B and ANAX2, using lysates from Huh7 cells transfected with Flag-FNDC3B for immunoprecipitation (IP) with anti-Flag. (B) Migration assay for FNDC3B overexpressed Huh7 cells treated with ANXA2 shRNA and ANXA2 overexpressed Huh7 cells treated with FNDC3B shRNA. (C) Vector only and FNDC3B overexpression Huh7 cells observed by confocal microscopy. F-actin and Flag-FNDC3B were visualized with TRITC-conjugated phalloidin (red) and anti-Flag antibody (green), respectively. (D) Migration assay for FNDC3B overexpressed Huh7 cells treated with 1 mM/mL Y27632. ***represents p < 0.005. (E) Overall survival curves of HCC patients categorized according to FNDC3B and ANXA2 expression.↑ and ↓ stand for high (T/N ≥ 1.5) and low (T/N < 1.5) expression groups, respectively.