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]).
- ARNTL Transcription Factors/genetics
- Bevacizumab/administration & dosage
- Cell Line, Tumor
- Colorectal Neoplasms/drug therapy
- Drug Resistance, Neoplasm/genetics
- Kaplan-Meier Estimate
- Neovascularization, Pathologic/drug therapy
- Progression-Free Survival
- Promoter Regions, Genetic/genetics
- Vascular Endothelial Growth Factor A/genetics