Targeted disruption of fibrinogen like protein-1 accelerates hepatocellular carcinoma development

Abstract

Fibrinogen like protein-1 (Fgl1) is a predominantly liver expressed protein that has been implicated as both a hepatoprotectant and a hepatocyte mitogen. Fgl1 expression is decreased in hepatocellular carcinoma (HCC) and its loss correlates with a poorly differentiated phenotype. To better elucidate the role of Fgl1 in hepatocarcinogenesis, we treated mice wild type or null for Fgl1 with diethyl nitrosamine and monitored for incidence of hepatocellular cancer. We find that mice lacking Fgl1 develop HCC at more than twice the rate of wild type mice. We show that hepatocellular cancers from Fgl1 null mice are molecularly distinct from those of the wild type mice. In tumors from Fgl1 null mice there is enhanced activation of Akt and downstream targets of the mammalian target of rapamycin (mTOR). In addition, there is paradoxical up regulation of putative hepatocellular cancer tumor suppressors; tripartite motif-containing protein 35 (Trim35) and tumor necrosis factor super family 10b (Tnfrsf10b). Taken together, these findings suggest that Fgl1 acts as a tumor suppressor in hepatocellular cancer through an Akt dependent mechanism and supports its role as a potential therapeutic target in HCC.

Keywords: Diethyl nitrosamine; Fibrinogen like protein-1; Hepatocellular carcinoma; Hepatoprotecatant; Mitogen.

Fig. 1

Fgl1 null mice have a higher incidence of carcinoma compared to wild type. A). Representative images at 8 months after administration of diethyl nitrosamine. H&E staining of a lesion (left). Arrows denote boundary with normal hepatic parenchyma. B) Ki-67 staining shows evidence of proliferating cells. C) Representative non-tumor liver from Fgl1WT mouse. D) Large tumor burden in an Fgl1KO mouse E) Marked decrease in weight of Fgl1KO mouse.

Fig. 2

Liver Tumors are HCCs. A and C) H&E stains (original magnification x100 and x600, respectively) of non-tumor liver from Fgl1WT. H&E stains B (x100) D and E (x600) of Fgl1KO tumors. Note the abnormal architecture in the tumors with nested and pseudoglandular patterns (B; arrows) as well as mitotic figures (D, arrow), variation in nuclear size and multinucleate cells (E, arrow). F and G) Immunohistochemistry showing positive nuclear staining of Ki-67 and PCNA respectively in tumors of Fgl1KO. H and I) Tumors from Fgl1KO have elevated transcripts for HCC biomarker alfa-feto protein and oncogene UHRF1 compared to non-tumor liver (n = 4 livers, n = 6 tumors). J) Expression of UHRF1 protein in normal liver compared to marked increase in liver tumors (graph is from n = 3 livers, n = 6 tumors) (*p < 0.05). K) Prevalence of HCC is approximately 2.5 times in Fgl1KO compared to Fgl1WT.

Fig. 3

Fgl1KO Tumors have increased activation of Akt dependent mTOR Signaling. A) Total and phospho p38 (top two panels) are unchanged in Fgl1WT compared to Fgl1KO tumors. Likewise total and phospho Yap levels (panels 3 and 4) pathways are not differentially activated in Fgl1KO and Fgl1WT tumors. B) Increased phosphorylation of Akt in tumors from Fgl1KO, compared to Fgl1WT tumors. Top). Representative blot (n = 4 for both Fgl1WT and Fgl1KO). Bottom). Quantitation shows nearly 6-fold increase in ratio of pAkt/Total Akt. C) Enhanced phosphorylation of 4EBP1 in tumors from Fgl1KO. Top). Representative blot. Bottom) quantitation shows a 2-fold difference in phosphorylated 4EPB1 to total 4EBP1 between Fgl1KO and Fgl1WT tumors. Data for graphs is from n = 6 Fgl1WT and n = 7 Fgl1KO tumors) (**p < 0.001). D) Increased phosphorylation on serine 371 of p70S6K in tumors from Fgl1KO. Graphs represent densitometric quantitation of p-P70S6K/total p70S6K. n = 4 for Fgl1WT and n = 5 for Fgl1KO. (*p < 0.05).

Fig. 4

Candidate genes from the human chromosome 8 tumor suppressor cluster are upregulated in Fgl1KO liver tumors. A) There are no differences in gene expression at 6 months after DEN administration when tumors and proliferative foci are absent (n = 6 in each group) B) Increased expression of Trim35 and Tnfrsf10b is exclusive to tumor tissue of Fgl1KO, with no enhancement in non-tumor liver tissue at 12 month of DEN + Pb treatment (left, n = 9 tumors, n = 12 livers). C) Increased expression of Trim35 and Tnfrsf10b expression is seen in Fgl1KO tumors only not those from Fgl1WT. (n = 5 in each group) (*p < 0.05).