G. Oshima1,2, S. P. Pitroda2,3, C. Zhang4, L. Huang4, A. Uppal1, S. C. Wightman1, M. E. Stack1, S. Lim1, X. Huang2,3, T. E. Darga2,3, J. Andrade4, M. C. Posner1,3, N. N. Khodarev2,3, R. R. Weichselbaum2,3 2University Of Chicago,Department Of Radiation And Cellular Oncology,Chicago, IL, USA 3University Of Chicago,Ludwig Center For Metastasis Research,Chicago, IL, USA 4University Of Chicago,Center For Research Informatics,Chciago, IL, USA 1University Of Chicago,Department Of Surgery,Chciago, IL, USA
Introduction: Oligometastasis is a clinically distinct patient subset characterized by a limited numbers of metastases, low rates of progression and favorable prognosis after resection or ablative local therapy. Recently we found oligometastatic lesions are associated with the expression of several microRNAs located in a distinct microRNA cluster in chromosome 14 (14q32 locus, see ref. 1). This locus and chromosomal locus 19q13 represent two unique microRNA clusters in the human genome. They both are located in imprinted regions of the genome, where gene activity is regulated by allele-specific DNA methylation. We therefore hypothesized that DNA methylation is implicated in regulation of microRNAs associated with oligometastatic disease and located in methylation-dependent loci 14q32 and 19q13. We suggest that suppression of DNA methylation will reactivate these microRNAs and achieve an oligometastatic state.
Methods: HCT116 cells were stably transfected by luciferase and tdTomato genes for in vivo and ex vivo monitoring of metastases development. Cells were treated by DNA-methyltransferase I (DNMT1) inhibitor 5′-Aza-C (5μM) for 48 hours. Whole-genome differentially methylated CpG loci were assessed with Illumina Infinium HumanMethylation450 BeadChip. For whole-genome expressional profiling of mRNAs and microRNAs we used Illumina HT-12 bead arrays and Affymetrix GeneChip arrays. For ectopic expression of selected microRNAs (miR-127-3p, miR-329-3p, miR-382-5p and miR-432-5p) that were up-regulated after DNMT1 inhibitor treatment in vitro, HCT116 cells were transfected by microRNA mimics with subsequent intrasplenic injection of transfected cells to generate liver metastases. Growths of liver metastases were monitored based on in vivo luminescence with subsequent ex vivo fluorescent imaging as described in (2).
Results: We found that limited demethylation of genomic DNA led to predominant reactivation of 14q32 and 19q13-encoded microRNAs. Using DIANA-microT and miRBase databases of predicted interactions between microRNAs and mRNAs we further found that ~80% of down-regulated genes can be explained by demethylation-dependent reactivation of microRNAs located in these two regions of the human genome. Methylation levels were significantly reduced in 78% of CpG loci in 14q32 after DNMT1 inhibitor treatment. Intrasplenic injection of HCT116 cells, ectopically expressing miR-382-5p led to the 1.5-fold reduction of liver metastases as compared with non-targeting control.
Conclusion: Our findings represent first indication that microRNAs implicated in the development of clinical oligometastases may be regulated epigenetically by differential DNA methylation and target genes involved in metastases development. Our results also suggest therapeutic potential of DNA methylation inhibitors and microRNAs in the treatment of liver metastases.
(1) Uppal A, et al. Oncotarget. 28;6(6):3540-52 (2015)
(2) Oshima G, et al. Sci Rep. 22;5:10946 (2015)