Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK

Philip W. Voorneveld, Liudmila L. Kodach, Rutger J. Jacobs, Nalan Liv, A. Christiaan Zonnevylle, Jacob P. Hoogenboom, Izak Biemond, Hein W. Verspaget, Daniel W. Hommes, Karien De Rooij, Carel J M Van Noesel, Hans Morreau, Tom Van Wezel, G. Johan A Offerhaus, Gijs R. Van Den Brink, Maikel P. Peppelenbosch, Peter Ten Dijke, James C H Hardwick

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalGastroenterology
Volume147
Issue number1
DOIs
StatePublished - 2014
Externally publishedYes

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Bone Morphogenetic Proteins
Colorectal Neoplasms
Neoplasm Metastasis
Bone Morphogenetic Protein Receptors
Cell Movement
Epithelial-Mesenchymal Transition
Transforming Growth Factors
Survival
Neoplasms
Lim Kinases
HT29 Cells
Growth Factor Receptors
Medical Genetics
Heterografts
Phosphotransferases
Immunohistochemistry

Keywords

  • Colon Cancer
  • Signal Transduction
  • Transcription
  • Tumor Progression

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Voorneveld, P. W., Kodach, L. L., Jacobs, R. J., Liv, N., Christiaan Zonnevylle, A., Hoogenboom, J. P., ... Hardwick, J. C. H. (2014). Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK. Gastroenterology, 147(1). https://doi.org/10.1053/j.gastro.2014.03.052

Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK. / Voorneveld, Philip W.; Kodach, Liudmila L.; Jacobs, Rutger J.; Liv, Nalan; Christiaan Zonnevylle, A.; Hoogenboom, Jacob P.; Biemond, Izak; Verspaget, Hein W.; Hommes, Daniel W.; De Rooij, Karien; Van Noesel, Carel J M; Morreau, Hans; Van Wezel, Tom; Offerhaus, G. Johan A; Van Den Brink, Gijs R.; Peppelenbosch, Maikel P.; Ten Dijke, Peter; Hardwick, James C H.

In: Gastroenterology, Vol. 147, No. 1, 2014.

Research output: Contribution to journalArticle

Voorneveld, PW, Kodach, LL, Jacobs, RJ, Liv, N, Christiaan Zonnevylle, A, Hoogenboom, JP, Biemond, I, Verspaget, HW, Hommes, DW, De Rooij, K, Van Noesel, CJM, Morreau, H, Van Wezel, T, Offerhaus, GJA, Van Den Brink, GR, Peppelenbosch, MP, Ten Dijke, P & Hardwick, JCH 2014, 'Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK', Gastroenterology, vol. 147, no. 1. https://doi.org/10.1053/j.gastro.2014.03.052
Voorneveld, Philip W. ; Kodach, Liudmila L. ; Jacobs, Rutger J. ; Liv, Nalan ; Christiaan Zonnevylle, A. ; Hoogenboom, Jacob P. ; Biemond, Izak ; Verspaget, Hein W. ; Hommes, Daniel W. ; De Rooij, Karien ; Van Noesel, Carel J M ; Morreau, Hans ; Van Wezel, Tom ; Offerhaus, G. Johan A ; Van Den Brink, Gijs R. ; Peppelenbosch, Maikel P. ; Ten Dijke, Peter ; Hardwick, James C H. / Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK. In: Gastroenterology. 2014 ; Vol. 147, No. 1.
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title = "Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK",
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.",
keywords = "Colon Cancer, Signal Transduction, Transcription, Tumor Progression",
author = "Voorneveld, {Philip W.} and Kodach, {Liudmila L.} and Jacobs, {Rutger J.} and Nalan Liv and {Christiaan Zonnevylle}, A. and Hoogenboom, {Jacob P.} and Izak Biemond and Verspaget, {Hein W.} and Hommes, {Daniel W.} and {De Rooij}, Karien and {Van Noesel}, {Carel J M} and Hans Morreau and {Van Wezel}, Tom and Offerhaus, {G. Johan A} and {Van Den Brink}, {Gijs R.} and Peppelenbosch, {Maikel P.} and {Ten Dijke}, Peter and Hardwick, {James C H}",
year = "2014",
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TY - JOUR

T1 - Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of rho and ROCK

AU - Voorneveld, Philip W.

AU - Kodach, Liudmila L.

AU - Jacobs, Rutger J.

AU - Liv, Nalan

AU - Christiaan Zonnevylle, A.

AU - Hoogenboom, Jacob P.

AU - Biemond, Izak

AU - Verspaget, Hein W.

AU - Hommes, Daniel W.

AU - De Rooij, Karien

AU - Van Noesel, Carel J M

AU - Morreau, Hans

AU - Van Wezel, Tom

AU - Offerhaus, G. Johan A

AU - Van Den Brink, Gijs R.

AU - Peppelenbosch, Maikel P.

AU - Ten Dijke, Peter

AU - Hardwick, James C H

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Colon Cancer

KW - Signal Transduction

KW - Transcription

KW - Tumor Progression

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