Cancer Initiation: March 2015

Saturday, March 14, 2015

The Methylome Maintenance Model, a medically relevant organizational paradigm for biological sciences based upon DNA promoter methylation status


Introduction
 This article introduces the concept of the "Methylome Maintenance Model".  It is proposed as an alternative to the current "central dogma of molecular biology" as proposed by Francis Crick.  Although the central dogma has provided an organizational framework for cancer research since the inception of molecular cancer research, it has ultimately proven to offer nothing in terms of insight or organizational characterization of clinical molecular observations.

Background
We start our discussion with a very brief review of the history, failure  and future of biological organizational frameworks, or paradigms, as I prefer to call them here. First was taxonomy, or classification based upon physical characteristics, followed by evolution, or the notion that taxonomy originates as a function of evolution.   As such Charles Darwin was recognized as the founder of modern biological science for his exhaustive, yet empirical argument that evolution was based upon small incremental changes that result from random mutations, and preferential adaptation to the environment.
  Although Mendel was a contemporary of Darwin, his work was temporarily lost, only to be rediscovered decades after his passing.  Upon rediscovery, Mendel's concept of a genetic factor seemed to reinforce Darwin's work. Mendel's description of the workings of the factors that came to be called genes led to a race for the identification of the genetic material. The finish line for the description of the genetic material seemed to be crossed when Watson and Crick elucidated the structure and function of DNA,  The case seemed closed as far as a search for organizational paradigms.
  Francis Crick stated the so called "central dogma" of molecular biology in 1956. It is summarized in the Wikipedia as follows:
 The central dogma has also been described as "DNA makes RNA and RNA makes protein,"[3] a positive statement which was originally termed the sequence hypothesis by Crick. However, this simplification does not make it clear that the central dogma as stated by Crick does not preclude the reverse flow of information from RNA to DNA, only ruling out the flow from protein to RNA or DNA. Crick's use of the word dogma was unconventional, and has been controversial.
Since the word "dogma" usually refers to a concept that is repeated automatically and religiously,  even when incorrect or not relevant, Crick's choice of the word was correct, and in fact prophetic. Today, the "central dogma" remains the operative paradigm for what molecular biology laboratories do.
 With respect to cancer, a landmark paper was delivered in 1971 by Alfred Knudson.[1] which described cancer ( retinoblastoma ) as a genetic event operating in adherence to the central dogma and Mendelian genetics. He proposed, that A first hit could be inherited, and a second hit ( to DNA sequence ) could case  cancer.  At least in terms of cancer, this was the first major event linking medical  (epidemiological)  observations to the genetic / central dogma paradigm.
  There was a gap of quite a few decades between the Knudson's proposal and the ability of clinical researchers to definitively evaluate the DNA sequence of pRB ( protein retinoblastoma, the predicted, and discovered  ) gene responsible for the observed cancer.
   Surprisingly,  ( and very quietly I might add)  in many, if not most cases, the DNA sequence of  pRB is intact. By strict interpretation of Crick's central dogma, this is impossible.
   Lets make clear that pRB is the right gene ( at least in the cases we are presently discussing ). What has happened is that although the DNA sequence is intact,  the promoter associated with pRB has become methylated, and consequently bound by methyl CpG binding protein 2 (MeCPB2 ), so the gene is not functional,  and is blocked from being expressed.
    Obviously, billions of dollars of research spent on potential problems with pRB function became dubious. What is even more breathtaking is that billions of dollars of research money continue to be spent on similar studies almost a decade after many of the particulars of pRB's function have became quaint, at least in clinical terms.
   For a description of the political inertia that continues, please browse Mastrangelo [2][3][4]. It is unfortunate that practitioners have to doubt that clinical evidence is even relevant, or that the research community has any interest in all at obtaining actual medical data. Consider the following from Mastrangelo[4]

The presumed genetic origin of Rb and its relationship with the Rb1 gene represent a clear example of how an entire body of prominent researchers may fail to question a flawed pathogenetic hypothesis (i.e., the “two-hit” theory), for the sake of personal, academic, or other interests. It was not by chance that we had to approach many different scientific journals to have access to the medical community about the role of aneuploidy and genomic instability in the genesis of Rb [4, 5, 39]. We are still optimistic, however, because our alternative pathogenetic explanation has finally appeared in recent ophthalmologic literature [40].
  
