Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo

Abstract

Background/aims: The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models.

Methods: Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts.

Results: Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone.

Conclusions: Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

Fig. 1

Flow chart of the samples analyzed in this study (n = 351). A training cohort of 155 samples including exploratory (n = 77) and replication sets (n = 78) was used to analyze molecular alterations of Ras signaling. Exploratory set: normal livers (n = 10), cirrhosis (n = 10), low (n = 10) and high (n = 7) grade dysplasia, very early (n = 10), early (n = 10), advanced (n = 10) and very advanced (n = 10) HCC. Replication set: 78 HCC samples. Clinical correlations of Ras pathway activation were investigated in clinical training (n = 82) and validation (n = 196) sets.

Fig. 2

Box plot of the fold changes in H-ras expression by qRT-PCR in the exploratory (A) and replication sets (B) compared with normal livers. Results are expressed in logarithmic scale as fold changes normalized to 1 (mean expression in normal liver). c, control (normal liver); ci, cirrhosis; lg, low-grade and hg, high-grade dysplasia; ve, very early; e, early; a, advanced and va, very advanced HCC. °Significant outliners, ^*non-significant outliners.

Fig. 3

(A) Box plot of the fold changes in B-Raf expression in the replication set compared with normal livers by qRT-PCR and (B) by oligonucleotide microarray. Results are expressed in logarithmic scale as fold changes normalized to 1 (mean expression in normal liver). c, control (normal liver); e, early; a, advanced HCC. °Significant outliners, ^*non-significant outliners. (C) Copy number gain of B-Raf. Each dot represents the copy number value for each HCC sample. Horizontal dashed lines indicate the copy number ranges found among non-tumoral counterparts. (D) Correlation between B-Raf mRNA expression levels (logarithmic scale) and B-Raf genomic copy number changes.

Fig. 4

(A) Immunostaining of phosphorylated ERK in tumor cells, (B) surrounding cirrhotic tissue, and (C) endothelial cells included in the tumor sections. (D) Vascular invasion within tumor was seen rarely.

Fig. 5

Kaplan–Meier curve showing correlation between H-ras expression levels (fold-change >3 compared with normal livers) and early recurrence in the replication set. Green line, H-ras >3; blue line: H-ras <3.

Fig. 6

(A) Immunoblotting for S6, ERK and phosphorylated forms after treatment with sorafenib (1 μM) and rapamycin (5.5 nM) in Huh7 cells. (B) Proliferation assay in Huh-7 treated with sorafenib (1 μM) and rapamycin (5.5 nM) and combination. Results are expressed as percentage of ³H-thymidine incorporation (counts per million) compared with control. (C) Immunoblotting for PARP cleavage in Huh7 treated with both the compounds. (D) Histogram representing the percentage of cells in different phases of the cell cycle after treatment with sorafenib (1 and 5 μM), rapamycin (5.5 nM) and combination in Huh7 by flow cytometry.

Fig. 7

(A) Tumor volume (mm³) in xenografts treated with sorafenib, rapamycin and combination compared to placebo ^*p < 0.05. (B) Kaplan–Meier curves showing tumor ulceration/necrosis as surrogate of response to sorafenib, rapamycin and combination. Tumor ulceration was not observed in the control (vehicle-treated) group.