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SCF(β-TRCP) suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2.

Shaik S, Nucera C, Inuzuka H, Gao D, Garnaas M, Frechette G, Harris L, Wan L, Fukushima H, Husain A, Nose V, Fadda G, Sadow PM, Goessling W, North T, Lawler J, Wei W - J. Exp. Med. (2012)

Bottom Line: Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC.We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells.These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCF(β-TRCP) promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)-dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP-depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

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β-TRCP regulates VEGFR2 protein levels in human PTC-derived cells, and thereby influences sensitivity to the VEGFR2 inhibitor sorafenib. (A and B) HMVECs were co-cultured with EV or β-TRCP–expressing BCPAP cells in the presence or absence of VEGF-A antibody. (B) Cells were photographed after 44 h. Bars, 5 µm. Data are representative of three independent experiments. (C) Quantitative measurement of migrated BCPAP cells shown in B. The error bars represent mean ± SD. ***, P < 0.001 (n = 3). (D) Immunoblot analysis of the BCPAP cells infected with the indicated shRNA lentiviral vectors. Data shown is representative of two independent experiments. (E) BCPAP cells shown in D were exposed to 200 ng/ml VEGF-A where indicated. Recruited cells were photographed after 5 h. Bars, 5 µm. Data shown is representative of two independent experiments. (F) Various PTC cell lines were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. Data shown is representative of three independent experiments. The error bars represent mean ± SD. *, P < 0.05; ***, P < 0.001 (n = 3). (G) EV or HA–β-TRCP1–expressing BCPAP cells were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. The error bars represent mean ± SD. **, P < 0.01; ***, P < 0.001 (n = 3).
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fig9: β-TRCP regulates VEGFR2 protein levels in human PTC-derived cells, and thereby influences sensitivity to the VEGFR2 inhibitor sorafenib. (A and B) HMVECs were co-cultured with EV or β-TRCP–expressing BCPAP cells in the presence or absence of VEGF-A antibody. (B) Cells were photographed after 44 h. Bars, 5 µm. Data are representative of three independent experiments. (C) Quantitative measurement of migrated BCPAP cells shown in B. The error bars represent mean ± SD. ***, P < 0.001 (n = 3). (D) Immunoblot analysis of the BCPAP cells infected with the indicated shRNA lentiviral vectors. Data shown is representative of two independent experiments. (E) BCPAP cells shown in D were exposed to 200 ng/ml VEGF-A where indicated. Recruited cells were photographed after 5 h. Bars, 5 µm. Data shown is representative of two independent experiments. (F) Various PTC cell lines were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. Data shown is representative of three independent experiments. The error bars represent mean ± SD. *, P < 0.05; ***, P < 0.001 (n = 3). (G) EV or HA–β-TRCP1–expressing BCPAP cells were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. The error bars represent mean ± SD. **, P < 0.01; ***, P < 0.001 (n = 3).

Mentions: However, this could not explain why elevated VEGFR2 expression in a subset of human thyroid cancer cells, by itself, could lead to increased angiogenesis which is typically associated with human endothelial cells. To address this question, we used a trans-well system of co-cultured BCPAP cells and human endothelial cells grown in the absence of serum to examine whether manipulation of the β-TRCP1–VEGFR2 signaling pathway in BCPAP cells could affect cellular migration of human endothelial cells (Fig. 9 A). Interestingly, we found that VEGFR2 levels in BCPAP cells play a critical role in determining migratory ability of the co-cultured HMVECs. Very few HMVECs migrated when they were co-cultured with β-TRCP1–expressing BCPAP cells compared with BCPAP-EV cells (Fig. 9, B and C). This is likely a result of the elevated secretion of VEGF-A from the BCPAP-EV cells because the use of VEGF-A blocking antibody could efficiently abolish cellular migration of the co-cultured HMVECs (Fig. 9, B and C). Furthermore, VEGFR2 knockdown BCPAP (shVEGFR2) cells showed a significant decrease in cell migration compared with control (shGFP) BCPAP cells (Fig. 9, D and E). Overall, these results indicated that elevated VEGFR2 expression in human thyroid cancer cells could not only regulate their own migratory ability via an autocrine mechanism but also regulate the migration of the surrounding human endothelial cells, presumably through a paracrine mechanism. The latter mechanism might offer a possible molecular mechanism for increased VEGFR2 expression that is frequently observed in various human cancers and its correlation with increased angiogenesis.


SCF(β-TRCP) suppresses angiogenesis and thyroid cancer cell migration by promoting ubiquitination and destruction of VEGF receptor 2.

Shaik S, Nucera C, Inuzuka H, Gao D, Garnaas M, Frechette G, Harris L, Wan L, Fukushima H, Husain A, Nose V, Fadda G, Sadow PM, Goessling W, North T, Lawler J, Wei W - J. Exp. Med. (2012)

β-TRCP regulates VEGFR2 protein levels in human PTC-derived cells, and thereby influences sensitivity to the VEGFR2 inhibitor sorafenib. (A and B) HMVECs were co-cultured with EV or β-TRCP–expressing BCPAP cells in the presence or absence of VEGF-A antibody. (B) Cells were photographed after 44 h. Bars, 5 µm. Data are representative of three independent experiments. (C) Quantitative measurement of migrated BCPAP cells shown in B. The error bars represent mean ± SD. ***, P < 0.001 (n = 3). (D) Immunoblot analysis of the BCPAP cells infected with the indicated shRNA lentiviral vectors. Data shown is representative of two independent experiments. (E) BCPAP cells shown in D were exposed to 200 ng/ml VEGF-A where indicated. Recruited cells were photographed after 5 h. Bars, 5 µm. Data shown is representative of two independent experiments. (F) Various PTC cell lines were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. Data shown is representative of three independent experiments. The error bars represent mean ± SD. *, P < 0.05; ***, P < 0.001 (n = 3). (G) EV or HA–β-TRCP1–expressing BCPAP cells were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. The error bars represent mean ± SD. **, P < 0.01; ***, P < 0.001 (n = 3).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: β-TRCP regulates VEGFR2 protein levels in human PTC-derived cells, and thereby influences sensitivity to the VEGFR2 inhibitor sorafenib. (A and B) HMVECs were co-cultured with EV or β-TRCP–expressing BCPAP cells in the presence or absence of VEGF-A antibody. (B) Cells were photographed after 44 h. Bars, 5 µm. Data are representative of three independent experiments. (C) Quantitative measurement of migrated BCPAP cells shown in B. The error bars represent mean ± SD. ***, P < 0.001 (n = 3). (D) Immunoblot analysis of the BCPAP cells infected with the indicated shRNA lentiviral vectors. Data shown is representative of two independent experiments. (E) BCPAP cells shown in D were exposed to 200 ng/ml VEGF-A where indicated. Recruited cells were photographed after 5 h. Bars, 5 µm. Data shown is representative of two independent experiments. (F) Various PTC cell lines were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. Data shown is representative of three independent experiments. The error bars represent mean ± SD. *, P < 0.05; ***, P < 0.001 (n = 3). (G) EV or HA–β-TRCP1–expressing BCPAP cells were treated with the indicated concentrations of sorafenib. Cell viability was measured at 48 h. The error bars represent mean ± SD. **, P < 0.01; ***, P < 0.001 (n = 3).
Mentions: However, this could not explain why elevated VEGFR2 expression in a subset of human thyroid cancer cells, by itself, could lead to increased angiogenesis which is typically associated with human endothelial cells. To address this question, we used a trans-well system of co-cultured BCPAP cells and human endothelial cells grown in the absence of serum to examine whether manipulation of the β-TRCP1–VEGFR2 signaling pathway in BCPAP cells could affect cellular migration of human endothelial cells (Fig. 9 A). Interestingly, we found that VEGFR2 levels in BCPAP cells play a critical role in determining migratory ability of the co-cultured HMVECs. Very few HMVECs migrated when they were co-cultured with β-TRCP1–expressing BCPAP cells compared with BCPAP-EV cells (Fig. 9, B and C). This is likely a result of the elevated secretion of VEGF-A from the BCPAP-EV cells because the use of VEGF-A blocking antibody could efficiently abolish cellular migration of the co-cultured HMVECs (Fig. 9, B and C). Furthermore, VEGFR2 knockdown BCPAP (shVEGFR2) cells showed a significant decrease in cell migration compared with control (shGFP) BCPAP cells (Fig. 9, D and E). Overall, these results indicated that elevated VEGFR2 expression in human thyroid cancer cells could not only regulate their own migratory ability via an autocrine mechanism but also regulate the migration of the surrounding human endothelial cells, presumably through a paracrine mechanism. The latter mechanism might offer a possible molecular mechanism for increased VEGFR2 expression that is frequently observed in various human cancers and its correlation with increased angiogenesis.

Bottom Line: Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC.We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells.These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cancer Biology and Angiogenesis, Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
The incidence of human papillary thyroid cancer (PTC) is increasing and an aggressive subtype of this disease is resistant to treatment with vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor. VEGFR2 promotes angiogenesis by triggering endothelial cell proliferation and migration. However, the molecular mechanisms governing VEGFR2 stability in vivo remain unknown. Additionally, whether VEGFR2 influences PTC cell migration is not clear. We show that the ubiquitin E3 ligase SCF(β-TRCP) promotes ubiquitination and destruction of VEGFR2 in a casein kinase I (CKI)-dependent manner. β-TRCP knockdown or CKI inhibition causes accumulation of VEGFR2, resulting in increased activity of signaling pathways downstream of VEGFR2. β-TRCP-depleted endothelial cells exhibit enhanced migration and angiogenesis in vitro. Furthermore, β-TRCP knockdown increased angiogenesis and vessel branching in zebrafish. Importantly, we found an inverse correlation between β-TRCP protein levels and angiogenesis in PTC. We also show that β-TRCP inhibits cell migration and decreases sensitivity to the VEGFR2 inhibitor sorafenib in poorly differentiated PTC cells. These results provide a new biomarker that may aid a rational use of tyrosine kinase inhibitors to treat refractory PTC.

Show MeSH
Related in: MedlinePlus