YKL-40, a secreted glycoprotein, promotes tumor angiogenesis

Abstract

Tumor angiogenesis is of paramount importance in solid tumor development. Elevated serum levels of YKL-40, which is a secreted heparin-binding glycoprotein, have been associated with a worse prognosis from various advanced human cancers. Yet the role of YKL-40 activity in these cancers is still missing. In this study, we showed that ectopic expression of YKL-40 in MDA-MB-231 breast cancer cells and in HCT-116 colon cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affinity-purified recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the effects of which are similar to the activities observed using MDA-MB-231 and HCT-116 cell-conditioned medium after transfection with YKL-40. Furthermore, YKL-40 was found to induce coordination of membrane-bound receptor syndecan-1 and integrin alpha(v)beta(3) and to activate an intracellular signaling cascade, including focal adhesion kinase and mitogen-activated protein kinase extracellular signal-related kinase1/2 in endothelial cells. Moreover, blockade of YKL-40 using small-interfering RNA gene knockdown suppressed tumor angiogenesis in vitro and in vivo. Immunohistochemical analysis of human breast cancer showed a correlation between YKL-40 expression and blood vessel density. These findings provide novel insights into angiogenic activities and molecular mechanisms of YKL-40 in cancer development.

Figure 1. Expression of YKL-40 in a variety of cell lines

A. Once all the cells were grown to be confluent, culture medium was replaced with serum-free medium and cultured for 24 hr. Conditioned medium was used for immunoblotting using polyclonal anti-YKL-40 antibody (rAY) and cells were lyzed for measuring mRNA levels of YKL-40 by RT-PCR. B. By retroviral infection, HCT-116 and MDA-MB-231 cells were engineered to express ectopic YKL-40 (Y) or control vector (C) and after 24-hr culture, the conditioned media in the absence of serum were collected for analyzing the levels of YKL-40 secreted from these cells by immunoblotting. Cell lysates were subjected to the testing of actin expression. MG-63 cells were used for the positive control.

Figure 2. Tumor cells expressing ectopic YKL-40 promote tumor growth and angiogenesis in xenograft models

A. HCT-116 and MDA-MB-231 cells over-expressing YKL-40 or vector were subcutaneously injected into right flank of mice and tumor growth was monitored for 6 weeks. n=6. *P<0.05 compared with the control group. B. A representative sample of each group from HCT-116 and MDA-MB-231 tumor models indicated that larger tumors and more blood vessels developed in mice bearing YKL-40-expressing cells relative to these seen in control animals. An arrow indicates blood vessels extended from host vessels. n=6. C and D. Blood vessel density in tumor tissue was evaluated by IHC staining with anti-CD31 antibody and quantified with an average of CD31 density from six to eight fields in each section using an NIH image analysis program. n=6. *P<0.05 compared with the control group. Bar:100 µm.

Figure 3. YKL-40 stimulates endothelial cell angiogenesis in culture

A. HMVEC were loaded into the upper chamber of a transwell and ySP (100 ng/ml), YKL-40 (100 ng/ml), or VEGF (10 ng/ml) was introduced into the lower chamber for migration assay. Cells migrated into the membrane were quantified. n=5. *P<0.05 compared with the control or ySP group. B. HMVEC were loaded on a layer of Matrigel and cultured for overnight in the presence of ySP (100 ng/ml), YKL-40 (100 ng/ml), or VEGF (10 ng/ml) for tube formation analysis. Vessel-like network was quantified. n=4–5. *P<0.05 compared with control or ySP group. C. Conditioned media (CM) derived from MDA-MB-231 and HCT-116 cells expressing YKL-40 or control vector were transferred into HMVEC culture in the presence of an anti-VEGF antibody (1 µg/ml) to evaluate their effects on endothelial cell migration and tube formation. n=6. *P<0.05 compared with the control.

