Intratumoral hypoxia is associated with greater risk of metastasis and less favorable prognosis. Hypoxia increases the expression in tumor cells of c-MET, the tyrosine kinase receptor for hepatocyte growth factor, through HIF-1α binding sites on the c-MET promoter. Activation of c-MET promotes tumor cell proliferation, motility, and invasion and is associated with tumor aggressiveness. Intratumoral hypoxia is increased by vascular pruning produced by angiogenesis inhibitors that block VEGF signaling.

We asked whether selective VEGF inhibition, which exaggerates intratumoral hypoxia and activates c-MET, is sufficient to increase tumor invasiveness and metastasis and whether selective inhibition c-MET is sufficient to block these effects. We found that treatment of pancreatic neuroendocrine tumors in RIP-Tag2 mice with a neutralizing anti-VEGF antibody or with the tyrosine kinase inhibitor sunitinib reduced tumor burden. Both treatments also increased intratumoral hypoxia, expression and activation of c-MET, tumor invasion into the exocrine pancreas, and metastasis to the liver. Increased intratumoral hypoxia was documented by increased HIF-1α and staining for pimonidazole, carbonic anhydrase-IX, and Glut1 in tumor cells. Treatment with either agent for only a week was accompanied by increased expression of c-MET mRNA and corresponding increases in c-MET protein and c-MET phosphorylation in tumor cells. Exposure to hypoxia also increased c-MET phosphorylation in freshly isolated RIP-Tag2 tumor cells in vitro. With these findings, the selective anti-VEGF antibody #AF-493-NA was added to the list, which already includes sunitinib, anti-VEGFR-2 antibody (DC101), and genetic deletion of VEGF, of interventions reported to promote tumor invasion and metastasis in preclinical models.

To test the involvement of c-MET in the exaggerated tumor aggressiveness, we used three complementary approaches to block c-MET signaling. The first used the tyrosine kinase inhibitor PF-04217903 because of its high selectivity for c-MET. The second used PF-02341066 (crizotinib), a tyrosine kinase inhibitor that has high potency against c-MET but also blocks anaplastic lymphoma kinase (ALK). The third used the tyrosine kinase inhibitor XL184 (cabozantinib), which simultaneously blocks c-MET and VEGFR among other receptor tyrosine kinases.

We found that RIP-Tag2 tumors treated with either PF-04217903 or PF-02341066 from age 14 to 17 weeks were about the same size as vehicle-treated controls but were less invasive and had fewer metastases. After this treatment, the tumors had a smoother contour, a distinctive ball-like shape, and less invasion into the exocrine pancreas. Liver metastases were significantly smaller and less numerous. Inhibition of c-MET and VEGF together, by administration of PF-04217903 or PF-02341066 together with anti-VEGF antibody or sunitinib, slowed tumor growth and had even more potent effects on invasion and metastasis than found with either c-MET inhibitor used alone. These findings in RIP-Tag2 tumors were corroborated by parallel experiments on orthotopic Panc-1 pancreatic adenocarcinomas. Expression of c-MET and invasiveness of Panc-1 tumors were increased by treatment with sunitinib, and the increase was blocked by concurrent administration of PF-04217903. XL184 given as a single agent reduced tumor invasion and metastasis in RIP-Tag2 mice as much as the drug combinations. The similarity of effects of XL184 on tumor invasion and metastasis to those of selective inhibitors given together argues for c-MET and VEGFR being relevant targets of XL184, although additional involvement of inhibition of AXL, KIT, RET or other targets cannot be excluded for this multi-targeted agent.

To test whether concurrent inhibition of c-MET and VEGF signaling can reverse tumor invasion, we compared tumors in RIP-Tag2 mice treated for 3 weeks with tumors in onset controls at age 14 weeks. The amount of invasion found after treatment with PF-042107903 plus sunitinib or with XL184 given alone was less than in onset controls. Similarly, the metastatic burden in the liver was less after XL184 for 3 weeks than in onset controls. Overall, the experiments revealed that, consistent with published reports, inhibition of VEGF signaling by function-blocking antibody or sunitinib causes vascular pruning and slowing of tumor growth but also increases invasion and metastasis in these preclinical models. The findings fit with a mechanism involving vascular pruning, intratumoral hypoxia, HIF-1α accumulation, and activation of c-MET in tumor cells. Invasion and metastasis that accompany VEGF inhibition can be blocked by concurrent inhibition of c-MET. PF-04217903 and PF-02341066 have similar efficacy in this regard when either is given together with anti-VEGF antibody or sunitinib. Inhibition of both c-MET and VEGF signaling by XL184 has potent anti-angiogenic activity and reduces tumor growth while also reducing invasion and metastasis. The findings indicate that tumor invasion and metastasis can be reduced along with tumor growth by blocking c-MET and VEGF signaling together to offset the consequences of intratumoral hypoxia resulting from angiogenesis inhibition and vascular pruning.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY41-02. doi:1538-7445.AM2012-SY41-02