Philip W Voorneveld1, Liudmila L Kodach1, Rutger J Jacobs1, Nalan Liv2, A Christiaan Zonnevylle2, Jacob P Hoogenboom2, Izak Biemond1, Hein W Verspaget1, Daniel W Hommes3, Karien de Rooij4, Carel J M van Noesel5, Hans Morreau6, Tom van Wezel6, G Johan A Offerhaus7, Gijs R van den Brink8, Maikel P Peppelenbosch9, Peter Ten Dijke10, James C H Hardwick1. 1. Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands. 2. Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands. 3. Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands; Center for Inflammatory Bowel Diseases, University of California Los Angeles Medical Center, Santa Monica, California. 4. Department of Radiology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands; Percuros B.V., Leiden, The Netherlands. 5. Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands. 6. Department of Pathology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands. 7. Department of Pathology, University Medical Center, Utrecht, The Netherlands. 8. Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands; Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands. 9. Department of Gastroenterology and Hepatology, Erasmus MC, Erasmus University Rotterdam, Rotterdam, The Netherlands. 10. Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Abstract
BACKGROUND & AIMS: SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor β signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in human tumor samples. METHODS: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial-mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in human colorectal tumors; these were related to patient survival times. RESULTS: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial-mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor β receptors. CONCLUSIONS: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.
BACKGROUND & AIMS:SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor β signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in humantumor samples. METHODS: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial-mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in humancolorectal tumors; these were related to patient survival times. RESULTS: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial-mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor β receptors. CONCLUSIONS: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.
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