Tumor-Protective Mechanism Identified from Premature Aging Disease
A cell nucleus from a Hutchinson-Gilford Progeria Syndrome patient labelled with the nuclear protein lamina A (green).
Hutchinson-Gilford Progeria Syndrome (HGPS) is an extraordinarily rare genetic disorder caused by a mutation in the LMNA gene, which encodes architectural proteins of the human cell nucleus. The mutation causes the production of a mutant protein called progerin. Patients with HGPS display signs of premature aging, such as hair loss, slowed growth, weakening of bone and joint integrity, and cardiovascular disease. Most die in their mid-teens of heart disease or stroke. Intriguingly, these patients do not develop another aging-related disease, cancer, despite having dramatically elevated levels of DNA damage. Tom Misteli, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues hypothesized that, rather than patients not living long enough to develop cancer, a resistance mechanism was operating in HGPS cells to prevent cancer formation.
To begin testing this idea, the researchers transformed fibroblasts from HGPS patients or age-matched, healthy controls with telomerase, constitutively-activated HRAS, and SV40 large and small T antigens. Transformed HGPS cells displayed morphological changes and increased proliferation similar to transformed controls but formed fewer colonies in soft agar and fewer tumors when injected into mice. When the investigators examined global gene expression in the two populations of cells, they found that transformed HGPS cells failed to activate many of the genes that are induced in response to transformation in controls, including oncogenic and proliferation pathways. In addition the transformed HGPS cells were unable to undergo oncogenic de-differentiation. Importantly, the tumor resistance in HGPS cells was due to the presence of the progerin protein, which was both necessary and sufficient to protect cells from oncogenic transformation. Together these results suggested that HGPS cells resist cancer-inducing stimuli by not undergoing the genetic reprogramming necessary for tumor initiation.
The scientists then set out to elucidate the molecular basis of this protective mechanism. The researchers used a genome-wide RNA interference screen to look for regulatory factors that affected colony and tumor formation. They observed the strongest effect with loss of BRD4, a bromodomain-containing protein that binds acetylated histones and modulates gene expression by recruiting transcriptional regulators. Previous studies have suggested that BRD4 has anti-metastatic and anti-proliferative activities in breast and colon cancers, respectively, and knocking-down BRD4 in the transformed HGPS fibroblasts restored colony and tumor formation. In contrast, BRD4 has been reported to have a cancer-promoting role in hematologic cancers.
When the scientists looked more closely at BRD4 in the transformed HGPS fibroblasts, they found no upregulation at the protein level but, instead, observed altered chromatin binding patterns compared to controls. They saw a significant decrease in BRD4 binding at promoters and an increase in binding between genes and near many genes involved in transformation and proliferation. The researchers also noted that BRD4 had a distinct nuclear distribution in the transformed HGPS fibroblasts with large clumps that overlapped with acetylated histone H4 and that BRD4 chromatin binding was stronger in these HGPS cells. Expressing progerin in transformed control fibroblasts increased BRD4 binding to chromatin, demonstrating that progerin alters BRD4 distribution and binding properties.
To determine which pathways are important for BRD4-mediated cancer resistance, the investigators compared the gene expression profiles of transformed control cells and HGPS cells with and without BRD4 knock-down. Loss of BRD4 led to reactivation of transformation-sensitive gene sets, re-expression of oncogene networks, and enrichment of stem cell gene signatures. In line with the notion that BRD4 affects oncogenic transformation by interfering with de-differentiation, transformed HGPS fibroblasts lacking BRD4 were able to grow as spheres in suspension, a hallmark of multipotent stem cells.
The scientists next asked whether BRD4 plays a more general role in cancer resistance other than in HGPS patients. Knocking down BRD4 in normal skin fibroblasts doubled their colony-forming ability and expression of BRD4-protected normal cells from transformation, suggesting the protein has a role in tumor protection in non-HGPS cells. Consistent with BRD4 being a tumor suppressor, the researchers used the Catalogue of Somatic Mutations in Cancer (COSMIC) to identify mutations in BRD4, the majority of which were missense mutations likely to lead to loss of function, at a mutation rate similar to established tumor suppressors, such as BRCA1.
The investigators then analyzed the expression of BRD4-sensitive genes in several types of cancer, both solid and hematologic, and identified a BRD4-loss-of-function signature. By measuring the signature’s expression in large patient cohorts, they found that patients clustered based on similarity to the signature and that the signature could predict patient survival. In breast and lung cancers, patients with expression patterns similar to transformed HGPS fibroblasts had better outcomes than those with patterns resembling HGPS cells lacking BRD4. In stark contrast, patients with lymphoma who had expression patterns like cells without BRD4 did better. In acute myeloid leukemia, however, the signature had no prognostic value.
These studies suggest that BRD4 functions in a context dependent manner. This conclusion has important implications since inhibitors of BRD4 are being explored for clinical use in various cancers. It will be important to thoroughly evaluate therapies targeting BRD4 to avoid off-target effects in noncancerous tissues.Summary Posted: 10/2014
Fernandez P, Scaffidi P, Markert E, Lee JH, Rane S, and Misteli T. Transformation Resistance in a Premature Aging Disorder Identifies a Tumor-Protective Function of BRD4. Cell Rep. October 2, 2014. PubMed Link