FAM21C drives hepatocellular carcinoma invasion and migration by regulating recycling endosome-mediated MT1-MMP transport to promote invadopodia formation

The highly invasive and metastatic characteristics of hepatocellular carcinoma (HCC) are the primary contributors to the extremely poor prognosis of the patients. Therefore, exploring the molecular mechanisms underlying the invasion and metastasis of HCC is of great clinical and scientific significance. This study aims to investigate the role and mechanism of FAM21C in promoting invadopodia formation through regulating recycling endosome-mediated transport of membrane-type 1 matrix metalloproteinase(MT1-MMP) during HCC invasion and migration.

① A retrospective cohort study was conducted on 96 patients who were pathologically diagnosed with HCC and underwent surgical treatment in Department of Hepatobiliary Surgery of the First Affiliated Hospital of Army Medical University between January 2010 and December 2012. Their HCC tissue specimens were collected and fabricated into tissue microarrays, and clinical pathological data and follow-up information were summarized. According to FAM21C expression, the 96 HCC patients were divided into high and low FAM21C expression groups. The relationship of FAM21C expression level with clinicopathological characteristics and prognosis of HCC patients was statistically analyzed. Immunohistochemical staining was used to detect the expression of FAM21C, MT1-MMP, and recycling endosome marker Rab11a in HCC tissues, and the correlation of their expression was analyzed. ② In in vitro experiments, FAM21C was knocked down and overexpressed in HCCLM3 and Huh7 cells using lentiviral vectors, and the expression of FAM21C was detected by Western blotting. ③ Transwell assay was performed to determine the effects of FAM21C on the invasive and migratory abilities of HCC cells. ④ Western blotting was applied to detect the effect of FAM21C expression changes on the content of filamentous actin(F-actin) in HCC cells. ⑤ A Flag-tagged FAM21C-Flag fusion protein was constructed, and co-immunoprecipitation(Co-IP) was employed to verify the interaction between FAM21C and CAPZA1. ⑥ A nonsense mutant of the FAM21C-CAPZA1 binding site(FAM21CΔ) was constructed. Western blotting and immunofluorescence staining were carried out to investigate its effects on the content and morphology of F-actin. ⑦ Western blotting was used to detect the impact of FAM21C expression changes on the expression of the recycling endosome marker Rab11a, and Co-IP was conducted to examine the interaction between MT1-MMP and Rab11a. ⑧ Immunofluorescence staining was performed to assess the effect of FAM21C expression changes on F-actin remodeling, as well as the levels and distribution of Rab11a, MT1-MMP, and the invadopodia marker Cortactin in HCC cells. ⑨ Matrix degradation assay was conducted to evaluate the impact of FAM21C expression on invadopodia formation. ⑩ A nude mouse model of liver orthotopic tumor(n=5) was constructed to observe the number of intrahepatic micrometastatic lesions and investigate whether FAM21C promotes HCC invasion and metastasis.

① The 5-year survival rate was significantly lower in the FAM21C-high group than in the FAM21C-low group (P=0.0097). Further analyses showed that FAM21C expression was positively correlated with Rab11a expression(r=0.3280, P<0.0001) and MT1-MMP expression(r=0.5384, P<0.0001). ② FAM21C-overexpressing and FAM21C-knockdown stable cell lines were constructed in HCCLM3 and Huh7 cells (P<0.05). ③ Transwell assay results showed that knockdown of FAM21C suppressed the invasive ability of HCCLM3 and Huh7 cells (P<0.05), whereas overexpression of FAM21C enhanced invasive ability of HCCLM3 and Huh7 cells (P<0.05). ④ Western blotting and immunofluorescence staining displayed that upregulation of FAM21C promoted the remodeling of cytoskeletal F-actin in HCCLM3 cells, while its downregulation inhibited this process (P=0.0131). ⑤ Co-IP indicated that FAM21C interacted with CAPZA1 in HCCLM3. ⑥ Western blotting and immunofluorescence results revealed no significant change in F-actin level in HCCLM3 cells overexpressing FAM21CΔ. ⑦ Western blot analysis demonstrated that FAM21C could regulate Rab11a expression. Co-IP confirmed the interaction between Rab11a and MT1-MMP. ⑧ Immunofluorescence results indicated that downregulation of FAM21C in Huh7 cells inhibited the transport of Rab11a-positive recycling endosomes toward the cell membrane, whereas FAM21C upregulation in HCCLM3 cells promoted the co-localization of MT1-MMP and Cortactin in invadopodia. ⑨ Matrix degradation assay further displayed that FAM21C downregulation inhibited invadopodia formation (P=0.0007), while FAM21C overexpression promoted the formation (P=0.0107). ⑩ In in vivo experiments, FAM21C downregulation significantly inhibited HCC invasion and metastasis in the orthotopic liver tumor model(P=0.0462).

FAM21C can regulate F-actin remodeling and transport MT1-MMP to invadopodia via recycling endosomes to promote invadopodium formation, thereby enhancing HCC invasion and metastasis.