CH3 News

Cancer, Epigenetics and Protein Arginine Methylation

Protein arginine methylation has been implicated in one way or another in 5 major disease categories: neurodevelopmental, autoimmune, cardiovascular, viral and neoplastic disease. The first blog posting in this series (PROTEIN Methylation; The Other Epigenetic Regulator) laid the theoretical groundwork for how protein arginine methylation might influence epigenetic molecular mechanisms that impact diverse targets and processes including genome stability, transcription factors, co-activators and co-repressors of gene expression.  Today’s post focuses in on how these protein methylation mechanisms may influence human cancers.  Several representative examples drawn from recent journal articles are offered for your review and comments.

Recently, estrogen receptor alpha (ERα) was identified as a target of protein arginine methyltransferase 1 (PRMT1).  Estradiol (E2) produces a rapid stimulation of the methylation of ERα in the DNA-binding domain of the receptor (Le Romancer et al., 2008).  The direct actions of the methylated form of ERα are thought to initiate a series of non-genomic interactions (Le Romancer et al., 2010).  Interestingly, though, these non-genomic actions have more far-reaching consequences.  Methylated ERα interacts with a cytoplasmic complex containing SRC, focal adhesion kinase (FAK) and the p85 sub-unit of phospoinositide-3-kinase (PI3K).  The end result of this signaling is activation of AKT, increased cell survival and cell proliferation, most likely due to nuclear integration of regulatory pathways that direct gene expression.

Another interesting finding in addition to the data on E2-induced methylation of ERα was the use of an antibody directed against methylated ERα in a series of human breast tumors (see Figure above).  Normal epithelial cells adjacent to the tumors exhibited only a weak reactivity and there was no reactivity of the antibody in myoepithelial cells.  A subset of the tumors (about half) displayed hypermethylated ERα.  Patient-dependent differences in methylation status could turn out to be very interesting in terms of both biological mechanisms and clinical relevance.

Vezzalini et al. used a broad-spectrum methylarginine-specific antibody to characterize methylation status in normal and tumor tissues.  The authors found that depending on the organ source of normal tissue, there are differences in the predominant subcellular location of protein methylarginine staining.  For example, methylarginine specific staining in endocrine tissues tends to be highest in nuclei, whereas intestinal epithelial cells have a cytoplasmic and Golgi-like pattern of staining.  Importantly, there is consistent staining from one normal specimen to another and staining is well correlated with immunostaining for PRMT1, the major protein arginine methyltransferase in mammalian cells.

Tumors, however, have a more heterogeneous staining from one specimen to the next with regard to intensity and sub-cellular location.  For instance, although normal thyroid gives a predominant nuclear stain, some papillary thyroid tumors instead can also display major cytoplasmic staining.  Table I (below) summarizes how very weak to negative methylarginine-reactivity is observed in a fraction of tumors analyzed.  Depending on tumor type, the reduced reactivity ranges from ~5% (breast) to ~30% (pancreatic duct).  The authors conclude that tumors may be stratified based on methylation patterns with regard to both staining intensity and subcellular location.

TABLE I

Lastly, two different tumor suppressor proteins are now known to be targets of protein arginine methylation that affects cellular biological responses.  BRCA1 is a multifaceted suppressor that is mutated in approximately half of all hereditary breast cancers.  Guendel et al. recently showed an association of BRCA1 with PRMT1.  They further showed that chemical inhibition of methylation or knockdown of PRMT1 decreases BRCA1 methylation and alters BRCA1 binding to promoter sites based on chromatin immunoprecipitation assays.  Interactions with specific transcription factors, such as SP1 and STAT1 could be either increased or decreased under conditions that reduce the methylation of BRCA1.  These results indicate that the methylation status of BRCA1 can influence transcription in a protein context-specific manner.

PDCD4 is another tumor suppressor protein that correlates well with better clinical outcomes when expressed at high levels particularly in lung, colon, ovarian and esophageal cancers.  There are, however, exceptions to this rule in breast tumors.  Some breast tumors that exhibit elevated levels of PDCD4 are associated with an unexpectedly poor clinical outcome.  The overexpression of the methyltransferase, PRMT5, in leukemia, lymphoma and gastric cancers encouraged Powers et al. to test whether PRMT5 might influence PDCD4 effects in tumor biology and growth.

For the assessment of tumor growth, the authors used an orthotopic NOD/SCID mouse transplantation model.  The MCF7e breast tumor cell line was stably transfected with PRMT5 and/or PDCD4.  Tumor growth in mice transplanted with empty vector control MCF7e cells, or cells over-expressing PRMT5, PDCD4 or both proteins was compared. Faster tumor growth and greater necrosis, edema and vascularization were observed in the transplants containing both proteins compared to vector, PRMT5, or PDCD4 alone transplants.

