Cancer Initiation: April 2014

Tuesday, April 15, 2014

The epidemiology of epigenetic pathologies

The title of this article contains two words that appear similar, but are not closely related. The first of these two words is epidemiology. Epidemiology is the social and laboratory science of the study of disease occurrence.  For a pattern of disease to be classified as an "epidemic", there must by some pattern in its occurrence which statistically implies that there is a cause related to each case of occurrence. Epidemiology is an important input to the medical science of etiology, or the study of causation of disease or pathology. In practice, epidemiological studies provide one input to determine the etiology of a particular set of symptomatically related pathologies. Of most interest in the field of health is the epidemiology and etiology of pathologies related to bacterial infections, viruses, and environmental or workplace toxins.
   An example of an epidemic and etiology is the occurrence of hepatitis C infection. Hepatitis C is known to be caused by an RNA virus. For quite a few reasons, RNA is difficult to deal with in the laboratory compared to DNA and proteins which are more environmentally stable, and as such, are easier to isolate from tissue and blood samples. As such, if Hepatitis C Virus (HCV) is suspected as a causation of a pathology, the first step is to test for antibodies to proteins produced by the patient to viral proteins.  Immunological diagnostics are relatively inexpensive. If a preliminary study implies that HCV infection is probable, then RNA can be isolated from patient samples, and then reverse transcribed into DNA. The now double stranded DNA sample can then be expanded, or amplified with the use of a common laboratory procedure called polymerase chain reaction ( PCR ). PCR is a fairly standard and sensitive procedure, but requires a piece of specialized equipment called a thermo-cycler, which makes the test expensive relative to immunology based preliminary tests. By careful choice of DNA primers in PCR, specific genes of interest, or specific viral DNA can be amplified, and this in turn can be an input into a DNA sequencer which will determine the exact sequence, and the exact strain of the pathogen.
  This is an example of a traditional study, but not what we are here to talk about today. The other big word in the title is epigenetic.  The etiology and diagnosis of epigenetic pathologies is a relatively recent phenomenon which relates to the ability of laboratories to determine the promoter state of particular genes as well as to isolate particular micro RNAs.

An example in breast cancer
  As an example of the type of etiological input promoter state provide lets look directly at a recent article related to some of the most trendy terms in breast cancer pathology. In Stefansson et. al. [1], the first three words of the title, CpG island hypermethylation, refers to the previously mentioned promoter state. In this case the related gene is the well known cancer prediction gene breast cancer 1 (BRCA1). A promoter is a region on DNA upstream of a particular genes start of translation, which is the site of the assembly of the genes transcription engine, RNA polymerase. In the case of genes that can be "switched off", the promoter region of the DNA contains numerous copies of the sequence CG. Since these DNA bases are connected by a phosphate ( phosphodiester bond ), and because they occur in bunches, they are referred to in literature as CpG islands. As the result of an gene expression blocking process I will touch on briefly later, a methyl group can be added to these sequences in the DNA. Where as the prefix hyper means "over" or "too much",  the word hypermethylation in the title of [1], means that the these CpG sequences in the promoter have been methylated to the point where they attract the binding of methyl CpG binding protein 2 . ( MeCP2 ). When MeCP2 is bound to a promoter, expression of the gene is blocked. When placed in the context described in Stefansson[1], the expression of the tumor suppressor BRCA1 is blocked, and as a result, the tissue has a greater propensity, or probability of becoming tumorogenic.
  Moving through the title of Stefansson, the next term is "loss of pRB as co-occuring events". The gene pRB refers to protein Retinoblastoma, and was one of the first genes studied in relation to the long time "two hit" theory of cancer developed by Knudson. It has long been known that pRB is a critical component of cell cycle checkpoint regulation machinery, and as such when its expression has been suppressed, its role in checkpoint management is eliminated, more specifically, the transcription factor E2F is not bound and inhibited from entry into the next stage in the cell cycle. I've done a whole discussion of that topic here:
  When two or more loss of expression events occur simultaneously, they first thing we presume, or suspect, is that a micro RNA has been aberrantly expressed, and that that particular micro RNA has targeted numerous genes.  In the case of BRCA1, one such candidate micro RNA is miR-9,  which according to Sun [2] regulates BRCA1 epigenetically as follows:
Reverse miR library screening revealed that miR-9 reduced the normalized luciferase activity to 60.3% (95% confidence interval [CI] = 52.0% to 68.5%; P < .001). miR-9 bound directly to the 3'-UTR of BRCA1 and downregulated BRCA1 expression in ovarian cancer cells.
What this short passage says is that the investigators created an assay in which a light emitting protein, luciferase, was used as a reporter to detect activity of micro RNA of micro RNAs in regulating targets, and miR-9 was found to bind to the 3' untranslated region of the messenger RNA of BRCA1. As such, translational activity was blocked, and the presumptive initiation of a silencing complex ( RISC) was initiated for BRCA1. A silencing complex, ( RISC), is the active tool that leads to promoter hypomethylation.
  In terms of the concept of cancer etiology, the implications are huge here. BRCA1 is commonly considered as one of the primary actors, or "causes" in terms of etiology, but in fact, a perfectly normal BRCA1 may be lost from cellular activity is it has been silenced by a micro RNA (miR-9). In this case, loss of BRCA1 decreases the cells ability to repair DNA, and increases the cells sensitivity to chemotherapy ( cisplatin ).
  Far from being unique in the role of cancer causation, epigenetic changes to cancer suppressors seems to be the rule, not the exception. That includes the "bedrock" gene of cancer theory itself, retinoblastoma.  Where as pRB became the foundation of the "two hit", primary structure ( DNA sequence) theory of cancer initiation, it is now known that the overwhelming evidence is that pRB itself is the subject of epigenetic changes. The summary from the abstract of Mastrangelo[3] is as follows:
Through the analysis of the specialised literature and a set of original epidemiological and biological data concerning retinoblastoma, the authors illustrate the evidences arguing against the 'two hit' hypothesis and propose that epigenetic factors and aneuploidy play central roles in the disease.

