Furin Overexpression Suppresses Tumor Growth and Predicts a Better Postoperative Disease-Free Survival in Hepatocellular Carcinoma

<div><p>Furin is a member of the pro-protein convertase family. It processes several growth regulatory proteins into their active forms, which are critical to tumor progression, metastasis, and angiogenesis. Furin over-expression could occur in liver cancer and a previous study showed that over-expression of furin promoted HepG2 cell invasion in tail vein xenograft models. However, the clinical relevance of furin expression in hepatocellular carcinoma (HCC) remained unknown. Surprisingly, in a postoperative survival analysis for HCC patients, it was found that the tumor/non-tumor (T/N) ratio of furin expression ≥ 3.5 in HCC tissues predicted a better postoperative disease-free survival (DFS) (<em>P</em> = 0.010; log-rank test). Furthermore, subcutaneous xenograft experiments demonstrated a significant suppression effect of tumor growth in the furin-overexpressed xenografts (Huh7-Furin) compared to the mock control. Administration of a synthetic furin inhibitor for inhibition of the pro-protein convertase activity, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decRVKR-CMK), to the Huh7-Furin xenograft bearing mice restored the repression effect of tumor growth. In contrast, administration of decRVKR-CMK to the mock Huh7 xenograft bearing mice showed no change in growth rate. In conclusion, furin overexpression inhibited HCC tumor growth in a subcutaneous xenograft model and predicted a better postoperative DFS in clinical analysis.</p> </div

Inhibition of furin activity by decRVKR-CMK in Huh7-Fruin cells.

<p>(A) Expression levels of furin, pro-TGFβ1 and TGFβ1 in Huh7-Neo and Huh7-Fruin cells were showed by immunoblot. Gelatin zymography was performed to detect the MMP2 activities. (B) Immunoblot was performed to determine the pro-TGFβ1 and TGFβ1 expression levels and gelatin zymography was performed to determine the MMP2 activities in the absence (-) and presence (+) of decRVKR-CMK.</p

Treatment with decRVKR-CMK promoted cell viability in Huh7-Furin xenograft tumor.

<p>(A) Animal experimental protocol was shown. (B to D) Comparisons of the tumor volumes between Huh7-Furin xenografts with or without decRVKR-CMK. (E) Gelatin zymography was performed to detect the MMP2 activities. (F) Immunohistochemistry analysis for xenograft tumor section. Immunostain for furin (i and ii) and Ki-67 (vii and viii); H&E stain (iii and iv); TUNEL assay (v and vi) (200X). The areas marked by yellow dashed lines in (iii) and (iv) were necrosis areas.</p

Differential expression of cell proliferation and cell death regulatory molecules.

<p>The expression level of furin, pro-TGFβ1, and several cell proliferation and cell death regulatory molecules between Huh7-Neo and Huh7-Furin xenograft tumors after administration with or without decRVKR-CMK was analyzed by immunoblot.</p

No effects of cell viability by treatment with decRVKR-CMK in Huh7-Neo xenograft tumor.

<p>(A to C) Comparisons of the tumor volumes between Huh7-Neo xenografts with or without decRVKR-CMK. (D) Immunohistochemistry analysis for xenograft tumor section. Immunostain for furin (i and ii) and Ki-67 (v and vi); TUNEL assay (iii and iv) (200X).</p

A dual and contradictory role of furin in HCC.

<p>(A) Furin overexpression in 20 pairs of representative cancerous (T) and adjacent noncancerous (N) tissues demonstrated by immunoblot analysis. (B) Furin underexpressed in the 8 representative paired HCC tissues. (C) Expression of furin assessed by IHC staining in noncancerous and cancerous liver tissue (i and ii). Specificity of the IHC staining was verified by blocking of the primary antibody with recombinant furin protein (iii and iv). (D) Disease-free survivals in HCC patients expressing different levels of furin (Kaplan-Meier survival analysis).</p

Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective autophagy

<p>Recent studies have demonstrated a critical association between disruption of cellular thyroid hormone (TH) signaling and the incidence of hepatocellular carcinoma (HCC), but the underlying mechanisms remain largely elusive. Here, we showed that disruption of TH production results in a marked increase in progression of diethylnitrosamine (DEN)-induced HCC in a murine model, and conversely, TH administration suppresses the carcinogenic process via activation of autophagy. Inhibition of autophagy via treatment with chloroquine (CQ) or knockdown of ATG7 (autophagy-related 7) via adeno-associated virus (AAV) vectors, suppressed the protective effects of TH against DEN-induced hepatic damage and development of HCC. The involvement of autophagy in TH-mediated protection was further supported by data showing transcriptional activation of DAPK2 (death-associated protein kinase 2; a serine/threonine protein kinase), which enhanced the phosphorylation of SQSTM1/p62 (sequestosome 1) to promote selective autophagic clearance of protein aggregates. Ectopic expression of DAPK2 further attenuated DEN-induced hepatoxicity and DNA damage though enhanced autophagy, whereas, knockdown of DAPK2 displayed the opposite effect. The pathological significance of the TH-mediated hepatoprotective effect by DAPK2 was confirmed by the concomitant decrease in the expression of THRs and DAPK2 in matched HCC tumor tissues. Taken together, these findings indicate that TH promotes selective autophagy via induction of DAPK2-SQSTM1 cascade, which in turn protects hepatocytes from DEN-induced hepatotoxicity or carcinogenesis.</p

Association between postoperative survivals and expression levels of prognostic miRNAs.

<p>(A) Comparison of postoperative RFS between patients with higher and lower expression levels of miR-15a, miR-486-3p, miR-381, miR-30c, miR-155, miR-432, miR-15b and miR-30b, respectively. (B) Comparison of postoperative OS between patients with higher and lower expression levels of miR-29a, miR-486-3p and miR-876-5p. (C) Combination of six miRNA (miR-155, miR-15a, miR-432, miR-486-3p, miR-15b and miR-30b) markers as a prognostic score for RFS in HCC. ‘n’ represents the number of samples in each miRNA marker. The <i>P</i> value from the log rank test is shown in each panel.</p

The expression levels of five miRNAs predict a high risk of tumor recurrence in HCC tumor and adjacent non-tumor liver tissues.

<p>The expression levels of miR-30c (A), miR-155 (B), miR-432 (C), miR-15b (D), and miR-30b (E) in 11 pairs of HCC tumor and non-tumor liver tissues (to the left). The expression levels of five miRNA in HCC non-tumor liver tissues (BP, n = 6; PP, n = 6) or tumor liver tissue (BP, n = 6; PP, n = 5) with known prognosis (to the right). *, <i>P</i><.05; **, <i>P</i><.01 (<i>^a</i> Wilcoxon Signed Ranks Test; <i>^b</i> Mann-Whitney test).</p

Inhibition of tumor growth by miR-155 expression knockdown in subcutaneous hepatoma xenografts.

<p>(A and B) Comparisons of the tumor volumes between hepatoma xenografts with or without miR-155 knockdown. Xenografts were derived from J7 (A) and Mahlavu (B) cells, respectively. Left panels, the final dissected xenograft tumors. Middle panels, comparisons of the growth curves of xenografts. Tumor volumes were measured at days 7 to 14 in J7 xenografts and at days 14 to 36 in Mahlavu xenografts after subcutaneous injection of cells. *<i>P</i><>\raster="rg1"<>.01; **<i>P</i><>\raster="rg1"<>.001; Red triangle, with miR-155 knockdown; Solid squares, without miR-155 knockdown. Right panels, comparisons of the mean tumor weights. Shaded bars, with miR-155 knockdown; Empty bars, without miR-155 knockdown. (C) Comparisons of cell apoptosis assessed by TUNEL assays between xenografts with (miRZip-155) or without (miRZip) miR-155 knockdown. Upper panels, J7 xenografts; Lower panels, Mahlavu xenografts. (D) Comparisons of Ki-67 expression assessed by immunohistochemistry between xenografts with (miRZip-155) or without (miRZip) miR-155 knockdown. Upper panels, J7 xenografts; Lower panels, Mahlavu xenografts. (E) Comparisons of CDK2, cyclin E and Bcl-2 expression levels assessed by immunoblot between xenografts with or without miR-155 knockdown. GAPDH served as a loading control. C1 and C2, xenografts without miR-155 knockdown; E1 and E2, xenografts with miR-155 knockdown. Quantitative data (by densitometer) were shown in the right.</p