P-221 - Acquired 5-FU resistance in CRC models is accompanied by upregulation of VEGF-VEGFR1 signaling, increased migration and invasion that can be attenua...

Date 04 July 2015
Event WorldGI 2015
Session Posters
Topics Anti-Cancer Agents & Biologic Therapy
Basic Science
Colon Cancer
Rectal Cancer
Presenter A. Larsen
Citation Annals of Oncology (2015) 26 (suppl_4): 1-100. 10.1093/annonc/mdv233
Authors A. Larsen1, A. De Gramont2, M. Ayadi3, T. André4, A. Bouygues5, P. Mesange6, M. Chiron7, E. Dochy8
  • 1Laboratory of Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Paris, France; Institut National de la Santé et de la Recherche Médicale (INSERM) U938, Paris, France; Université Pierre et Marie Curie (UPMC), Paris/FR
  • 2Institut Hospitalier Franco-Britannique, Levallois-Perret/FR
  • 3Cancer Biology and Therapeutics; Centre de Recherche Saint-Antoine; INSERM U938 and Université Pierre et Marie Curie, Paris/FR
  • 4Hopital Saint-Antoine, Paris/FR
  • 5Laboratory Of Cancer Biology And Therapeutics, Centre de Recherche Saint-Antoine, Paris/FR
  • 6Laboratory of Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Paris/FR
  • 7Sanofi Aventis, Vitry-sur-Seine/FR
  • 8Sanofi, Diegem/BE

Abstract

Introduction

Colorectal cancer (CRC) cells express VEGF as well as functional VEGF receptor 1 (Flt-1) thereby mediating both paracrine and autocrine VEGF-signaling. Autocrine VEGF-signaling in CRC cells has been associated with resistance to 5-FU and hypoxia as well as with increased migration and invasion. Aflibercept, also known as ziv-aflibercept in the United States, is a soluble recombinant protein approved for treatment of metastatic CRC that selectively neutralizes all known VEGFR1 ligands including VEGF-A, VEGF-B and PlGF. We here wanted to establish if aflibercept can attenuate the migration and invasion of chemo-naïve and 5-FU resistant CRC cells.

Methods

Ten well-characterized CRC cell lines and two 5-FU resistant variants were used in these studies. The Boyden chamber was used to establish the influence of aflibercept on CRC cell migration and invasion. The cytotoxic effects of 5-FU in the absence or presence of aflibercept was characterized by the MTT viability assay. The expression of VEGF ligands in CRC cells and tumor xenografts were determined by ELISA, while the active, autophosphorylated forms of VEGFR1 and VEGFR2 (Flk-1/KDR) in CRC xenografts were characterized by quantitative fluorescent immunohistochemistry.

Results

Aflibercept attenuated the migration of all ten CRC cell lines under both normoxia and hypoxia (p < 0.01 and p < 0.001). The migration was mediated by VEGFR1 since i) the migration was stimulated by VEGFR1 ligands (VEGF-A, VEGF-A and PlGF) but not by VEGF-E, a specific VEGFR2 ligand and ii) was attenuated by blocking antibodies to VEGFR1, but not to VEGFR2. Two independently selected 5-FU resistant CRC models showed increased expression of VEGF-A (p < 0.001) and the activated form of VEGFR1 (p < 0.01) in vitro and in tumor xenografts without upregulation of VEGFR2. The increased VEGF-A levels were associated with up to 5-fold increased migration of both 5-FU resistant cell lines, compared to the parental cells, under both normoxia and hypoxia (p < 0.001) and could be attenuated by aflibercept (p < 0.001). Invasion was increased 6- to 11-fold for both 5-FU resistant cell lines under normoxia and hypoxia (p < 0.001) and could be attenuated by aflibercept (p < 0.05 and p < 0.001). Aflibercept by itself had no detectable influence on the viability of parental or 5-FU resistant cells under normoxia or hypoxia, but was able to increase the sensitivity of 5-FU resistant cells to 5-FU under hypoxia.

Conclusion

Aflibercept directly attenuated the migration of CRC cells under all conditions examined without any influence on the viability. Cells with acquired 5-FU resistance showed up-regulation of the VEGF-VEGFR1 axis associated with increased migration and invasion, especially under hypoxic conditions. Aflibercept inhibited these functions. Our results suggest that aflibercept have direct anti-invasive properties in addition to its well-established antiangiogenic activities.