Case Made
   It is time to start from scratch in terms of an organizing paradigm. The new paradigm is based on the concept of cellular differentiation. An organism begins its existence as a clump of similar, undifferentiated stem cells.
    These stem cells have a few important characteristics.
  • The first of these cells are totipotent, they can differentiate to any somatic tissue.
  • The earliest of these cells are immortal, either telomerase ( hTERT) or other mechanisms exist to maintain telomere ends. relatively speaking, the promoters of respective genes are unmethylated, so genes can respond to any transcription factor which will interact with its promoter
From the "stem cell" stage, the process of development is directed by two major factors:
  •  Signals received from outside the cell in the form of chemical or electrical signals.
  • DNA methylation patterns inherited from the cells predecessor(s)
Yes, the amount of signals that a cell can receive is immense, but since we are talking about organizational paradigms, here, we can choose to simplify here, and detail another day. That is the function of an organizational paradigm.

The major advance of the MMM is that we have long known that all cells have the same DNA. Each cell works from a complete genome, and yet, each cell has an accounting system which integrates its current signaling environment with its inherited cell type. In more common terms different tissues may have unique response to common signals.
   The currency of this cellular accounting system is DNA methylation.  Methylation of the promoters of  the DNA of a cells genome can give the cell a unique response to signals.
   In stead of speaking of a cells genome, which is static, and the same for all cells of an organism, we speak now of a genome, a transcriptome, and a methylome. As such, these cumbersome terms are sometimes referred to as "omics".
    Due to advances in technology, a cells DNA can be sequenced relatively rapidly. Likewise for those genes that are currently being transcribed, messenger RNA can be recovered, and rapidly processed with "gene chips" to give relatively automatic printout of those genes that are currently being transcribed. The "transcriptome", unlike the genome is unique for each cell, or more practically, tissue. The methylome refers to those genes that may be behind responsive promoters for the particular cellular environment, but for which transcription is blocked by methylation, ( and subsequent binding by MeCPB2. Increasingly, it is the methylome that is of interest in cancer research.
   In fact our ability to collect automatic data about cancer progression has outstripped our ability to process effectively.
  Perhaps as an aid to automatic data analysis ( informatics ) of automatically generated data (omics) we really need to advance our organizational paradigm. Neither genomics or the central dogma address the present problem.
  At the core of historic cancer research are the concepts of "cell fate" and apoptosis, or programmed cell death. Failure of apoptosis is seen as a common clinical feature in cancerous clones of cells. Likewise, according to the cell fate model, a cell differentiates into its terminal somatic tissue type, and then functions in support of metabolism until it is senescent and goes through the previously mentioned programmed cell death.
  What I am going to do here is no less than re-define metabolism. Metabolism in the maintenance of fidelity of the methylome. When the cell detects a failure, which questions this fidelity, the first response should be apoptosis. Failing that, we go into additional defenses, which as I have mentioned are telomere length related, or immunologic. These additional defenses are what we commonly call cancer.

Conclusion
   All models need a name. I feel "methylome maintenance model" is as descriptive as we can get for our exercise of redefining metabolism as methylome fidelity maintenance.    In the MMM, it is the goal, not only of every cell, but of the organism as a system to maintain it's methylome in a fully differentiated state.
  We need to recognize that this  maintenance system operates both at the level of the individual cell, and at the level of the organism. At the cellular we have many ( hundreds at least ) biochemical pathways that lead to apoptosis. Failing that, we have what I have previously defined as the Rattlesnake Hypothesis in which loss of methylation fidelity causes  Wingspan's antigens to become expressed, and signal an immune response against the offending ( cancer ) cell.

