IMPaCT Investigator Highlights (Text Version) - Dr. Bernard Kwabi-Addo

Title: The Role of DNA Methylation in Prostate Cancer Health Disparity

Investigator: Bernard Kwabi-Addo, Ph.D., Howard University

My current grant is to really investigate DNA methylation changes in aging prostate tissues samples and also to study whether DNA methylation changes may be important in prostate cancer disparity.

Now when we look at the pathways from normal prostate cells to prostatic adenocarcinoma, the first step in the disease pathway of transformation from normal to prostate cancer is actually referred to as proliferative inflammation atrophy or PIA. And we notice that in these PIA cells, expression of key genes, one referred to as glutathione S-transferase 2, or GST pi and cyclooxygenase 2, or COX-2 are increased. And the protein product of these genes are increased in response to stress and inflammation.

But in about 5% of these PIA cells, GST-Pi is silenced okay and it’s predicted that this 5% of the PIA cells and those that progress to high-grade pain and prostatic adenocarcinoma—now the mechanism that causes the inactivation of silencing of the GST-Pi is due to DNA methylation, so we now know that disruption of DNA methylation is a very important mechanism that is involved in prostate cancer disease initiation.

It’s now known that hyper-methylation of GST-Pi is the most common observed event in prostate cancer and over 90% of prostate cancer disease have shown this hyper-methylation and so it is very common and perhaps more consistent than either genetic alterations that have been shown to occur in prostate cancer. So really this provides us with a very important opportunity to explore the DNA methylation as potential biomarkers for disease detection at an early stage and perhaps used alone or in conjunction with PSA screening to improve the sensitivity and specificity of prostate cancer detection.

Now DNA methylation changes per se does not change the primary DNA sequences. What it does is add a methyl group to a nucleotide called cytosine. So the approach we use is kind of helping us to really distinguish between methylated and unmethylated cytosine. And we can really quantify this mechanism using a technique called pyrosequencing, which is a robust high-throughput approach for quantitative DNA methylation studies.

And this Figure A is DNA samples—blind samples from a normal 52-year old male and we can basically see very, very little methylation at three CTG sites that we investigated in this sample. And B, it’s actually DNA samples from a 58-year old man with prostate cancer and we begin to see very, very high methylation frequency. So this approach really allows us to quantify and get an idea of the percentage of methylation that is occurring in the DNA samples that we are studying.

So one other interest of my lab is to really see if we can explore methylated genes as potential biomarkers for disease detection. So a useful candidate would show very little methylation than normal tissues, and the methylation will be significantly increased in the kinds of tissues. And some of the genes that we analyze show very, very low DNA methylation changes in the normal prostate tissues that we use and at least two- to threefold higher in the cancer tissues, to suggest that yes, indeed, we can really explore some of these methylated genes as novel biomarkers for disease detection, which we can use perhaps in conjunction with a PSA screening that is currently used for prostate cancer detection.

So the next thing we did was to investigate if there are differences in the methylation pattern. We looked at matched pairs of the normal, referred to as NL, and cancer tissues from individual patients. So again we see very little methylation in the normal tissues compared to the cancer tissues in the Caucasian samples and also in the African American samples. But it’s very interesting that we begin to see significantly higher methylation in the African American samples when we compare to the Caucasian.

We see that in African Americans the average methylation is significantly higher. So we use a regression analysis to do age, race interaction, and we observe significantly increased methylation levels in the tissue samples from African Americans compared to Caucasians.

We also know that there’s different PSA thresholds for African Americans compared to Caucasians so we really need additional biomarkers which are perhaps racial ethnic sensitive. So we—this slide shows a ROC curve analysis that really we used to investigate the sensitivity and specificity of DNA-methylated changes and distinguishing cancer from normal tissues. And in Caucasian samples the use of retinoic acid receptor and perhaps GST-Pi is really sensitive in detecting the cancer tissues in Caucasians. But when it comes to African American, we notice that perhaps other genes may be more sensitive in really detecting the prostate cancer. So our data indicate we can potentially explore the DNA-methylated genes to really not only to as a biomarker for disease detection but perhaps we may be able to generate DNA-methylated genes that are more sensitive to one racial group for prostate cancer disease detection.