Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo.
OBJECTIVE
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.
Newell
Pippa
P
Mount Sinai Liver Cancer Program, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA.
Toffanin
Sara
S
Villanueva
Augusto
A
Chiang
Derek Y
DY
Minguez
Beatriz
B
Cabellos
Laia
L
Savic
Radoslav
R
Hoshida
Yujin
Y
Lim
Kiat Hon
KH
Melgar-Lesmes
Pedro
P
Yea
Steven
S
Peix
Judit
J
Deniz
Kemal
K
Fiel
M Isabel
MI
Thung
Swan
S
Alsinet
Clara
C
Tovar
Victoria
V
Mazzaferro
Vincenzo
V
Bruix
Jordi
J
Roayaie
Sasan
S
Schwartz
Myron
M
Friedman
Scott L
SL
Llovet
Josep M
JM
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage*; D001557:Benzenesulfonates / Q000008:administration & dosage*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D004357:Drug Synergism; D005260:Female; D018628:Gene Dosage / Q000187:drug effects; D011905:Genes, ras / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009536:Niacinamide / Q000031:analogs & derivatives; D010671:Phenylurea Compounds; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011494:Protein Kinases / Q000187:drug effects; D011725:Pyridines / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000008:administration & dosage*; D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases; D014183:Transplantation, Heterologous; D018631:ras Proteins / Q000378:metabolism*
D001557:Benzenesulfonates; D010671:Phenylurea Compounds; D011725:Pyridines; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D009536:Niacinamide; D000077157:Sorafenib; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D018631:ras Proteins; D020123:Sirolimus