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

Pippa Newell, Sara Toffanin, Augusto Villanueva, Derek Y. Chiang, Beatriz Minguez, Laia Cabellos, Radoslav Savic, Yujin Hoshida, Kiat Hon Lim, Pedro Melgar-Lesmes, Steven Yea, Judit Peix, Kemal Deniz, M. Isabel Fiel, Swan Thung, Clara Alsinet, Victoria Tovar, Vincenzo Mazzaferro, Jordi Bruix, Sasan RoayaieMyron Schwartz, Scott L. Friedman, Josep M. Llovet

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)725-733
Number of pages9
JournalJournal of Hepatology
Volume51
Issue number4
DOIs
Publication statusPublished - Oct 2009

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Sirolimus
Hepatocellular Carcinoma
Methylation
Neoplasms
DNA Copy Number Variations
Cell Line
Polymerase Chain Reaction
Oligonucleotide Array Sequence Analysis
Tumor Suppressor Genes
Oncogenes
Heterografts
Antineoplastic Agents
Single Nucleotide Polymorphism
sorafenib
Theoretical Models
Necrosis
Immunohistochemistry
Cell Proliferation
Apoptosis
Gene Expression

Keywords

  • HCV
  • Liver cancer
  • Molecular therapies
  • mTOR
  • Ras
  • Signaling pathway

ASJC Scopus subject areas

  • Hepatology

Cite this

Newell, P., Toffanin, S., Villanueva, A., Chiang, D. Y., Minguez, B., Cabellos, L., ... Llovet, J. M. (2009). Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. Journal of Hepatology, 51(4), 725-733. https://doi.org/10.1016/j.jhep.2009.03.028

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. / Newell, Pippa; Toffanin, Sara; Villanueva, Augusto; Chiang, Derek Y.; Minguez, Beatriz; Cabellos, Laia; Savic, Radoslav; Hoshida, Yujin; Lim, Kiat Hon; Melgar-Lesmes, Pedro; Yea, Steven; Peix, Judit; Deniz, Kemal; Fiel, M. Isabel; Thung, Swan; Alsinet, Clara; Tovar, Victoria; Mazzaferro, Vincenzo; Bruix, Jordi; Roayaie, Sasan; Schwartz, Myron; Friedman, Scott L.; Llovet, Josep M.

In: Journal of Hepatology, Vol. 51, No. 4, 10.2009, p. 725-733.

Research output: Contribution to journalArticle

Newell, P, Toffanin, S, Villanueva, A, Chiang, DY, Minguez, B, Cabellos, L, Savic, R, Hoshida, Y, Lim, KH, Melgar-Lesmes, P, Yea, S, Peix, J, Deniz, K, Fiel, MI, Thung, S, Alsinet, C, Tovar, V, Mazzaferro, V, Bruix, J, Roayaie, S, Schwartz, M, Friedman, SL & Llovet, JM 2009, 'Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo', Journal of Hepatology, vol. 51, no. 4, pp. 725-733. https://doi.org/10.1016/j.jhep.2009.03.028
Newell, Pippa ; Toffanin, Sara ; Villanueva, Augusto ; Chiang, Derek Y. ; Minguez, Beatriz ; Cabellos, Laia ; Savic, Radoslav ; Hoshida, Yujin ; Lim, Kiat Hon ; Melgar-Lesmes, Pedro ; Yea, Steven ; Peix, Judit ; Deniz, Kemal ; Fiel, M. Isabel ; Thung, Swan ; Alsinet, Clara ; Tovar, Victoria ; Mazzaferro, Vincenzo ; Bruix, Jordi ; Roayaie, Sasan ; Schwartz, Myron ; Friedman, Scott L. ; Llovet, Josep M. / Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. In: Journal of Hepatology. 2009 ; Vol. 51, No. 4. pp. 725-733.
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T1 - Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo

AU - Newell, Pippa

AU - Toffanin, Sara

AU - Villanueva, Augusto

AU - Chiang, Derek Y.

AU - Minguez, Beatriz

AU - Cabellos, Laia

AU - Savic, Radoslav

AU - Hoshida, Yujin

AU - Lim, Kiat Hon

AU - Melgar-Lesmes, Pedro

AU - Yea, Steven

AU - Peix, Judit

AU - Deniz, Kemal

AU - Fiel, M. Isabel

AU - Thung, Swan

AU - Alsinet, Clara

AU - Tovar, Victoria

AU - Mazzaferro, Vincenzo

AU - Bruix, Jordi

AU - Roayaie, Sasan

AU - Schwartz, Myron

AU - Friedman, Scott L.

AU - Llovet, Josep M.

PY - 2009/10

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N2 - 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.

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KW - Liver cancer

KW - Molecular therapies

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KW - Signaling pathway

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