Abstract 2508: Blockade of Raf/MEK/ERK pathway by Raf265 inhibits tumor growth in colorectal cancer

Autor: Benjamin Cy Wong, Roberta Wc Pang, Andrew Cy Chu, Ronnie T.P. Poon, Ariel Km Chow
Rok vydání: 2010
Předmět:
Zdroj: Cancer Research. 70:2508-2508
ISSN: 1538-7445
0008-5472
DOI: 10.1158/1538-7445.am10-2508
Popis: Deregulation of the Raf/MEK/ERK signalling pathway is commonly observed in colorectal cancer (CRC). Since this signalling pathway plays a central role in controlling cell proliferation, apoptosis and differentiation; therefore, a number of therapeutics targeting on the Raf/MEK/ERK pathway has been established recently. Raf265 is an orally bioavailable small molecule which is a potent inhibitor of wild-type and mutant (e.g. V600E) B-raf kinases. The study of Raf265 has entered Phase I clinical trial in subjects with locally advanced or metastatic melanoma. However, its therapeutic efficacy in CRC has not been established. The objective of this study is to examine the functional effects of Raf265 in CRC cells in vitro and in vivo. CRC cell lines of different B-raf status were used for this study. Cell proliferation upon Raf265 treatment (0-50 μM) was determined using MTT cell proliferation assay. Cell cycle distribution and cell apoptosis upon Raf265 treatment (0, 1, 5, and 10 μM) were assessed by flow cytometry. Phosphorylation of molecules including MEK and ERK 1/2, and eIF4E, and expression of Mcl-1 and cyclin D1 was analyzed using Western blot. For in vivo animal studies, subcutaneous tumors were established by subcutaneous injections of 1×106 cells into nude or SCID mice, and tumor growth was monitored. Mice were sacrificed week 16 or when tumor sizes exceeded 30% of their body weight. Raf265 significantly inhibited cell proliferation in a dose-dependent manner with IC50 at 0.83 to 5.54 μM. Increased annexin V positive cells were observed with escalating dose of Raf265, which is indicative of induction of apoptosis in CRC cells. Dose-dependent increase in G1 and decrease in S phase population (cell cycle arrest at G1 phase) was also observed after treatment with Raf265. This was accompanied by the reduction of phosphor-MEK and phosphor-ERK 1/2. Down-regulation of Mcl-1 and cyclin D1, which regulate apoptosis and cell proliferation, respectively, was also observed. Intraperitoneal injections of Raf265 four times weekly demonstrated significant anti-tumor activity in established tumors of xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of ERK 1/2 phosphorylation in the xenograft tumors, consistent with inhibition of the RAF/MEK/ERK pathway. Additional analyses of microvessel density in the same tumour sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in xenograft models. Furthermore, repeated sequential use of Raf265 and chemotherapy with 5-Fu and/or cisplatin demonstrated synergistic effect of inhibition of tumor growth compared with Raf265 or chemotherapy alone. These pre-clinical data demonstrate robust anti-tumour activity of Raf265 in CRC, providing the basis for exploiting its potential use as a therapeutic for Raf-driven CRC tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2508.
Databáze: OpenAIRE
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