Figure 4. Coordination between S1 and integrin α_υβ₃ is required for YKL-40-induced angiogenesis

A. YKL-40 (1 µg) was used for heparin-Sepharose affinity binding assay. YKL-40 was finally eluted with sodium phosphate buffer containing 1 N NaCl. The first lane indicates YKL-40 recombinant protein used in immunoblot. B. HMVEC were treated with heparitinase (Hep 0.1U/ml)/chondroitinase (Chon 0.1 U/ml) or those heat-inactivated enzymes (HI) for 2 hr prior to stimulation with YKL-40 or ySP (100 ng/ml) for 10 min. Cell lyastes were subjected to imunoprecipitation with anti-integrin β₃ or β₅ antibody followed by immunoblotting using anti-S1 antibody. C. After treatment with Hep/Chon or HI as indicated in B, cells were introduced to the plates pre-coated with YKL-40 or ySP (10 µg/ml) in the absence or presence of LM609 or P1F6 (10 µg/ml, Chemicon) for 2 hr. The cells were then fixed and stained with rhodamine-conjugated phalloidin. Bar: 10 µm. D. After treatment with Hep/Chon or HI as indicated in B, cells were applied to Matrigel for analysis of tube formation in the absence or presence of YKL-40 (100 ng/ml), ySP (100 ng/ml), LM609 (10 µg/ml), or P1F6 (10 µg/ml). n=4. *P<0.05 compared with corresponding control cells treated with ySP. ⁺P<0.05 compared with cells treated with YKL-40 alone.

Figure 5. siRNA knockdown of S1 and angiogenic signaling cascade in endothelial cells

A. Western blot analysis showed S1 gene knockdown in the cells. siRNA targeted the ectodomain of S1. B. Control and siRNA S1 endothelial cells were stimulated with YKL-40 or ySP (100 ng/ml) for 10 min and then cell lysates were subjected to immunoprecipitation and immunoblotting as described in Figure 4. C. Control and siRNA S1 cells were used for tube formation in the presence of YKL-40 or ySP (100 ng/ml). n=3. *P<0.05 compared with control cells treated with ySP. ⁺P<0.05 compared with cells treated with YKL-40 alone. D. HMVEC pre-treated with serum-free medium were stimulated with YKL-40 (100 ng/ml) from 5 to 30 min. Cell lysates were collected for analyzing phosphorylated levels of FAK and Erk1/2 by immunoblotting using anti-FAK, FAK pY³⁹⁷, FAK pY⁸⁶¹, Erk, and pErk1/2. In some conditions, HMVEC were pre-treated with LM609 or P1F6 (10 µg/ml) for 15 min and then stimulated with YKL-40 (100 ng/ml) for 10 min. E. HMVEC were measured for tube formation in the presence of YKL-40 (100 ng/ml) and/or PD98059 (10 µM). n=3. *P<0.05 compared with control cells. ⁺P<0.05 compared with cells treated with YKL-40 alone.

Figure 6. YKL-40 gene knockdown inhibits angiogenesis

A. Western blot analysis displayed YKL-40 gene knockdown in U87 cells. Cell cultured serum-free media were collected for immunoblotting. MG-63 cells as a positive control. B. The conditioned medium of U87 cells was transferred to HMVEC for measuring tube formation and migration. *P<0.05 compared with the control. n=4. C. The time course for the development of xenografted tumors in mice. U87 cells expressing vector control, YKL-40 siRNA 1 or siRNA 2 were subcutaneously injected into SCID/Beige mice. *P<0.05 compared with control group. n=5. D. Immunohistochemistry with CD31 staining showed higher levels of microvasculature in YKL-40 control tumors than those seen in YKL-40 siRNA1 and siRNA2 tumors. *P<0.05 compared with control group. n=5. Bar: 100 µm.

Figure 7. YKL-40 expression correlates with blood vessel density in breast cancer tissue

A. Human breast cancer specimens were performed for IHC YKL-40 (top panel) and CD34 staining (low panel). The images from the left to the right represent the levels of YKL-40 and CD34 from negative/low to high in cancer tissues. Bar: 100 µm. B. Cancer cases were categorized into three groups according to the expression levels of YKL-40 (negative/low: n=20; medium: n=9; and high: n=9) as described in the Methods. Blood vessel density in each case was quantified with an average of CD34 arbitrary density numbers from six to eight fields using an NIH image analysis program. Red bars indicate the average levels of CD34 density. C. All of cancer cases were plotted to analyze the relationship between expression levels of YKL-40 and CD34 density by the software of Significance of a Correlation Coefficient.