Abstract 3265: Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors
Authors: Carbone Carmine , Piro Geny , Simionato Francesca , Loupakis Fotios , Cremolini Chiara , Fontanini Gabriella , Di Nicolantonio Federica , Moratti Elisa , Ligorio Francesca , Zanotto Marco , Santoro Raffaela , Mina Maria Mihaela , Scarpa Aldo , Bardelli Alberto , Tortora Giampaolo , Melisi Davide 
Published: July 2016

Abstract
Resistance to anti-angiogenic therapies poses one of the greatest challenges in gastrointestinal tumors research. We recently identified several proinflammatory secreted factors, including interleukin-1 (IL1), CXC ligand (CXCL) 1 and 8, Transforming Growth Factor β (TGFβ)1, and Angiopoietin-like Protein 2 (ANGPTL2), that were overexpressed in murine models of tumors resistant to the anti-VEGF antibody bevacizumab (BEV). These factors induced epithelial-to-mesenchymal transition (EMT) and increased aggressiveness. HOXB9 has been identified as a key transcription factor in common for these factors. Here, we hypothesized that HOXB9 might be responsible for BEV resistance in gastrointestinal tumors.
HOXB9 expression and activation were measured in BEV-sensitive COLO357FG and in their BEV-resistant counterpart FGBR pancreatic cancer cell lines, and in LOVO, MDST8, LIM2099, CCK81, GP5D, and SNUC4 colon cancer cell lines by EMSA and DAPA. Serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 in nude mice bearing tumors were measured by multiplex xMAP technology. Immunohistochemical analyses were performed in pre- vs. post-progression biopsies from colorectal cancer patients receiving BEV.
HOXB9 protein was more expressed and activated in FGBR than did in COLO357FG cells. shRNA to knock down HOXB9 significantly (P&lt;0.01) reduced proinflammatory factors and EMT markers expression, and migration in FGBR cells. Mice bearing HOXB9-silenced tumors had significantly (P&lt;0.01) lower serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 than did their respective controls. BEV significantly reduced tumor burden (P≤0.0001) and prolonged survival duration (P = 0.0006) in mice bearing HOXB9-silenced FGBR tumors, whereas it was ineffective on FGBR scramble tumors. HOXB9-positive LOVO, MDST8, and LIM2099 cells had significantly higher expression levels of proinflammatory factors and EMT markers, and migration rates than did HOXB9-negative CCK81, GP5D, and SNUC4 colon cancer cell lines. In vivo, BEV significantly reduced tumor burden and prolonged survival duration in mice bearing CCK81 or GP5D tumors (P&lt;0.05), whereas it was ineffective on MDST8 and LIM2099 tumors. HOXB9 shRNA significantly reduced proinflammatory factors and EMT markers expression, and migration in MDST8 cells. BEV significantly prolonged survival duration in mice bearing HOXB9-silenced MDST8 tumors (P = 0.0025), but did not on MDST8 scramble tumors. Validation of the differential expression of HOXB9 in biopsies from colorectal patients progressing under BEV therapy will be available at the time of the meeting.
In conclusion, we identified HOXB9 as crucial transcription factor to sustain BEV resistance in gastrointestinal tumors. Silencing of HOXB9 is a promising approach to modulate this resistance. Our results candidate HOXB9 as potential biomarker for selecting patients with colorectal and pancreatic cancer for antiangiogenic therapy.
Citation Format: Carmine Carbone, Geny Piro, Francesca Simionato, Fotios Loupakis, Chiara Cremolini, Gabriella Fontanini, Federica Di Nicolantonio, Elisa Moratti, Francesca Ligorio, Marco Zanotto, Raffaela Santoro, Maria Mihaela Mina, Aldo Scarpa, Alberto Bardelli, Giampaolo Tortora, Davide Melisi. Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3265.