ORIGINAL ARTICLE Type IV collagen-initiated signals provide survival and growth cues required for liver metastasis JV Burnier 1 , N Wang 2 , RP Michel 3,4 , M Hassanain 2 , S Li 1 , Y Lu 2 , P Metrakos 2 , E Antecka 3 , MN Burnier 1,3,4,5 , A Ponton 6 , S Gallinger 7 and P Brodt 1,2,4 1 Department of Medicine, McGill University and the McGill University Health Center–Royal Victoria Hospital, Montreal Quebec, Canada; 2 Department of Surgery, McGill University and the McGill University Health Center–Royal Victoria Hospital, Montreal, Quebec, Canada; 3 Department of Pathology, McGill University, Montreal, Quebec, Canada; 4 Department of Oncology, McGill University, Montreal, Quebec, Canada; 5 Department of Opthalmology, McGill University, Montreal, Quebec, Canada; 6 McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada and 7 Department of Surgery, Mt Sinai Hospital, University Health Network Toronto, Toronto, Ontario, Canada The liver is a major site of metastasis for human malignancies, yet the factors that regulate tumor cell survival and growth in this organ remain elusive. Previously, we reported that M-27 IGFÀIR murine lung carcinoma cells with ectopic insulin-like growth factor-1 (IGF-I) receptor overexpression acquired a site-specific, liver-metastasizing potential. Gene expression profiling and subsequent RNA and protein analyses revealed that this was associated with major changes to the expression of extracellular matrix (ECM) protein-encoding genes including type III, IV and XVIII collagen genes, and these changes were also observed in the respective tumors in vivo. Because type IV collagen was the most prominently altered ECM protein in this model, we further analyzed its functional relevance to liver metas- tasis. M-27 cells stably overexpressing type IV collagen a1 and a2 chains were generated and their growth and metastatic properties investigated. We found that these cells acquired a site-selective growth advantage in the liver and this was associated with cell rescue from anoikis in a collagen IV/a2 integrin/FAK-dependent manner and increased responsiveness to IGF-I. Conversely, collagen IV or focal adhesion kinase (FAK) silencing by small- interfering RNA in highly metastatic tumor cells enhanced anoikis and decreased liver metastases formation. More- over, analysis of human surgical specimens revealed uniformly high collagen IV expression in 65/65 hepatic metastases analyzed, regardless of tissue of origin, whereas it was variable and generally low in 50/50 primary colo- rectal carcinoma specimens examined. The results suggest that collagen IV-conveyed signals are essential cues for liver metastasis in diverse tumor types and identify mediators of collagen IV signaling as potential therapeutic targets in the management of hepatic metastases. Oncogene (2011) 30, 3766–3783; doi:10.1038/onc.2011.89; published online 11 April 2011 Keywords: metastasis; liver; extracellular matrix; col- lagen IV; anoikis Introduction Tumor cell dissemination from the primary site to distant organs, such as the liver, involves complex phenotypic changes that enable cell detachment from the primary mass, cell migration, invasion of host tissues, extravasation and tumor cell adaptation to its changing microenvironment (reviewed in Steeg, 2006 and Brooks et al., 2009). The phenomenon of prefer- ential homing of tumor cells (site-specific metastasis) has been recognized for decades but its molecular basis, particularly as it pertains to liver metastasis, is not completely understood. As predicted by the ‘seed and soil’ hypothesis (Paget, 1989), the ability of tumor cells to grow in a secondary site depends on a complemen- tarity between tumor cell properties and the unique microenvironment of the target organ. The identifica- tion of genetic changes that facilitate tumor cell growth in specific organ sites may therefore lead to improved predictive tools and targeted therapeutic intervention. The basement membrane (BM) is a specialized extracellular matrix (ECM) separating the epithelium from the stromal components of the tissue and the underlying vascular structures. It consists mainly of type IV collagen and laminin, as well as heparan-sulfate proteoglycans and nidogens (Miner et al., 2004; Poschl et al., 2004). Cell adhesion to the ECM is mediated mainly by integrins, a family of cell surface hetero- dimeric receptors consisting of noncovalently linked a- and b-subunits (reviewed in van der Flier and Sonnenberg, 2001). The acquisition of a migratory/ metastatic phenotype is often associated with changes to the expression and activation states of integrins that affect cell attachment to ECM, regulate the balance between cell adhesion and motility and enable cell survival under conditions of detachment, a critical requirement for metastasis (reviewed in Felding-Habermann, 2003). The expression of a2b1 integrin, a type IV collagen receptor Received 14 July 2010; revised 8 February 2011; accepted 20 February 2011; published online 11 April 2011 Correspondence: Dr P Brodt, McGill University Health Center, Royal Victoria Hospital, Room H6.25, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: pnina.brodt@mcgill.ca Oncogene (2011) 30, 3766–3783 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc