Twist Is Required for Thrombin-Induced Tumor Angiogenesis as Well as Migration.

Twist, a master regulator of embryonic morphogenesis, induces functions that are also required for tumor invasion and metastasis. A recent study (Cell117: 927, 2004) defined a requirement of Twist for tumor metastasis unrelated to tumor growth and invasion. We now report a requirement of Twist for angiogenesis. It has been speculated that Twist expression might be derepressed during tumor progression. Indeed, high levels of Twist correlate with invasive lobular breast carcinoma, gastric carcinoma, prostate cancer and metastatic nasopharyngeal carcinoma. Since thrombin contributes to the malignant phenotype by upregulating tumor metastasis (as well as growth, invasion and angiogenesis) we proposed that it might be operating through the upregulation of Twist. Five different tumor cell lines [(Human MCF7 Breast, DU145 Prostate and murine 4T1 breast, B16F10 melanoma, undifferentiated cell line (UMCL)] and 2 different endothelial cell lines [HUVEC and Brain microvascular endothelial cell (BMEC)] were examined. Thrombin upregulated Twist mRNA and protein (>2 fold) in all 7 cell lines, indicating a general biologic effect. In Twist knock down (KD) studies of B16F10 tumor growth on a chick choriallantoic membrane we noted an ∼45% decrease in angiogenesis (neoangiogenesis branching), n=8. This suggested that B16F10 cells might contain inducible vascular growth factors and receptors that are present in endothelial cells. Twist was therefore further examined in both HUVEC and tumor cells and compared with known effects of thrombin on angiogenesis. Twist KD in HUVEC led to a 2.8 fold impaired thrombin-induced chemotaxis and 5.6 fold impaired vascular tubule formation in matrigel compared to wild type, empty vector and scrambled vector, n=3. Similar Twist KD results were noted in BMEC. We next introduced a tetracycline-dependent Twist-inducible plasmid into B16F10 cells and vascular factors analyzed by immunoblot. A 3-8 fold upregulation was noted for VEGF and its KDR receptor, Ang-2 and its Tie 2 receptor and the growth-regulated oncogene (GRO-α) chemokine, n=2–3. GRO-α has recently been shown to be responsible for thrombin-induced upregulation of angiogenesis and tumor growth (Cancer Res66:4125, 2006). In studies on the effect of thrombin on a tetracycline-dependent Twist-inducible plasmid, the mRNA and protein of several vascular growth factors and receptors were shown to be identically upregulated (by Twist or thrombin) 2–3 fold in tumor cells as well as HUVEC (GRO-α, VEGF, KDR, Ang-2, MMP-1 and CD31), n=3–5. These data suggest that the positive effect of thrombin on angiogenesis requires the upregulation of Twist. Since the chemokine GRO-α upregulates the identical vascular growth factors and receptors as Twist, this prompted an examination of the relationship of Twist and GRO-α with thrombin. Utilizing a GRO-α inducible plasmid system in B16F10 cells, Twist mRNA and protein were both shown to be upregulated ∼2 fold. Other vascular growth factor proteins were also upregulated 2–5 fold (Ang-2, KDR, VEGF and CD31), n=2–3. Thus Twist regulates angiogenesis as well as metastasis. Thrombin upregulates Twist which is required for thrombin-induced angiogenesis as measured by tumor angiogenesis index, endothelial cell migration and matrigel tubule formation. Twist upregulates GRO-α which can upregulate Twist resulting in positive feedback. It is suggested that the positive effect of thrombin on angiogenesis is via its upregulation of both Twist and GRO-α.