Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Extracellular matrix protein ␤ig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation Chaoyu Ma,1 Yu Rong,1 Daniel R. Radiloff,1 Michael B. Datto,2 Barbara Centeno,3 Shideng Bao,4 Anthony Wai Ming Cheng,2 Fumin Lin,1 Shibo Jiang,5 Timothy J. Yeatman,3 and Xiao-Fan Wang1,6 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA; 2Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA; 3Department of Surgery, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA; 4Department of Radiation Oncology and Department of Neurosurgery, University of Colorado at Denver and Health Science Center, Aurora, Colorado 80045, USA; 5Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, USA Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of ␤ig-h3/TGFBI (transforming growth factor, ␤-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the ␤ig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of ␤ig-h3 expression dramatically reduced metastasis. Mechanistically, ␤ig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of ␣ ␤ ␤ the integrin v 5–Src signaling pathway. Thus, cancers associated with overexpression of ig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients. ␤ ␣ ␤ [Keywords: ig-h3/TGFBI; extravasation; metastasis; extracellular matrix; colon cancer; integrin v 5] Supplemental material is available at http://www.genesdev.org. Received November 7, 2007; revised version accepted December 6, 2007. Tumor metastasis consists of a series of complex steps, vessel wall (Weiss 1990; Chambers et al. 2002; MacDon- all of which must be successfully completed to give rise ald et al. 2002; Weis and Cheresh 2005). As a major com- to clinically detectable metastatic tumors at distal or- ponent of the tumor microenvironment, ECM proteins gans (Woodhouse et al. 1997; Hanahan and Weinberg secreted by cancer and/or stromal cells potentially affect 2000; Chambers et al. 2002; Bierie and Moses 2006; Go- the dissemination process through modulation of the in- mis et al. 2006). To complete the journey, primary cancer teraction between cancer and endothelial cells (Bern- cells have to invade through the basement membrane, stein and Liotta 1994). Recently, large-scale analyses of intravasate into the bloodstream, disseminate through gene expression profiles of human cancers have revealed the circulation, and extravasate to distal tissues/organs. that aberrant expression patterns of ECM proteins are The entire process, regulated by interactions between common among a wide range of cancer types (Buck- cancer cells and stromal cells and between cancer cells haults et al. 2001; Cooper et al. 2003), suggesting that and extracellular matrix (ECM), remains poorly under- alterations in the expression profiles of ECM proteins stood at the level of molecular mechanism. during tumor development can consequently exert a Extravasation, the process by which tumor cells leave functional impact on the metastatic process, including a blood or lymphatic vessel and invade the surrounding extravasation (Gupta et al. 2007; Karnoub et al. 2007). tissue parenchyma, requires dynamic interaction be- Previously we demonstrated that periostin, a secreted tween cancer cells and endothelial cells lining the blood ECM protein that is overexpressed by several cancer types, promotes tumor angiogenesis and metastasis (Bao et al. 2004; Shao et al. 2004). ␤ig-h3/TGFBI (transforming 6Corresponding author. ␤ ␤ E-MAIL [email protected]; FAX (919) 681-7152. growth factor, -induced; hereinafter referred to as ig- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1632008. h3), a structural homolog of periostin, was found by se- 308 GENES & DEVELOPMENT 22:308–321 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press ␤ig-h3 enhances tumor cell extravasation rial analysis of gene expression (SAGE) to be one of the sisting of two colon cancer cell lines, SW480 and SW620, genes that are highly elevated in various types of cancers derived from the primary tumor and lymph node metas- (Zhang et al. 1997; Argani et al. 2001). ␤ig-h3 contains an tasis of the same patient, respectively (Leibovitz et al. N-terminal secretory signal peptide, followed by a cys- 1976). Previous studies with xenograft tumor growth in teine-rich domain, four internal homologous repeats immunocompromised mice showed that the primary tu- (FAS1 domain), and a C-terminal RGD motif (Supple- mor-derived SW480 cell line exhibits a limited meta- mental Fig. S1; Skonier et al. 1992). The FAS1 domain of static potential, whereas