Cancer Initiation: Kidney cysts are shown to arise from an epigenetic "second hit"

Wednesday, December 18, 2013

Kidney cysts are shown to arise from an epigenetic "second hit"

Autosomal Dominant Polycystic Kidney Disease has always been observed to be an inherited disorder.  But, as the name implies, there has always been a puzzle as to the nature of onset of kidney cysts.
Recently, in the past five years or so, there has been a surge of interest in what has been called epigenetic contributions to cancer causation. Traditional cancer research has historically been almost completely based upon what is known as the "central dogma" of molecular biology, which implies that cellular information flows in one direction, DNA provides a template for messenger RNA, and mRNA is in turn translated into proteins, the actual workhorses of cellular metabolism.
  As a result of strict adherence by researchers to the central dogma, research has focused almost exclusively on DNA sequence as a causal foundation for loss of cell cycle control and neoplasm. In other words, somatic mutations, where somatic refers to cells of the body as differentiated from germ line or directly inherited sequences.
  In recent years, there has been comprehensive acknowledgement of the observation that in many cases, the somatic sequence of the causative gene is intact, but gene expression, the result of the previously mentioned central dogma, has been blocked. If the expression of a so called cancer suppressor gene is blocked, the cell is at high risk to become cancerous.
  Epigenetic modifications are numerous, but the primary method, and easiest to detect using laboratory methods is called changes to DNA methylation. Methylation applies to the portion of a gene called a promoter. In order to initiate transcription of DNA to RNA, a complex of proteins including a polymerase must assemble on the DNA strand upstream of the start of transcription.This location is called a promoter.
  A promoter for a particular gene may contain a large number of the dinucleotide CG. These are referred to as CpG islands, where the p refers to the phosphate that joins two nucleotides in DNA structure. As the result of a cellular differentiation process, the CpG islands can become methylated, meaning a methyl group has been has been added. Once a promoter has been methylated, a protein known as methyl CpG binding protein can bind to the promoter. At this stage, expression is suppressed.
 Methylation as a result of differentiation  or expression constitutes what is known as "paradigm change". This is because cellular information flow in an epigenetically regulated gene expression system is fundamentally different than that implied by the central dogma.
  In a typical gene that is regulated, or blocked by promoter methylation, the messenger RNA interacts with a complementary micro RNA, or short sequence with which it can combine to form a helix. That short helix can be processes by the cell into a RISC which will target the promoter of that specific gene for repression by promoter methylation. A RISC, or gene suppression complex makes use of a particular strand of RNA to identify the proper gene in the nucleus of the cell, and bind to and methylate the promoter of that gene.
  This is why the recent announcement by a team in South Korea qualifies as a "paradigm shift" in our understanding of the genesis of a particular medical condition, kidney cysts [1] .  It seems that epigenetic analysis, as opposed to traditional genetics [2]  provided the data to advance understanding of cyst causation and formation.
  What Bae and Woo and are saying then, is that an observed lack of expression of PKD1 is due to promoter methylation, presumably as a metabolic incident which triggered the formation of a RISC which targeted PKD1 expression at the DNA level, and blocked it.
   We should make a quick note here that this explanation is significantly different than that offered by current researchers sponsored by the NIH. Current views are summarized in an NIH sponsored view provided by Harris[2]. The first line of the abstract summarizes puzzling nature of PKD presentation. It is as follows:
    A 50% dosage reduction of gene product, known as haploinsufficiency, as found in heterozygotes with an inactivating mutation, is not usually associated with a detectable physiologic effect. 
 In other words, the "autosomal dominant" nature of PKD is in itself unusual, we usually expect genetic syndromes to occur in the homozygous case. In most cases, of a patient has one good allele, health is maintained.  This has inspired the "second hit" hypothesis, or loss of heterozygosity. Indeed, the two hit hypothesis is attractive because cysts are observed to be "clonal", or a large number of cells having decended from a single cell, as identified by genetic analysis.
   But, we should note here, that epigenetic changes such as promoter methylation are also passed from parent to progeny across mitosis by a process of methylation pattern copying. Clonality of cysts in no way excludes epigenetic considerations.
  Because, in a relatively high percentage of cysts,  heterozygosity seems to be preserved, Harris moves on to an alternative hypothesis, "haploinsufficiency". This if a large word to imply an affect which is actually almost never observed. That is, on allele is insufficiently "powerful" enough to produce enough protein to reach a threshold level to maintain healthy expression. Based upon this hypothesis, Harris concludes:
The emerging model in ADPKD is of loci that are dosage sensitive, which likely alone explains some disease phenotypes.
As such, they seem ready to close the book on ADPKD. Harris then acknowledges:

    This study was supported by National Institute of Diabetes and Digestive and Kidney Disease grant DK058816.
Given the recent publication year, One would assume that his views reflect the views of this division of the National Institutes of Health - NIH.

