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. 2014 Jan 10;9(1):e85340.
doi: 10.1371/journal.pone.0085340. eCollection 2014.

High expression of IGFBP7 in fibroblasts induced by colorectal cancer cells is co-regulated by TGF-β and Wnt signaling in a Smad2/3-Dvl2/3-dependent manner

Cui Rao  1 , Shan-Li Lin  1 , Wen-Jing Ruan  1 , Huan Wen  1 , Dan-Ju Wu  2 , Hong Deng  1
Affiliations

High expression of IGFBP7 in fibroblasts induced by colorectal cancer cells is co-regulated by TGF-β and Wnt signaling in a Smad2/3-Dvl2/3-dependent manner

Cui Rao et al. PLoS One. .

Abstract

Fibroblasts in the tumor microenvironment are a key determinant in cancer progression and may be a promising target for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is known as a tumor suppressor in colorectal cancer (CRC). The present study investigated the inductive mechanism of IGFBP7 expression in fibroblasts by supernatant from the CRC cell line, SW620. The results showed that the expression of IGFBP7 was up-regulated in the fibroblasts when treated with SW620 supernatant and exogenous TGF-β1. The IGFBP7 induced by SW620 supernatant or TGF-β1 was partially inhibited by the TGF-β1 specific antibody AF and TGF-β1 receptor antagonist SB431542. The Wnt signaling-targeted genes, c-Myc, CCND1 and the proteins Dvl2/3, were all up-regulated in fibroblasts expressing high levels of IGFBP7, and the up-regulation could be inhibited both by the Wnt signaling antagonist Dickkopf-1 (DKK1) and by the TGF-β1 receptor antagonist SB431542. In conclusion, CRC cells promote the high expression of IGFBP7 in fibroblasts, most likely through the co-regulation of TGF-β and Wnt signaling in a Smad2/3-Dvl2/3 dependent manner. Taken together, these data suggest that the fibroblasts could be a novel therapeutic target in tumor therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SW620-S induces high IGFBP7 expression in fibroblasts.
A. B. IGFBP7 mRNA expression determined by RT-PCR (A) and Q-PCR (B) in fibroblasts exposed to SW620-S for 0, 2, 4 and 6 days respectively. C. D. IGFBP7 mRNA expression determined by RT-PCR in fibroblasts exposed to HT29-S (C) and Lovo-S (D) for 0, 2, 4 and 6 days. The mRNA level was normalized to that of GAPDH in the same cell extracts. *P <0.05 between SW620-S-treated fibroblasts and control group (0 day).
Figure 2
Figure 2. TGF-β1 in SW620-S is the main factor inducing high IGFBP7 expression in fibroblasts.
A. B. IGFBP7 mRNA expression determined by RT-PCR (A) and Q-PCR (B) in fibroblasts exposed to RPMI1640 or 5 ng/ml TGF-β1 for 0, 2, 4 and 6 days. C. D. IGFBP7 mRNA expression in fibroblasts exposed to 0, 5, 10 and 20 ng/ml TGF-β1 for 6 days determined by RT-PCR (C) and Q-PCR (D). E. F. Fibroblasts were treated with SW620-S or 5 ng/ml TGF-β1 in the presence of 0, 20, 200 ng/ml AF for 6 days, IGFBP7 mRNA in fibroblasts was detected by RT-PCR (E) and Q-PCR (F). The mRNA level was normalized to that of GAPDH in the same cell extracts. *P <0.05 between SW620-S/TGF-β1-treated fibroblasts and control group. + P <0.05 between SW620-S-treated fibroblasts with or without 20 ng/ml AF. ++ P<0.05 between SW620-S-treated fibroblasts with or without 200 ng/ml AF. # P <0.05 between TGF-β1-treated fibroblasts with or without 20 ng/ml AF. ## P<0.05 between TGF-β1-treated fibroblasts with or without 200 ng/ml AF.
Figure 3
Figure 3. High expression of IGFBP7 in fibroblasts is mainly through the TGF-β/ALK5/Smad2 pathway.