[1] Alfred G. Knudson, Jr.
Mutation and Cancer: Statistical Study of Retinoblastoma
Proc Natl Acad Sci U S A. 1971 Apr; 68(4): 820–823. [PubMed]

[2] 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. [PubMed]

[3]  Mastrangelo D, De Francesco S, Di Leonardo A, Lentini L, Hadjistilianou T
Does the evidence matter in medicine? The retinoblastoma paradigm.
Int J Cancer. 2007 Dec 1;121(11):2501-5.[PubMed]


[4] Mastrangelo D1, Hadjistilianou T, De Francesco S, LorĂ© C   Retinoblastoma and the genetic theory of cancer: an old paradigm trying to survive to the evidence. J Cancer Epidemiol. 2009;2009:301973. [PubMed Central]






Thursday, March 12, 2015

Towards a better terminology for somatically repressed antigens

To this point I have been using the term "Cancer/Testis antigens"  (CTA) to refer to genes which are behind promoters that respond to ubiquitous transcription factors, and yet are completely repressed in somatic cells. These antigens seem to have no other function than to mark stem and germ line cells ( testis, placenta), and then become cryptic ( behind methylated promoters ) essentially forever, in the life of a somatic cell.
  The exception, of course, is the abnormal somatic cell, one that has lost control of its cell cycle, has begun to loose methylation of promoters ( global hypomethylation in oncology terms ). These cryptic antigens re-emerge, and signal the immune system. We seemingly have defined an important function. So then, we look to see what current reviews [1] say about these reclusive coyotes.

Rather than go back to primary, or first observations of these repressed antigens, which I believe I have already done,  I am going to start from a relatively modern  (2011),  highly regarded ( I found it first on Google under the operative keywords ) review paper. The purpose is to demonstrate, that although extensively studied, their biological role has not been clearly stated. According to Frattaa [1].
Most of these CTA are expressed during spermatogenesis, but their function is still largely unknown. Epigenetic events, particularly DNA methylation, appear to be the primary mechanism regulating CTA expression in both normal and transformed cells, as well as in cancer stem cells. (Fratta et. al.)
While Frattaa et. al. go on to produce a comprehensive and useful article on what is known, it remains descriptive rather than insightful.
    Here it is, the promised insight. In order for a species to be immortal, germ line cells must be imortal. That is, they express telomerase ( human Telomere Reverse Transcriptase , hTERT ) because they must. If germ line cells ( sperm ) are subject to the Hayflick limit, that is a limited number of cell divisions as allowed by progressively shortening telomeres, then the species is no more. I have referred to this overall systems biology relationship between somatically repressed antigens and telomerase as the Rattlesnake Hypothesis
  The cells of the testis must be enclosed in a immunologically privileged compartment. If, the barrier of that compartment is breached, an immunological reaction occurs. This has long been observed. Once the cells germline and stem cell mission is complete, The telomerase, as well as the CTA are repressed for the length of the somatic cells life time.
   Sort of. As it turns out, many of our systems need continuous supplies of new cells to function. The blood supply is a good example. The bone marrow, or core of our bones provides the hematopoetic ( blood producing ) stem cells a place to work, that is divide. As it turns out, it appears that these osteoid ( bony ) compartments also seem to be immunologically protected from the immune system. That was in fact the point of the first post in this blog. Don't you wish you hadn't missed that one?

 So there it is, from here in our cumbersome old Cancer/Testis/Placenta/etc antigens are to be called either  somatically repressed antigens ( SRA ) or Wingspans antigens in appreciation for this explanation.

References
[1]  Elisabetta Frattaa, Sandra Corala,  Alessia Covrea,    Giulia Parisia,  Francesca Colizzia,    Riccardo Daniellia,     Hugues Jean Marie Nicolaya,     Luca Sigalottia, Michele Maioa,
The biology of  cancer testis antigens: Putative function, regulation and therapeutic potential
 Molecular Oncology Volume 5, Issue 2, April 2011, Pages 164–182 [Full Text]