Long-term exposure to sorafenib of liver cancer cells induces resistance with epithelial-to-mesenchymal transition, increased invasion and risk of rebound growth.

Sorafenib leads to a survival benefit in patients with advanced hepatocellular carcinoma but its use is hampered by the occurrence of drug resistance. To investigate the molecular mechanisms involved we developed five resistant human liver cell lines in which we studied morphology, gene expression and invasive potential. The cells changed their appearance, lost E-cadherin and KRT19 and showed high expression of vimentin, indicating epithelial-to-mesenchymal transition. Resistant cells showed reduced adherent growth, became more invasive and lost liver-specific gene expression. Furthermore, following withdrawal of sorafenib, the resistant cells showed rebound growth, a phenomenon also found in patients. This cell model was further used to investigate strategies for restoration of sensitivity to sorafenib.

[1]  M. Kudo,et al.  Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma , 2011, Molecular Cancer Therapeutics.

[2]  S. Forsburg,et al.  Eukaryotic DNA replication in a chromatin context. , 2006, Current topics in developmental biology.

[3]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[4]  A. Oza,et al.  Phase I study to determine the safety and pharmacokinetics of the novel Raf kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced, refractory solid tumors. , 2005, Annals of oncology : official journal of the European Society for Medical Oncology.

[5]  John M L Ebos,et al.  Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. , 2009, Cancer cell.

[6]  D. Auclair,et al.  BAY 43-9006 Exhibits Broad Spectrum Oral Antitumor Activity and Targets the RAF/MEK/ERK Pathway and Receptor Tyrosine Kinases Involved in Tumor Progression and Angiogenesis , 2004, Cancer Research.

[7]  Y. Nakanishi,et al.  Twist expression promotes migration and invasion in hepatocellular carcinoma , 2009, BMC Cancer.

[8]  M. Bhasin,et al.  Resistance of Renal Cell Carcinoma to Sorafenib Is Mediated by Potentially Reversible Gene Expression , 2011, PloS one.

[9]  Jeffrey W. Clark,et al.  Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: a review of four phase I trials in patients with advanced refractory solid tumors. , 2007, The oncologist.

[10]  D. Amadori,et al.  Phase II study of sorafenib in patients with advanced hepatocellular carcinoma. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[11]  Kevin Truong,et al.  Cadherins in embryonic and neural morphogenesis , 2000, Nature Reviews Molecular Cell Biology.

[12]  Dirk Strumberg,et al.  Phase I clinical and pharmacokinetic study of the Novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[14]  B. Daniele,et al.  Design and Endpoints of Clinical Trials in Hepatocellular Carcinoma , 2008 .

[15]  Tracy T Batchelor,et al.  AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. , 2007, Cancer cell.

[16]  M. Büchler,et al.  Sorafenib modulates the gene expression of multi-drug resistance mediating ATP-binding cassette proteins in experimental hepatocellular carcinoma. , 2010, Anticancer research.

[17]  Kuen-Feng Chen,et al.  Sorafenib Overcomes TRAIL Resistance of Hepatocellular Carcinoma Cells through the Inhibition of STAT3 , 2010, Clinical Cancer Research.

[18]  K. Csiszȧr,et al.  A molecular role for lysyl oxidase‐like 2 enzyme in Snail regulation and tumor progression , 2005, The EMBO journal.

[19]  J. Ferlay,et al.  Global Cancer Statistics, 2002 , 2005, CA: a cancer journal for clinicians.

[20]  J. Llovet,et al.  Second-Line Therapies in Hepatocellular Carcinoma: Emergence of Resistance to Sorafenib , 2012, Clinical Cancer Research.

[21]  A. Ullrich,et al.  PRKX, TTBK2 and RSK4 expression causes sunitinib resistance in kidney carcinoma‐ and melanoma‐cell lines , 2012, International journal of cancer.

[22]  S Detre,et al.  A "quickscore" method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. , 1995, Journal of clinical pathology.

[23]  R. Kalluri,et al.  Mechanisms of metastasis: Epithelial‐to‐mesenchymal transition and contribution of tumor microenvironment , 2007, Journal of cellular biochemistry.

[24]  Sagar Agarwal,et al.  The Role of the Breast Cancer Resistance Protein (ABCG2) in the Distribution of Sorafenib to the Brain , 2011, Journal of Pharmacology and Experimental Therapeutics.

[25]  S. Hirohashi,et al.  Cadherin intercellular adhesion molecule in hepatocellular carcinomas: loss of E-cadherin expression in an undifferentiated carcinoma. , 1991, Cancer letters.