the lymph node metastasis-de- ␤ig-h3 shares high homology with fasciclin I, an axon rived SW620 cell line displays an aggressive metastatic guidance protein that is involved in neural development pattern (Hewitt et al. 2000). Importantly to this study, in invertebrates (Bastiani et al. 1987). Normally, the ex- the two cell lines exhibit a different ␤ig-h3 expression pression of ␤ig-h3 is mainly found in fibroblasts, kera- profile (Fig. 1C). To determine whether the difference in tinocytes, and muscle cells (LeBaron et al. 1995; Bae et ␤ig-h3 expression might be one of the reasons underlying al. 2002; Ferguson et al. 2003). The physiological func- the different metastatic potentials of this isogenic pair of tion of ␤ig-h3 has been postulated to be involved in cell– cell lines, we stably expressed ␤ig-h3 in the poorly meta- matrix interaction and cell migration (Bae et al. 2002; static SW480 cells to a level comparable with that of the Kim et al. 2003), and ␤ig-h3 overexpression has been as- aggressively metastatic SW620 cells (Fig. 1C). Equal sociated with atherosclerotic and restenotic vascular le- numbers of SW480-␤ig-h3 or SW480-VEC control cells sions in humans (O’Brien et al. 1996; Lee et al. 2006). In were then injected intravenously via the tail vein into 20 this report, we demonstrate that acquired expression of SCID-Beige mice, respectively. Twelve weeks after in- ␤ig-h3 by colon cancer cells leads to a more aggressive oculation of the tumor cells, mice were sacrificed and phenotype of metastasis. ␤ig-h3 induces the dissociation examined for metastases in the lung and other organs. As of VE-cadherin junctions between endothelial cells via shown in Figure 1, D and E, metastatic growth was easily ␣ ␤ the integrin v 5–Src signaling pathway, which results detected in the lungs of all 20 mice injected with SW480- in enhanced cancer cell extravasation. ␤ig-h3 cells. A majority of these animals bore large num- bers of tumors covering almost the entire lung. In con- Results trast, only nine out of 20 mice injected with the SW480- ␤ig-h3 expression is up-regulated in human colon VEC cells developed a small number of tumor nodules in cancers the lung (Fig. 1D,E). The average number of visible meta- static nodules increased from 1.6 ± 2.1 per lung in mice Previous studies using the SAGE strategy have revealed carrying SW480-VEC control tumors to 23.2 ± 11.6 per that TGFBI expression is substantially elevated in colon lung in mice carrying SW480-␤ig-h3 tumors. Ectopic ␤ig- and pancreatic cancers in comparison with correspond- h3 expression remained present in the metastatic tu- ing normal tissues (representative results are summa- mors, which was confirmed by immunohistochemical rized in Supplemental Table S1). Several subsequent staining (Fig. 1F). Interestingly, however, there was no studies using gene array analyses have also demonstrated significant difference in the average size of the meta- that TGFBI is overexpressed in a majority of tumor static tumors derived from the SW480-␤ig-h3 and samples in comparison with their normal tissue coun- SW480-VEC cells (Supplemental Fig. S2A; Supplemental terparts in colorectal cancer patients (summarized in Table S5). This suggests that ␤ig-h3 expression in the Supplemental Table S2). To further confirm the associa- SW480 cells significantly increased the probability of ␤ tion between human colon cancer and ig-h3 overex- metastatic colonization in the lung without affecting the ␤ pression, we conducted a survey of ig-h3 expression us- growth rate of metastasized tumors. Overall, our data ing immunohistochemical staining of tissue microarray suggest that ␤ig-h3 overexpression is sufficient to pro- (TMA) samples derived from the colon tissue of 66 pa- mote the metastatic potential of colon cancer cell line ␤ tients. ig-h3 overexpression was more frequent in high- SW480 in vivo. grade (Stage III and IV) tumors (32.2%, n = 31) than in low-grade (Stage I and II) tumors (18.5%, n = 27), whereas no ␤ig-h3 expression was detected in the normal epithe- Suppression of ␤ig-h3 expression in SW620 cells lial tissues (n = 8) (Fig. 1A,B). Furthermore, ␤ig-h3 over- significantly decreases their metastatic potential in vivo expression is closely correlated with high-grade tumors We next performed loss-of-function experiments to test derived from patients who were metastatic-prone or had whether ␤ig-h3 expression is required for the metastatic already developed metastatic disease, indicating that high potential of the aggressive isogenic line of SW620 cells.