  Most of the cases of autism are classified as "idiopathic" or of unknown causes. An exception to that rule is Rett syndrome which is classified as an autism spectrum disorder, and is known to arise from mutations to the gene MeCP2 [4]. As we have mentioned previously, methyl-CpG binding protein 2 ( MeCP2 ) plays an important role in the course of development as it is in charge of actuating promoter blockage once a promoter has been hyper-methylated. Presumable mutations is MeCP2 could corrupt the entire epigenetic aspect of development. As such, Rett syndrome is only found in girls since the MeCP2 gene is found on the X chromosome, and patients only survive in the heterozygous case. Since males have one X chromosome, if they carry the mutation, they are not viable. Although, as the reference shows, Rett is the subject of considerable study,  the only take away point that I want to make here is that autism type symptoms result from processes that interfere with the maintenance of epigenetic promoter markers on genes.
   The implication with respect to epidemiology of the previous point is that is the case of idiopathic autism, on logical place to start looking is processes  and factors that relate to maintenance of epigenetic markers ( methylation patterns ) on DNA. THere are two ways DNA can become methylated, de-novo methylation as a result if cellular processes, likely involving micro RNAs and RNA Induced Silencing Complexes ( RISC), and inherited methylation patterns which result from duplication of methylation patterns over cell division.
  In terms of inherited mechanisms, these are to be considered the most important where the pathology occurs in an area of  high cellular proliferation. Areas of cellular proliferation are breast tissue, testicular tissue, hippocampus ( short term memory ) portion of the brain, skin and intestinal lining.
  The loss of methylation pattern fidelity over mitosis can logically be attributed to one of two general ( pathological ) processes, where it is not the result of random error ( small, and correctable ).  The first potentially pathological process is the introduction of toxins that chemically interfere with the duplication process,  The second potentially pathological process is the incomplete duplication which may occur as a result of the loss of a checkpoint in the regulation of the cell cycle.
   These to mechanisms are not mutually exclusive, but rather potentially synergistic. For example, a toxin from the environment may interfere with the correct methylation pattern duplication of a gene responsible for regulation of the cell cycle. As a result, the cell may loose an important checkpoint. From that point, additional epigenetic alterations may "snowball" as a result of the loss of the checkpoint and incomplete methylation pattern duplication. This "snowball" effect is commonly observed in tumors, and commonly characterized in cancer research literature as "global hypomethylation". ( Note here the prefix "hypo" or under, as opposed to "hyper", or over used before) .
   Just quick note here, for one of the main points of this article, related to epidemiology, heavy metal toxins such as mercury, arsenic, and other environmental heavy metals are known to interfere with this exact epigenetic process, and are also associated with symptoms that parallel those, or are indistinguishable from autism.
Parkinsons Disease
   The epigenetic revolution in molecular biology has extended to Parkinson Disease.  It has recently been shown that not only can epigenetic changes be detected in tissue recovered from deceased patients, by those changes
  As a background lets review a short introductory passage from Masliah[5] to place it into the context of our discussion.
Epigenetic processes control several neurobiological and cognitive functions, from early brain development and neurogenesis7 to memory formation, learning and synaptic plasticity.8 Altered epigenetic mechanisms have also been associated with neurological disorders, including Rett syndrome, autism, schizophrenia and Alzheimer, Huntington and Parkinson diseases.9 We recently reported a decay on DNA methylation in the brain of PD subjects, associated with the interaction of α-syn with DNA methyltransferase 1 (DNMT1) that results in sequestration of DNMT1 in the cytoplasm.
This is a good time to bring up the topic of DNMT1, or DNA methyltransferase 1.  We discussed previously two methods by which DNA methylation patterns could be disrupted over cell division, ( the duplication process is mediated by DNMT1 ) Here, the job of methylation maintenance is interrupted because the mediator, is "sequestered by α-syn in the cytoplasm", and as a result cannot accomplish its purpose in the nucleus of the cell, where of course, the DNA is.