[26]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[27]  C. Pirker,et al.  A Human Model of Epithelial to Mesenchymal Transition to Monitor Drug Efficacy in Hepatocellular Carcinoma Progression , 2011, Molecular Cancer Therapeutics.

[28]  S. Hirohashi,et al.  E‐cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma , 1996, Hepatology.

[29]  Jeffrey W. Clark,et al.  Safety and Pharmacokinetics of the Dual Action Raf Kinase and Vascular Endothelial Growth Factor Receptor Inhibitor, BAY 43-9006, in Patients with Advanced, Refractory Solid Tumors , 2005, Clinical Cancer Research.

[30]  Raghu Kalluri,et al.  The basics of epithelial-mesenchymal transition. , 2009, The Journal of clinical investigation.

[31]  S. Wilhelm,et al.  Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. , 2006, Cancer research.

[32]  F. Ciardiello,et al.  Antitumour efficacy of MEK inhibitors in human lung cancer cells and their derivatives with acquired resistance to different tyrosine kinase inhibitors , 2011, British Journal of Cancer.

[33]  M. Kudo.  Signaling Pathway and Molecular-Targeted Therapy for Hepatocellular Carcinoma , 2011, Digestive Diseases.

[34]  D. Sargent,et al.  Bevacizumab beyond first progression is associated with prolonged overall survival in metastatic colorectal cancer: results from a large observational cohort study (BRiTE). , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[35]  M. Zern,et al.  Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations. , 2011, Journal of hepatology.

[36]  Zhong Chen,et al.  Suppression of ABCG2 inhibits cancer cell proliferation , 2009, International journal of cancer.

[37]  Alberto Martin,et al.  Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas , 2011, EMBO molecular medicine.

[38]  A. Jemal,et al.  Global Cancer Statistics , 2011 .

[39]  R. Kerbel,et al.  Tumor and Host-Mediated Pathways of Resistance and Disease Progression in Response to Antiangiogenic Therapy , 2009, Clinical Cancer Research.

[40]  K. Sugimachi,et al.  Transcriptional repressor snail and progression of human hepatocellular carcinoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[41]  C. Desbois-Mouthon,et al.  Epidermal growth factor receptor and HER-3 restrict cell response to sorafenib in hepatocellular carcinoma cells. , 2012, Journal of hepatology.

[42]  J. Tabernero,et al.  Targeting the PI3K/Akt/mTOR Pathway – Beyond Rapalogs , 2010, Oncotarget.

[43]  J. Thiery.  Epithelial–mesenchymal transitions in tumour progression , 2002, Nature Reviews Cancer.

[44]  Kuen-Feng Chen,et al.  Activation of Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Mediates Acquired Resistance to Sorafenib in Hepatocellular Carcinoma Cells , 2011, Journal of Pharmacology and Experimental Therapeutics.

[45]  Yoon-Koo Kang,et al.  Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. , 2009, The Lancet. Oncology.

[46]  Sun Young Park,et al.  Epithelial–mesenchymal transition gene signature to predict clinical outcome of hepatocellular carcinoma , 2010, Cancer science.

[47]  S. Kaye,et al.  Combining Antiangiogenics to Overcome Resistance: Rationale and Clinical Experience , 2012, Clinical Cancer Research.

[48]  Sonja Loges,et al.  Silencing or fueling metastasis with VEGF inhibitors: antiangiogenesis revisited. , 2009, Cancer cell.

[49]  J. Thiery,et al.  Complex networks orchestrate epithelial–mesenchymal transitions , 2006, Nature Reviews Molecular Cell Biology.

[50]  Qian Wang,et al.  Activation of the hedgehog pathway in human hepatocellular carcinomas. , 2006, Carcinogenesis.

[51]  Dieter Häussinger,et al.  Sorafenib in advanced hepatocellular carcinoma. , 2008, The New England journal of medicine.

[52]  Masahiro Inoue,et al.  Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. , 2009, Cancer cell.

[53]  A. G. Herreros,et al.  The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells , 2000, Nature Cell Biology.

[54]  M. Pomper,et al.  Hedgehog pathway inhibitor HhAntag691 is a potent inhibitor of ABCG2/BCRP and ABCB1/Pgp. , 2009, Neoplasia.

[55]  D. McDonald,et al.  Rapid vascular regrowth in tumors after reversal of VEGF inhibition. , 2006, The Journal of clinical investigation.

[56]  Ziqiu Wang,et al.  Involvement of receptor tyrosine phosphatase DEP‐1 mediated PI3K‐cofilin signaling pathway in Sorafenib‐induced cytoskeletal rearrangement in hepatoma cells , 2010, Journal of cellular physiology.