Sorafenib treatment during partial hepatectomy reduces tumorgenesis in an inflammation-associated liver cancer model

Abstract

The long-term prognosis after resection of hepatocellular carcinoma (HCC), which is one of the treatment options for early-stage HCC, remains unsatisfactory as a result of a high incidence of disease recurrence. Recent studies performed in murine models revealed a link between liver regeneration under chronic inflammation and hepatic tumorigenesis. Sorafenib is a potent drug for advanced HCC with multikinase inhibition activity. We propose that inhibition of signal transduction pathways which are activated during hepatectomy, using Sorafenib, will reduce accelerated tumorigenesis. To test this hypothesis, we studied the Mdr2-knockout (KO) mouse strain, a model of inflammation-associated cancer, which underwent partial hepatectomy (PHx) at three months of age, with or without Sorafenib.Here we show that Sorafenib treatment during PHx inhibited different signal transduction pathways at the multikinase levels, but did not result in increased morbidity or mortality. At the early stages after PHx, Sorafenib treatment had no effect on the course of proliferation, apoptosis and DNA repair in the regenerating liver, but resulted in decreased stellate cells activation and inflammatory response. Finally, we show that Sorafenib treatment during PHx at three months of age resulted in decreased fibrosis and tumor formation at 8.5 months.In conclusion our study indicates that short-term Sorafenib treatment during PHx is safe and effective in inhibiting inflammation-associated cancer, and is therefore a potential strategy for recurrence prevention in patients with early-stage HCC treated with PHx.

Keywords: Mdr2 knockout; Sorafenib; hepatectomy; liver cancer; stellate cells.

Figure 1. Short-term Sorafenib treatment during PHx inhibits different signaling pathways in the chronic inflamed liver

Mdr2- KO mice were subjected to PHx at three months of age and treated with Sorafenib or Cremophor immediately and two hours following surgery. A. Experimental schedule B. Down-regulated intracellular signaling pathways in livers of short-term Sorafenib treated mice four hours post PHx compared to controls. RNA from short-term Sorafenib or Cremophor treated mice four hours post PHx was analyzed using RNA sequencing. The graph shows the fold reduction of signaling pathways obtained by short-term Sorafenib treatment compared to the values in the control Cremophor-treated mice. C. Total hepatic proteins from short-term Sorafenib or Cremophor treated mice four hours and four days post PHx were analyzed using reverse phase protein array (RPPA). The graph shows the percentage reduction of signaling proteins obtained by short-term Sorafenib treatment. The data are presented as percentage decrease compared with the values in control Cremophor-treated mice. Four mice of each group were subjected to RPPA analysis with 166 antibodies. D–E Western blot analysis (D) and quantification (E) of total hepatic protein levels of phosphorylated (p)STAT3, phosphorylated (p)JNK, phosphorylated (p) MAPK1/2, and phosphorylated (p)AKT from mice before and four hours following PHx. Each band represents one single mouse sample in the indicated group. For all experiments n=4-5 per time point per group: **P < 0.01; *P < 0.05.

Figure 2. The cellular response of hepatocytes is not directly affected by short-term Sorafenib treatment during PHx

A–C IHC staining for Ki67-positive cells (A) and PH3-positive cells (B) in mice before and on day 4 post PHx, and for γ-H2AX-positive cells in mice before and on day 2 post PHx C. (A′-C′) quantification of Ki67-positive cells (A′), PH3-positive cells (B′) at the indicated time points, and γ-H2AX-positive cells (C′) on day 2 following PHx in liver tissue sections of mice for 10 randomly selected fields. D. IHC staining for apoptosis by TUNEL assay in mice before and on day 2 following PHx. (DAPI, blue; apoptosis, green). For all experiments: n=4-5 per time point per group, *P < 0.05.

Figure 3. Short-term Sorafenib treatment during PHx resulted in decreased inflammatory signaling pathways

A. RNA from short-term Sorafenib or Cremophor treated mice four hours post PHx was analyzed using RNA sequencing. A marked reduction of many inflammatory signaling pathways in livers of short-term Sorafenib treated mice four hours post PHx compared to controls. For all pathways the FDR is ≤ 0.05 B. Heatmap of the inflammatory chemokine genes, which almost all of them are down-regulated in short-term Sorafenib treated mice, but not in short-term Cremophor-treated mice four hours post PHx (n=2-3/group). C–E Expression of (C) CCR5 (D) CCR2 and (E) CCl2 in liver extracts of short-term Sorafenib or Cremophor treated three- month-old mice, as determined by real-time PCR before and four hours following PHx (n=6-8/group). For all experiments: **P < 0.01; *P < 0.05.

Figure 4. Short-term Sorafenib treatment during PHx resulted in a decreased inflammatory response and stellate cells activation

A. Gr-1 IHC staining of neutrophils in mice before and four hours following PHx (n=5-6/group). B. CD3 IHC staining of CD3 T-cells in mice before and on day 4 following PHx (n=4/group). Quantification of the Gr-1- and CD3-positive cells (A′ and B′, respectively) was done at the indicated time points for 10 randomly selected fields. C. Expression of TGF-β in liver extracts of three- month-old mice, as determined by real-time PCR before and four hours following PHx (n=7-8/group). D. HGF levels in the livers of three- month-old mice determined by ELISA assay before and four hours following PHx (n= 3/group). E. Immunohistochemistry staining of α -SMA in liver tissue sections of mice before and on day 2 following PHx (n=4-5/group). For all experiments: **P < 0.01; *P < 0.05. F. Assessment of extracellular collagen deposits indicated by Masson trichome staining at 8.5 months (n =6-7/group).

Figure 5. Short-term Sorafenib treatment during PHx reduces tumorigenesis in the Mdr2-KO model

Mdr2-KO mice were subjected to PHx at three months of age and treated with Sorafenib or Cremophor immediately and two hours following the surgery. Tumors were measured and counted in naïve (no PHx), short-term Sorafenib (during PHx at three months) and short-term Cremophor (during Phx at three months) treated mice at 8.5 months of age. A. Total tumor volume per mouse was calculated from all tumors larger than or equal to 2 mm (n=9/group). B. Number of dysplastic nodules smaller than or equal to 1 mm per mouse. (n=9/group). C. Representative H&E liver sections revealing internal tumors and dysplastic nodules in short-term Cremophor treated mice. D. Representative images of harvested livers and tumors from short-term Cremophor treated mice.