Discovery and validation of pan cancer epigenetic biomarkers

2017 Fiscal Year Research-status Report

• Project/Area Number: 17K18366
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: カチコフスキー ボグミル 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 研究員 (50648136)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Keywords: translational research / cancer / genome / epi-genome / transcriptome

Outline of Annual Research Achievements

In collaboration with researchers from Tokyo University, we performed the integrative analyses of gene expression and DNA methylation in lung cancer cell lines and clinical tumors (as described in stages 1-3 of the research plan). The results were published in Molecular Cancer Research (Horie, M., Kaczkowski, B.*, et al., 2017. Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC.: MCR, 15(10), *co-corresponding author.) In 2016, we showed that REP522 repeats are active as bi-directional promoters in multiple cancer types (Kaczkowski et al. 2016, Cancer Research). Here, we observed the that multiple copies of the REP522 DNA repeat family are, in fact, epigenetically activated in lung cancer by DNA hypomethylation and histone modification typical to active promoters (H3K4me3). The activated REP522 repeat elements act as bi-directional promoters for cancer-specific lncRNAs, e.g. RP1-90G24.10, AL022344.4, and PCAT7.
In the second step, I performed the pan-cancer analyses using RNA-Seq data from 21 tumor types profiled by The Cancer Genome Atlas (TCGA). I calculated the frequency (%) of activation/expression of REP522 promoters across 21 cancer types (7916 primary tumors and 725 normal tissue controls). The results were presented as a poster at Human Genomics meeting (Kaczkowski B.*, et. al. 2018, Human Genomics 2018, 12(Suppl 1):9).

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

The concept behind this grant - to integrate the epigenetic and transcriptomic data to find epigenetically regulated in cancer - was successfully implemented in lung cancer study and published in collaboration with the researchers from Tokyo University which allowed for an early publication/dissemination of the results (Horie, M., Kaczkowski, B.*, et al., 2017). The pan-cancer analysis is ongoing and preliminary result look promising, especially regarding the REP522 repeat family (Kaczkowski B.*, et. al. 2018, Human Genomics 2018, 12(Suppl 1):9, poster abstract).
Additionally, based on the results obtained so far, we designed new experiments and already started generating experimental data. Specifically, we are perturbing the DNA methylation and histone acetylation in normal cells, which is followed by promoter level transcriptomic profiling using CAGE technology. This way we aim to understand the direct link between the epigenetic aberrations and transcription and if hypomethylation on its own is enough to activate the transcription from repeat elements including REP522. This work is done in collaboration with Dr. Kazuhide Watanabe from RIKEN IMS.

Strategy for Future Research Activity

Stage 1,
As described above, the integrative analyses have been already performed in lung cancer and in FY2018 we plan to perform the analyses across multiple cancer types (pan-cancer analysis). This part will be performed as planned due to satisfactory preliminary results.
Stage 2,
The plan for wet-lab experiments will be updated because we now believe that it is more important to understand how the epigenetic alteration impact the gene expression. Thus, we are perturbing the DNA methylation and histone acetylation in normal cells (using DNA-demethylating agent and HDAC inhibitor), which is followed by promoter level transcriptomic profiling using CAGE technology. In FY2018, we will analyze the first data (currently in production) and we will perform the same perturbation in cancer cell lines to understand the differences of how normal and cancer cells respond to epigenetic drugs.
Depending on timeline and resources, we may perform some knock-down of especially promising gene targets/epi-drivers, followed by functional assays and CAGE/RNA-seq.

Causes of Carryover

Because most of the activities performed in FY2017 were computational, the wet-lab experiments were postponed in time. Additionally, we wanted to obtain and analyze the results in normal cells first, before performing the experiments in cancer cell lines.

1,100,000 yen - CAGE library preparation and deep sequencing for 8 samples; 150,000 yen - Cell culture consumables; 200,000 yen - Travel to international conference; 300,000 yen - Publication costs

Research Products

• [Journal Article] Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC. (2017)
  • Author(s): Horie M, Kaczkowski B, Ohshima M, Matsuzaki H, Noguchi S, Mikami Y, Lizio M, Itoh M, Kawaji H, Lassmann T, Carninci P, Hayashizaki Y, Forrest ARR, Takai D, Yamaguchi Y, Micke P, Saito A, Nagase T.
  • Journal Title: Molecular Cancer Research Volume: 15 Pages: 1354-1365
  • DOI: 10.1158/1541-7786.MCR-17-0191
  • Peer Reviewed / Open Access / Int'l Joint Research