   So then, the questions of interest to researchers are fundamentally different in this new paradigm. The first question is a primarily a computational problem, at least initially. That is to find any and all micro-RNAs which regulate our gene of interest.
interaction of micro RNAs and their substrates is dependent upon the secondary structure of the substrate. This also is primarily a computational problem, and for large transcripts, that is, most of the genes we are interested in, it is considered an unsolved problem. In fact, for large transcripts, there is no single structure which best represents our target, but a family of structures referred to as a Boltzman ensemble by one of the prefered computational tools SFOLD.
  Renal cysts can be caused by many abnormalities, but two of the must common sources of cysts are abnormalities to the genes PKD1 and PKD2.   PKD 1 is known to be a trans membrane receptor which has a lectin domain on the extracellular portion of the transcript.  A lectin is a protein which recognizes and interacts with a polysaccaride or a "sugar" group. Many proteins, particularly those which make up the extracellular matrix, are "decorated" with sugar groups in a process called glycosylation, which takes place in the Golgi after the polypeptide exits the endoplasmic reticulum.
   In the case of PKD 1, the lectin domain is called a "sea urchin receptor for egg jelly" or an suREJ domain, after the model in which it was first identified. The polysaccaride substrate for suREJ is fucose, which is also found in all epithelium basement membranes.
    In the sea urchin sperm - egg interaction, recognition results in calcium release as a sperm activation signal. Perhaps not coincidentally, PKD2 is known to be a calcium channel.
   So then, we get, finally, to Woo and Bae [1] to discuss the impact of epigenetic paradigm shift on our understanding of kidney cyst formation. They report that using a laboratory method to sequence and obtain a promoter methylation profile for somatic sequences ( those cells associated with cyst development in patients ) They observed promoter methylation in those cells where expression of PKD1 was reduced or eliminated.

Further, they performed a cell culture using Madin Darby Canine Kidney (MDCK) cells, which simulated cyst formation in a laboratory model system, and observed a reduction in cyst formation tendency when the cells were treated with therapeutic agents which are known DNA methylation inhibitors. Somewhat shockingly, Bae and Woo show how a paradigm shift in viewing a pathology can lead to a proposed therapeutic regimen where there was none.
  Bae and Woo certainly does not provide a close to the issue of autosomal dominant polycystic kidney disease (ADPKD). It certainly provides a new avenue of research. An "avenue" of research, has in the past, been guided by the principles of the central dogma. In abbreviated terms, locate DNA mutations, map them on to their amino acid in each protein, engineer affected proteins, and evaluate their change of function in terms of binding to their substrate, or enzymatic activity.
   In the new paradigm, there is no further interest in protein function. What is of interest is making a working map of the genes messenger RNA secondary structure, and seeing if it can be differentiated from that of the mutated gene. This itself is a tall computational task. To my knowledge, success in differentiating wild type and mutant (WT) and (MT) secondary structure has been claimed once, in Alcorn.
  Beyond that, DNA promoter methylation is the result of the interaction of an mRNA and its antisense micro-RNA partner. As a measure of complete systemic knowledge, PKD1's antisense micro-RNA should be identified. This is initially a computational problem to identify candidates from genomics, and then a verification step to profile gene expression and see which micro-RNAs are actually expressed in affected kidney cysts.
  All of this leaves the question of the calcium channel, PKD2 wide open. All of Bae and Woo's work could be repeated in cysts affected by PKD2 mutations. Since Bae and Woo seem to have used general methylation inhibitors in their follow up lab experiment with MDCK cells, the same results might be expected from cysts that arise as a result of PKD1 or 2.


[1] Woo YM, Bae JB, Oh YH, Lee YG, Lee MJ, Park EY, Choi JK, Lee S, Shin Y, Lyu J, Jung HY, Lee YS, Hwa Genome-wide methylation profiling of ADPKD identified epigenetically
 regulated genes associated with renal cyst development.

Hum Genet. 2013 Oct 16.  [Abstract]

[2]  Peter C. Harris What Is the Role of Somatic Mutation in Autosomal Dominant Polycystic Kidney Disease?  JASN July 1, 2010 vol. 21 no. 7 1073-1076  [Full Text]

[3] Jjingo D, Conley AB, Yi SV, Lunyak VV, Jordan IK.
 On the presence and role of human gene-body DNA methylation.
Oncotarget. 2012 Apr;3(4):462-74.  [Abstract] [Full Text]

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