Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of Rho and ROCK.
OBJECTIVE
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.
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Philip W
PW
Voorneveld
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Liudmila L
LL
Kodach
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Rutger J
RJ
Jacobs
Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
Nalan
N
Liv
Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
A Christiaan
AC
Zonnevylle
Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
Jacob P
JP
Hoogenboom
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Izak
I
Biemond
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Hein W
HW
Verspaget
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.
Daniel W
DW
Hommes
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.
Karien
K
de Rooij
Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands.
Carel J M
CJ
van Noesel
Department of Pathology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Hans
H
Morreau
Department of Pathology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Tom
T
van Wezel
Department of Pathology, University Medical Center, Utrecht, The Netherlands.
G Johan A
GJ
Offerhaus
Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands; Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands.
Gijs R
GR
van den Brink
Department of Gastroenterology and Hepatology, Erasmus MC, Erasmus University Rotterdam, Rotterdam, The Netherlands.
Maikel P
MP
Peppelenbosch
Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Peter
P
Ten Dijke
Department of Gastroenterology and Hepatology, Cancer Genomics Centre Netherlands and Centre for Biomedical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
James C H
JC
Hardwick
D000368:Aged; D000577:Amides / Q000494:pharmacology; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000502:physiology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D015179:Colorectal Neoplasms / Q000401:mortality / Q000473:pathology / Q000503:physiopathology*; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology; D005260:Female; D064593:Heterografts; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008875:Middle Aged; D009362:Neoplasm Metastasis / Q000473:pathology / Q000503:physiopathology*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D051901:Smad4 Protein / Q000172:deficiency*; D015996:Survival Rate; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*
D000577:Amides; D019485:Bone Morphogenetic Proteins; D004791:Enzyme Inhibitors; D011725:Pyridines; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C108830:Y 27632; D054460:rho-Associated Kinases
Colon Cancer; Signal Transduction; Transcription; Tumor Progression