Aryl hydrocarbon receptor nuclear translocator-like (ARNTL/BMAL1) is associated with bevacizumab resistance in colorectal cancer via regulation of vascular endothelial growth factor A

Elke Burgermeister; Francesca Battaglin; Fagr Eladly; Wen Wu; Frank Herweck; Nadine Schulte; Johannes Betge; Nicolai Härtel; Jakob N Kather; Cleo-Aron Weis; Timo Gaiser; Alexander Marx; Christel Weiss; Ralf Hofheinz; Ian S Miller; Fotios Loupakis; Heinz-Josef Lenz; Annette T Byrne; Matthias P Ebert

doi:10.1016/j.ebiom.2019.07.004

Aryl hydrocarbon receptor nuclear translocator-like (ARNTL/BMAL1) is associated with bevacizumab resistance in colorectal cancer via regulation of vascular endothelial growth factor A

EBioMedicine. 2019 Jul:45:139-154. doi: 10.1016/j.ebiom.2019.07.004. Epub 2019 Jul 9.

Authors

Elke Burgermeister¹, Francesca Battaglin², Fagr Eladly³, Wen Wu³, Frank Herweck³, Nadine Schulte³, Johannes Betge³, Nicolai Härtel³, Jakob N Kather⁴, Cleo-Aron Weis⁵, Timo Gaiser⁵, Alexander Marx⁵, Christel Weiss⁶, Ralf Hofheinz⁷, Ian S Miller⁸, Fotios Loupakis⁹, Heinz-Josef Lenz¹⁰, Annette T Byrne¹¹, Matthias P Ebert³

Affiliations

¹ Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany. Electronic address: elke.burgermeister@medma.uni-heidelberg.de.
² Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, CA, United States; Unit of Medical Oncology 1, Clinical and Experimental Oncology Department, Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.
³ Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
⁴ Division of Gastroenterology, Hepatology and Hepatobiliary Oncology, University Hospital RWTH Aachen, Aachen, Germany.
⁵ Institute of Pathology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
⁶ Department of Medical Statistics, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
⁷ Department of Medicine III, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
⁸ Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.
⁹ Unit of Medical Oncology 1, Clinical and Experimental Oncology Department, Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.
¹⁰ Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, CA, United States.
¹¹ Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland; UCD School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.

Abstract

Background: The identification of new biomarkers and the development of novel, targetable contexts of vulnerability are of urgent clinical need in drug-resistant metastatic colorectal cancer (mCRC). Aryl-Hydrocarbon-Receptor-Nuclear-Translocator-Like (ARNTL/BMAL1) is a circadian clock-regulated transcription factor promoting expression of genes involved in angiogenesis and tumour progression. We hypothesised that BMAL1 increases expression of the vascular endothelial growth factor A VEGFA gene and, thereby, confers resistance to anti-angiogenic therapy with bevacizumab (Beva), a clinically used antibody for neutralization of VEGFA.

Methods: PCR and immunohistochemistry were employed to assess BMAL1 expression in mice (C57BL/6 J ^Apcmin/+; BALB/c ^nu/nu xenografts) and CRC patients under combination chemotherapy with Beva. BMAL1 single nucleotide gene polymorphisms (SNPs) were analysed by DNA-microarray in clinical samples. BMAL1 functions were studied in human CRC cell lines using colorimetric growth, DNA-binding and reporter assays.

Findings: In murine CRCs, high BMAL1 expression correlated with poor preclinical response to Beva treatment. In CRC patients' tumours (n = 74), high BMAL1 expression was associated with clinical non-response to combination chemotherapy with Beva (*p = .0061) and reduced progression-free survival (PFS) [*p = .0223, Hazard Ratio (HR) = 1.69]. BMAL1 SNPs also correlated with shorter PFS (rs7396943, rs7938307, rs2279287) and overall survival (OS) [rs11022780, *p = .014, HR = 1.61]. Mechanistically, Nuclear-Receptor-Subfamily-1-Group-D-Member-1 (NR1D1/REVERBA) bound a - 672 bp Retinoic-Acid-Receptor-Related-Orphan-Receptor-Alpha-responsive-element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the VEGFA gene promoter, resulting in increased VEGFA synthesis and proliferation of human CRC cell lines.

Interpretation: BMAL1 was associated with Beva resistance in CRC. Inhibition of REVERBA-BMAL1 signalling may prevent resistance to anti-angiogenic therapy. FUND: This work was in part supported by the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]).

Keywords: ARNTL; BMAL1; Bevacizumab; Colorectal cancer; REVERBA; VEGFA.

MeSH terms

ARNTL Transcription Factors / genetics*
Animals
Bevacizumab / administration & dosage*
Bevacizumab / adverse effects
Cell Line, Tumor
Colorectal Neoplasms / drug therapy*
Colorectal Neoplasms / genetics
Colorectal Neoplasms / pathology
Drug Resistance, Neoplasm / genetics
Female
Heterografts
Humans
Kaplan-Meier Estimate
Male
Mice
Neovascularization, Pathologic / drug therapy*
Neovascularization, Pathologic / genetics
Neovascularization, Pathologic / pathology
Progression-Free Survival
Promoter Regions, Genetic / genetics
Vascular Endothelial Growth Factor A / genetics*

Substances

ARNTL Transcription Factors
BMAL1 protein, human
Vascular Endothelial Growth Factor A
Bevacizumab