CircRNAs: novel biomarkers for early detection of non-small cell lung cancer

What is the problem to be addressed?

The DNA in our body generates messages in the form of RNA (ribonucleic acid) to create, maintain and regulate our cells. Altered levels of specific RNAs are observed in the cells of people with lung cancer. This study aimed to improve early detection by investigating a novel type of RNA, called circular RNA (circRNA). circRNAs that change in the cancers of patients can potentially be detected in blood and may allow us to identify and manage patients earlier, giving them a better chance of long-term survival.

Background

The human genome is the genetic blueprint for building a human being. It is made up of deoxyribonucleic acid (DNA), and was sequenced in 2000. To make our protein, the cell generates messages from this DNA into ribonucleic acid (RNA).

Called messenger RNA (mRNA), these are generated from our genomic DNA by a process called transcription, and these mRNAs are used by the cell to make our proteins. We now know that only 2% of our total DNA is required to make these mRNAs used by the cell to make protein. However, it is also known that 75% of our genomic DNA is actually made into some form of RNA by transcription (also known as the transcriptome). This additional 73%, which is not used by the cell to make protein is known as non-coding RNA (ncRNA), and we are learning that these play important additional roles within our cells. Various different types of ncRNAs have been discovered including microRNAs (miRNAs), long non-coding RNAs (lncRNAs). Both of these ncRNAs have been shown to play important roles in cancer (including lung cancer). Another ncRNA, called circular RNA (circRNA), has been described which plays important roles in both the regulation of mRNA transcription and disease progression, but there is currently very little known about whether circRNAs are important in lung cancer.

What we do know is that circRNAs can be tissue specific (that is they are found only in a particular tissues, for example, the lung), and some circRNAs have recently been shown to show altered expression in cancer. We believe that there may be a number of circRNAs which are specific to lung, and may show altered expression during lung cancer development. These circRNAs might therefore allow us to detect lung cancer.

Taken together, these factors suggest that circRNAs could potentially be important biomarkers in early lung cancer diagnosis.

In this pilot study we sought to sequence the circRNA transcriptomes from a series of early stage non-small cell lung cancers (NSCLCs) with matched normal lung tissue taken from the same individuals at surgery to:

• establish the circRNAs that are specific for normal lung tissue;
  • identify the circRNAs that are altered in early stage NSCLC compared to normal lung;
  • confirm these observations in a larger number of patient samples and test if these circRNAs can be detected in patient blood samples.

Methods

Total RNAs isolated from a panel of matched normal/tumour NSCLC adenocarcinoma (stages 1A/1B) samples (n=6) were probed for circRNAs using the Arraystar circRNA microarray. Survival was assessed on linear RNAs with associated circRNAs using KM-Plot.

Findings and Outcomes

The project, which ran for one year, January to December 2016, achieved the following:

• It demonstrated a clear separation between the tumours and normal lung in terms of circRNAs for early stage NSCLC Adenocarcinomas.
• We have also demonstrated that for n=8 of the top altered circRNAs, the genes associated with these circRNAs are (a) specific to the Adenocarcinoma subset; and (b) linked to patient survival.
• We have validated the expression of one of these circRNAs in the same tumour samples using a different methodology.
• We have demonstrated that the expression of a gene associated with one of these circRNAs is upregulated in NSCLC Adenocarcinomas and have identified a candidate drug that can target this gene.
• We have identified a number of candidate circRNAs which could potentially detect early stage lung Adenocarcinomas.
• We have identified a candidate gene which is overexpressed in NSCLC adenocarcinomas, for which drugs exist. This gene has not yet been examined in detail in lung cancer and represents a potentially novel therapeutic target.

One of the difficulties we encountered with circRNAs is that frequently there are many circRNAs of different sizes generated from the same gene. This has led to some problems with respect to PCR validation strategies as often multiple PCR products are generated. This is obviously non-optimal, and we continue to optimise our lead candidate circRNAs.

A second difficulty emerged as the main chiller unit for our research institute broke down. This meant that our Biobank was inaccessible for the last third of the project timeline, which has meant that validation of circRNAs in separate cohorts (and blood samples) has not been conducted.

This project produced data and reports published in several journals, contributing considerably to the literature surrounding this particular aspect of lung cancer research.

Publications:

How presented (oral, poster or journal publication)	Title	Year	Authors	Journal publication name / conference venue	Status (in submission, in press or published)
Poster	circRNAs: Potential novel biomarkers for the early detection of lung cancer	2016	Lin, R.1,2, Reid, G.1, Mutti, L.3 , Ryan, A.W.4, Nicholson S.5, Leonard N.5, Young V.6, Ryan R.6, Finn, S.P.7, Cuffe, S.8, and Gray, S.G.9	Journal of Thoracic Oncology Volume 12 Issue S1: S323 January 2017	Published Abstract
Poster	Are circRNAs potentially useful for the early detection of lung cancer?	2017	Lin, R.1,2, Reid, G.1, Mutti, L.3 , Ryan, A.W.4, Nicholson	Lung Cancer Volume 103 Issue S1: S4- S5 January 2017 S.5, Leonard N.5, Young V.6, Ryan R.6, Finn, S.P.7, Cuffe, S.8, and Gray, S.G.9	Published Abstract

Lead researcher: Dr Steven G. Gray | Location: St James’s Hospital,Dublin | Type of research: Early detection