Regorafenib (BAY 73-4506): antitumor and antimetastatic activities in preclinical models of colorectal cancer

Abstract

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.

Keywords: CRC; antimetastasis; antitumorigenesis; multikinase inhibitor; regorafenib.

© 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Figures

Figure 1

Regorafenib inhibits growth-factor-stimulated VEGFR2 and VEGFR3 autophosphorylation in human umbilical vascular endothelial cells (HuVECs) and intracellular signaling and migration in lymphatic endothelial cells (LECs). Western blot analysis of (a) VEGFR2 and (b) VEGFR3, ERK1/2 and AKT from total cell lysates from (a) HuVECs and (b) LECs. All cells were treated with the indicated concentrations of regorafenib and subsequently stimulated with (a) VEGF-A or (b) VEGF-C. (c) Migration inhibition analyzed by scratch assay in LECs. Images are taken after 40 hr of incubation with 200 ng/mL VEGF-C. Boundaries of cell growth are marked by black lines. * indicates nonspecific signals.

Figure 2

Inhibition of proliferation and intracellular signaling by regorafenib in human colorectal cancer (CRC) cell lines. (a) Dose-dependent regorafenib-mediated inhibition of proliferation in 25 human CRC cell lines and mutational status of key oncogenic CRC driver genes taken from the COSMIC database of the Wellcome Trust Sanger Institute. (b) Western blot analyses of phosphorylated ERK1/2 from total cell lysates treated with the indicated concentrations of regorafenib.

Figure 3

Antitumor activity of regorafenib alone and in combination with irinotecan in oxaliplatin- and bevacizumab-refractory patient-derived colorectal cancer xenografts. Tumor growth curves (a, c) and time-to-event probability curves (b, d) of xenograft Co8183 (a, b) and xenograft Co8469 (c, d) are shown. Respective data for xenografts Co8541 and Co8213 are provided in Supporting Information Fig. 1. For tumor growth curves, data show arithmetic mean (±standard deviation) tumor volumes after treatment with oral vehicle or regorafenib, or intraperitoneal oxaliplatin or irinotecan according to the schedule depicted in a and c. Hatched areas indicate combination treatments. n = 8 animals/group. Arrows in a and c indicate the endpoints and dotted horizontal lines the defined tumor volume (event) used for the time-to-event probability calculations. In figures b and d (+) indicates censored events and (o) indicates nondrug-related death.

Figure 4

Tumor growth inhibition of patient-derived colorectal cancer (PD CRC) xenografts by regorafenib is mediated by antiangiogenic effects. Frozen sections of xenografts of model Co5896 grown in mice and treated orally with vehicle or regorafenib at a dose of 10 mg/kg per day for 22 days were stained immunohistochemically with antibodies against the endothelial cell marker protein CD31. (a) The percentage of CD31-positive area was determined and statistically evaluated as described in Material and Methods (n = 4; p = 0.0028). (b) Representative images of CD31 staining. The inset depicts an isotype IgG2a control image.

Figure 5

Antimetastatic activity of regorafenib in a murine CRC liver metastasis model. (a) Kaplan–Meier plot showing animal survival after treatment with vehicle or regorafenib (n = 10; p = 0.009). (b) Liver weights of mice killed at the time when the first animal in the vehicle group was sacrificed (n = 6; p < 0.05). (c) Representative images of metastases of the (left) liver and (right) mesenterium of vehicle-treated animals.