The authors of this study also examined clinical data from a large series of patient tumors with high expression of PDCD4 that were segregated into 4 categories based on PRMT5 expression levels (see Table II).  Those tumors with the highest levels of PRMT5 had the poorest outcomes, similar to a cohort of patients with low PDCD4.  When the levels of PRMT5 associated with tumors are lower, the probability of patient survival is increased in a statistically significant analysis.  Those tumors with high PDCD4 and the lowest levels of PRMT5 have a 20-year survival rate of 80% versus 43% for those patients in the top category with the relatively highest PRMT5 expression.

TABLE II

The precise understanding of the role of protein methylation in carcinogenesis clearly requires, and deserves, much greater study to enable advances in diagnosis and treatment.  However, based on the information currently available from animal and human studies on protein methylation, biomarkers based on protein methylation status may be useful in the future for determining clinical categories and prognosis.

Mitotic Bookmarking - More Epigenetic Control by Protein Arginine Methylation

The last posting at this site described the evidence for the role of protein arginine methylation as a key epigenetic regulator in addition to DNA methylation.  Since protein arginine methylation can produce important direct regulatory control over gene expression, it shouldn’t be surprising that it may also play a role in mitotic bookmarking, the latest epigenetic mechanism observed to regulate gene expression during normal cell development and carcinogenesis.  Mitotic bookmarking is a heritable form of epigenetic control that maintains cell identity following mitosis through a combination of histone modifications, DNA methylation and the retention of specific transcription factors at specific phenotypic promoter sites.  

An excellent review of bookmarking from Gary Stein’s lab at U. Mass. Cancer Center came out recently in Nature Reviews Genetics (Zaidi et al., 2010).  The Opinion piece does not directly reference protein arginine methylation, but it is intriguing that many of the effector molecules that carry out the bookmarking functions described in the review turn out to be methylarginine-modified proteins.  A few brief provocative examples are offered below to stimulate some brainstorming.  To understand the mechanism of the gene expression control exerted by bookmarking, it’s necessary to recognize that the compartmentalization and focal organization of protein complexes in nuclear microenvironments depends on diverse elements, such as RNA Pol I and Pol II, scaffold proteins, splicing factors and nuclear receptors.

Dynamic nuclear micro-domains tend to be organized around splicing factors (such as the serine/arginine rich (SR) proteins that process transcribed RNA) and scaffolding proteins (such as the Runt-related transcription factors, Runx) at specific sites of target-gene promoters to facilitate co-regulator recruitment.  These micro-processing domains are known to harbor PRMTs (protein arginine methyltransferases, Yanigida et al., 2004) and Runx1 transcriptional activity is regulated by arginine methylation mediated by PRMT1 (Zhao et al., 2008).  Krainer’s lab at Cold Spring Harbor recently reported on the potentially complex relationships between protein charge, methylation and the intracellular targeting and functions of a prototypical SR splicing protein (Sinha et al., 2010).

Zaidi et al. describe several gene bookmarking functions in development, lineage commitment and disease.  DNA sequence-specific protein associations, that exhibit accessibility to single strand nucleases in promoter regions only during mitosis, mark target genes for rapid transcription following mitosis.  For example, the single strand DNA binding protein, hnRNPK, can modulate transcription from the MYC promoter in this manner.  The basis for the preferential association/disassociation of hnRNPK from DNA is not clear, but perhaps the interaction depends on protein methylation state (Chen et al., 2008).  Similarly, Runx1 associates with Pol I transcribed ribosomal RNA (rRNA) genes and Pol II phenotype-specific genes involved in growth and differentiation, marking those genes for repression during early G1.

The recruitment of the methylarginine co-activator, CREB-binding protein (CBP), by specific transcription factors to the globin gene locus serves to enforce lineage-restricted expression of globin genes via rapid post-mitotic gene activation (Xin et al., 2007).  CCAAT/enhancer-binding proteins (C/EBPα, β, δ) also associate in a phenotype-specific way with mitotic chromosomes to mediate a program of growth and differentiation (Ali et al., 2008).  What role may be played by the arginine methylation of C/EBPβ (Kowen-Leutz et al., 2010) in lineage commitment and gene bookmarking, like the methylation of CBP, remains to be determined.

Alterations in gene bookmarking may also form the basis for some human diseases.  The association of the leukemogenic fusion protein, Runx1-ETO, with mitotic chromosomes up-regulates rRNA synthesis and may act as an epigenetic switch for unregulated cell proliferation (Bakshi et al., 2008).  Mixed lineage leukemia (MLL) protein, which is associated with the blood cell cancer, can also occupy mitotic chromatin to promote transcriptional reactivation after mitosis (Blobel et al., 2009).  Interestingly, there is a requirement for catalytically active PRMT1 in a mouse model of MLL-mediated leukemic transformation (Cheung et al., 2007).

The work on mitotic bookmarking is expanding our understanding of the increasing range of the epigenetic mechanisms that operate in biological systems.  Stay tuned for more links to how protein arginine methylation contributes to the epigenetics of mitotic bookmarks and methyl marks discovery!