  It has recently been shown by Mastroeni et. al. [6] that progression in alzheimers disease is associated with the failure of the ability of affected neurons to maintain methylation patterns. The following is a short passage from the abstract[6]
We evaluated immunoreactivity for two markers of DNA methylation and eight methylation maintenance factors in entorhinal cortex layer II, a region exhibiting substantial Alzheimer's disease (AD) pathology in which expression changes have been reported for a wide variety of genes. We show, for the first time, neuronal immunoreactivity for all 10 of the epigenetic markers and factors, with highly significant decrements in AD cases.
The markers that show decrements are members of the maintenance complex that binds to  CpG islands in the promoters of affected genes. As mentioned, a complex binds to these regions that can orchestrate a large number of related gene expression changes. As described in the introduction in [6]:
MeCP1 is not bound directly to methylated DNA, but rather to a single methyl-CpG-binding domain protein, MBD2. The resulting MeCP1/MBD2 complex is composed of 10 known proteins that include the complete nucleosome remodeling and histone deacetylase (NuRD) core, as well as MBD2. This group of proteins, in conjunction with CDK2AP1 (Doc1), make up a complex capable of nucleosome remodeling and histone deacetylation (Feng and Zhang, 2001, Feng and Zhang, 2003).
This is a pretty important passage, at least as far as understanding the primal nature of methylation patterns in understanding epigenetics as it relates not only to Alzheimers, but to various diseases. The term "epigenetics" brings back many responses when put into a medical search engine such as PubMed. Many of them are related to "nucleosome remodeling and histone remodeling". This passage is describing, and giving a reference, to a system where the "MeCP1/MBD2 complex" is the dominant machinery controling other nucleosome remodeling and histone deacetylation. It is the methylation patterns that are copied carried over cell division, and subservient modifications, nucleosome remodeling and histone deacetylation, result from the reassembly of an associated complex on the duplicate DNA strand.

An example of epigenetic pathology in the Kidney
  As I have mentioned previously here,  polycystic kidney disease (ADPKD) has also been shown to arise from loss of expression of genes required to maintain cellular polarity in the collecting ducts of the kidneys of affected patients. Of note here, is this model, on a molecular basis, fits into our understanding of a wide array of pathologies that are similar on a molecular level, but only differ in the symptoms that result from these often repeated pathologies.


  Here we have discussed how common epigenetic mechanisms underly the etiology of cancer, autism,  polycystic kidney disease, Parkinson disease and Alzheimer's disease. The implications going forward is that any factor that impacts maintenance proper DNA methylation can lead to a broad spectrum of seemingly unrelated pathologies. These pathologies are unrelated only in the sense that a different medical specialist is required for each one. They are similar in that the medical specialist that one would seek attention from would likely have no knowledge of "epigenetic" factors as they relate to their organ. what is needed is a broad reeducation in the public health system concerning environmental and dietary toxins that impact DNA methylation maintenance.

[1] Stefansson OA, Jonasson JG, Olafsdottir K, Hilmarsdottir H, Olafsdottir G, Esteller M, Johannsson OT, Eyfjord JE. CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer. Epigenetics. 2011 May;6(5):638-49. [PubMed Central]

[2]Sun C1, Li N, Yang Z, Zhou B, He Y, Weng D, Fang Y, Wu P, Chen P, Yang X, Ma D, Zhou J, Chen G.  miR-9 regulation of BRCA1 and ovarian cancer sensitivity to cisplatin and PARP inhibition. J Natl Cancer Inst. 2013 Nov 20;105(22):1750-8. [PubMed]

[3] Mastrangelo D1, De Francesco S, Di Leonardo A, Lentini L, Hadjistilianou T. Retinoblastoma epidemiology: does the evidence matter?  Eur J Cancer. 2007 Jul;43(10):1596-603. Epub 2007 May 31.[Abstract]

[4]LaSalle JM1, Yasui DH. Evolving role of MeCP2 in Rett syndrome and autism. Epigenomics. 2009 Oct;1(1):119-30. [PubMed Central]

[5] Masliah E, Dumaop W, Galasko D, Desplats P. Distinctive patterns of DNA methylation associated with Parkinson disease: identification of concordant epigenetic changes in brain and peripheral blood leukocytes.  Epigenetics. 2013 Oct 1;8(10):1030-8. [PubMed Central]

[6] Mastroeni D, Grover A, Delvaux E, Whiteside C, Coleman PD, Rogers J. Epigenetic changes in Alzheimer's disease: decrements in DNA methylation. Neurobiol Aging. 2010 Dec;31(12):2025-37. [PubMed Central]