A. The expression of TβRII was detected by Western blot. B. Fibroblasts were exposed to SW620-S for 0, 2, 4 and 6 days, and p-Smad2 was detected by Western blot. The protein levels were normalized to that of β-actin in the same cell extracts. C.D. Fibroblasts were exposed to SW620-S or TGF-β1 in the presence of 10 µM SB431542 for 6 days, and IGFBP7 mRNA expression was detected by RT-PCR (C) and Q-PCR (D). The mRNA level was normalized to that of GAPDH in the same cell extracts. E. Fibroblasts were exposed to SW620-S or TGF-β1 with 10 µM SB431542 for 6 days, and the expression of Smad2, p-Smad2 (E) was detected by Western blot. The protein levels were normalized to that of β-actin in the same cell extracts. * P<0.05 between SW620-S/TGF-β1-treated fibroblasts and control group. + P<0.05 between SW620-S-treated fibroblasts with or without SB431542, # P<0.05 between TGF-β1-treated fibroblasts with or without SB431542.
Figure 4
Figure 4. IGFBP7 up-regulation in fibroblasts is partially through the activation of Wnt signaling.
A. B. Fibroblasts were treated with SW620-S for 0, 2, 4 and 6 days, and the mRNA expression of the Wnt signaling target genes c-Myc (A) and CCND1 (B) mRNA expression was assessed by Q-PCR. C-E. Fibroblasts were treated with SW620-S in the presence of 50 ng/ml DKK1 (Wnt antagonist) for 6 days, and IGFBP7 mRNA expression was detected by RT-PCR(C) and Q-PCR (D). The mRNA expression was normalized to that of GAPDH in the same cell extracts. The Wnt signaling protein Dvl3 was detected in fibroblasts by Western blot (E). Protein expression was normalized to that of β-actin in the same cell extract. *P<0.05 between SW620-S-treated fibroblasts and control group. **P<0.05 between DKK1-treated fibroblasts and control group. + P<0.05 between SW620-S-treated fibroblasts with or without DKK1.
Figure 5
Figure 5. TGF-β and canonical Wnt signaling co-operate in regulating the high expression of IGFBP7 in fibroblasts.
A-D. Fibroblasts were exposed to SW620-S or TGF-β1 in the presence of 10 µM SB431542 for 6days, and the mRNA expression of the Wnt signal target genes c-Myc (A), CCND1 (B) and DKK1 (C) were assessed by Q-PCR. mRNA expression was normalized to that of GAPDH in the same cell extracts. The expression of Dvl3 was detected by Western blot (D). Protein expression was normalized to that of β-actin in the same cell extract. *P<0.05 between SW620-S-treated fibroblasts and control group. **P<0.05 between TGF-β1-treated fibroblasts and control group. + P<0.05 between SW620-S-treated fibroblasts with or without SB431542. # P<0.05 between TGF-β1-treated fibroblasts with or without SB431542. E. Model of high expression of IGFBP7 induced by the tumor cell-fibroblast interactions. F. TGF-β signal activation up-regulates Smad2/3 and Dvl2/3, accompanied by up-regulation of the Wnt target genes c-Myc, CCND1 and DKK1, such that IGFBP7 is over-expressed.
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References

    1. Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA Cancer J Clin 63: 11–30. - PubMed
    1. Dvorak HF (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315: 1650–9. - PubMed
    1. Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6: 392–401. - PubMed
    1. Liu M, Xu JJ, Deng H (2011) Tangled fibroblasts in tumor-stroma interactions. Int J Cancer 129: 1795–805. - PubMed
    1. Mueller MM, Fusenig NE (2004) Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 4: 839–49. - PubMed

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This work was supported by grants from the Natural Science Foundation of Zhejiang Province (LY12H16027), the National Natural Science Foundation of China (30870971) and the Major Program of the National Natural Science Foundation of China (81090420, 81090421). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.