Histone Methylation as a Marker of Breast Cancer Progression

Institution: University of Southern California
Investigator(s): Judd Rice, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0085 Award: $162,500
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2005)
The fundamental molecular mechanisms that contribute to sporadic breast cancer progression remain unknown. Discrete genetic factors that predispose an individual to the disease appear to be lacking, indicating that mechanisms other than DNA mutation must be involved. Increasing evidence demonstrates that the disruption of epigenetic mechanisms (i.e., changes in gene expression without changes in the DNA) contribute to cellular transformation and cancer progression, including breast cancer. We recently reported that methylation of histone H4 lysine 20 (H4 Lys20) is an epigenetic event that is critical for the formation of heterochromatin, or transcriptionally silent (inactive) regions of the genome. We have also found that global changes in H4 Lys20 methylation levels are associated with aging, differentiation defects and genomic instability; all of which are hallmarks of aggressive cancers.

Based on our findings, we hypothesize that aberrant genome-wide alterations in heterochromatin, as measured by H4 Lys20 methylation, plays a direct role in sporadic breast cancer progression. We further hypothesize that the regions where changes are detected may likely serve as future epigenetic markers for the enhanced detection, prognosis and, perhaps, treatment of sporadic breast cancer. Our aims are to: (1) determine and compare the genomic patterns of H4 Lys20 methylation of normal breast cells and breast cancer cells using an innovative “ChIP” technique (chromatin immunoprecipitation assay), (2) analyze the data to identify regions with consistent significant changes in H4 Lys20 methylation (heterochromatin "markers") during progression, and (3) verify each "marker" using standard ChIPs, RT-PCR and chromatin accessibility assays. Established normal breast and cancer cell lines (non-metastatic and metastatic) will be used for comparison purposes. The genomic regions containing H4 Lys20 methylation will be isolated from each cell line using a novel panel of H4 Lys20 methyl-specific antibodies that we have developed. The resulting H4 Lys20 methyl-enriched DNA will be used as hybridization probes on a unique DNA microarray chip containing 6800 annotated human CpG islands. Analysis of the different groups will lead to information on the normal state of heterochromatin in breast epithelial tissue and how these patterns change during sporadic breast cancer progression (i.e., potential markers).

The potential impact of this research on breast cancer and breast cancer patients will be relatively immediate. The identification and verification of novel epigenetic markers will quickly translate into high-throughput screening of these regions from patients to aid physicians in the detection, assessment and treatment of the disease. In addition, these experiments will enhance the current understanding of the molecular mechanisms that underlie normal breast cell physiology and the pathways that lead to malignant transformation.

Final Report (2006)
We recently discovered that a normal cellular event, known as histone methylation, is frequently and significantly altered during breast cancer progression. Histones are a group of evolutionarily conserved proteins that package and organize the DNA in each of our cells. Different chemical modifications, such as methylation, on the histones results in aberrant gene regulation, DNA damage, cell cycle defects and genomic instability – all of which are hallmarks of cancer. Based on these observations, we hypothesized that histone methylation could be a novel molecular biomarker for the diagnosis and prognosis of breast cancer.

To test this hypothesis, we proposed to identify specific genomic locations where dramatic changes in histone methylation were occurring in a cell line model of breast cancer progression with the ultimate goal of using these genetic elements as diagnostic biomarkers. The genomic locations from each cell line rich in histone methylation were isolated by employing a panel of extremely selective antibodies that we had developed in a technique known as chromatin immunoprecipitation (ChIP). The histone methylation-associated DNA was then hybridized to a DNA microarray chip containing 6800 known genomic regions allowing for the high throughput direct comparison of “normal” cells to breast cancer cell lines. The regions that differed from “normal” (either increased or decreased) would be candidate biomarkers and would be further analyzed. While this approach took us longer than anticipated due to technical issues, we did successfully perform the ChIP-chip experiments using a histone methylation antibody. However, comparison between “normal” cells and a panel of breast cancer cells revealed no statistical differences. It became clear that our narrow examination of only a very small fraction of the human genome would not permit detection of breast cancer specific histone methylation changes. While the technology is available to perform genome-wide experiments, this approach is not financially feasible at this time. Based on our initial findings, the aims of this proposal could not be carried forward.

However, in a related pilot experiment, we used our panel of histone methylation-specific antibodies to determine if global levels of histone methylation were changed in our cell line model of breast cancer progression. Surprisingly, we discovered that specific methylated forms of the histones were dramatically altered compared to the “normal” cells. These findings suggest that the antibodies, themselves, could be used as putative molecular tools to detect changes in histone methylation that could be directly correlated to the degree of breast cancer progression. To test this further, we first optimized the use of the antibodies in a technique known as immunohistochemistry (IHC) using the breast cancer cell lines as positive and negative controls. We are currently in the process of using IHC in conjunction with a high-throughput technique, known as tissue microarray (TMA), to determine the use of the antibodies on breast cancer specimens. The pilot TMA consists of 20 breast cancer patient samples. Once the IHC conditions are optimized for the pilot TMA, we will then use TMA to screen between 400-500 breast cancer patient samples with known pathological data and detailed patient history. The immediate goal is to correlate cancer grade/stage with a specific degree of histone methylation. However, the detailed patient information may also yield valuable clinical insights including successful treatment regiments and predicting the chance of recurrence. Importantly, our initial findings on bladder cancer specimens predict that the observed alterations in histone methylation may be an early and common event in all cancers.