Cancer Initiation: May 2014

Thursday, May 29, 2014

An emerging role for cancer/testis antigens in cancer progression and innate defences

The family of cancer/testis antigens ( CTA ) represents a class of several families of surface antigens that are expressed on the surface of cancer cells, testis cells, and only in very limited quantities, some somatic tissues. Here we propose a developmental and cancer defense function of these mostly epigenetically suppressed antigens. Cancer/testis antigens are a significant component of the Rattlesnake hypothesis ( alternatively, Wingspans rattlesnake ) model for cancer detection and immunological defense by the host organism. The emergence of CT antigens on the surface carcinogenic clones of cells is regarded by the host immune system as a warning ( rattle ) of a progression of global hypomethylation , in turn a sign that a cell cycle checkpoint has been lost and the cell (or colony)  has lost control of it's division process and is carcinogenic.
   CT antigens are currently a portion of the  "stem cell model" for carcinogenisis.[1]. It is summarized as follows( also from Costa):

Cancer stem cells were first identified in acute myeloid leukemia when surface markers were used to distinguish the stem cell population from the remaining cells with limited proliferative potential [5]. In solid tumors, cancer stem cells have been identified in breast cancer [6, 7], glioblastomas [8], lung cancer [9], ovarian cancer [4], prostate cancer [3], and epithelial gastric cancer [10]. Based on these observations, a cancer stem cell model has been proposed, and it is based on the concept that the great majority of the tumor cells have a limited proliferative potential, but a small cell population—the cancer stem cells—are able to self-renew and proliferate, maintaining the tumor cell mass. In this model, cancer is a disease of deregulated self-renewal of normal stem cells. Thus, in the cancer stem cell model, tumor recurrences and even metastases may occur due to residual cells—probably chemotherapy-resistant—that are able to expand to form secondary tumors [11]      ( Costa et. al. [1] )
The cancer stem cell model as it stands ( stood in 2006, and remains without significant change ) does not tie together loss of a checkpoint with immune system cancer defences. Costa does recognize the potential importance of CT antigens.
  To date, nearly 40 distinct CTAs have been identified based on immunogenic properties [41], expression profiles [42], and by bioinformatic methods [43]. However, little is known about their specific functions, and their functional connection with stem cell biology and cancer is widely unexplored. In this regard, it was recently reported that some CTAs such as N-RAGE, NY-ESO, MAGE-1, and SSX are expressed in human mesenchymal stem cells of the bone marrow, suggesting that CTA expression may not only be a hallmark of gametogenesis but also a stem cell marker [28] (Fig. 1).
We see the statement " little is known about their specific functions, ...", where as in fact under the  Rattlesnake hypothesis, their specific function is to tell the immune system that global hypomethylation is under way in a cell, or clone of cells, and to activate the immune system.  CTAs are a critical component of the hosts defense against cancer. They are a "dead mans switch" in case that a sells methylation "fidelity" has been lost and the cell is careening out of control.

It is also recognized that there is a significant difference between mesenchymal stem cells ( MSC) in that they do not stimulate an immune response where as cancer cells do.  It is evident then that there is some type of "fidelity" involved in normal mesenchymal cells that is lost in cancer cells.  The  following is also from Costa and describes experimental differences betwen MSCs and cancer cells with respect to interaction with cytolytic T  lymphocytes ( CTLs).
It is also possible that MSCs differ from cancer cells and escape recognition by therapeutically infused CTA-specific CTLs. This is supported by the fact that MSCs are not immunostimulatory in vitro when cultured with allogeneic lymphocytes [53, 54]. Furthermore, MSCs can escape lysis by allogeneic cytotoxic CD8+ T cells (CTLs). After transplantation of fully HLA-mismatched MSCs into an immunocompetent fetus, the cells persisted for a long term [55]. The transplanted MSCs did not induce any immune response in the child, again indicating that MSCs have immunoevasive properties

CTAs and the Germ Line Cells
  It has long been known that the cells of the testis generate an immune reaction, not only in foreign hosts, but in the hosts own immunologically environment. As such, the testes must be
compartmentalized in an immunologically privileged area. If the containment of the testicular area is broken, an immune reaction occurs, presumably mediated by cytolytic T cells. The concept of immunologic privilege is one that has been consistently overlooked, particularly given the prevalence of testicular and prostate cancer incidence. The prostate is an organ that must function in contact with cells of the testes, and as such, we might guess ( hypothesize ) that the prostate exists within the immunologically privileged area associated with the testes.

Research history
  C/T antigens were first identified by Van Der Bruggen [3]  ( Boon corresponding author ) and their first characterization noted that when expressed, they attracted the defensive and destructive attention of cytotoxic T lymphocytes( CTLs );
  The behaviorial observations are as follows in  Van Der Bruggen [3]:
 For human tumors, autologous mixed cultures of tumor cells and lymphocytes can generate CTLs that lyse tumor cells(4). These anti-tumor CTLs do not lyse targets of natural killer cells and autologous control cells such as fibroblasts or EBV-transformed B lymphocytes. However, it is difficult to evaluate to what extent the antigens recognized on human tumors by autologous CTLs are relevant for tumor rejection.
So we see that even though the property of tumor attack by CTLs , before Van Der Bruggen,  ( their reference 4 ), and families of specific signaling antigens ( CT antigens ) were identified from 1991 on, researchers have still not described their significance and roll in the larger picture of cancer defenses. That is, they have not connected the central components of the Rattlesnake Hypothesis, that is:
  •  telomerase expression status, ( repression is defense 1 - Hayflick )
  •  loss of a component of a checkpoint, and resulting
  •  progressive global hypomethylation,
  •  emergence of CT antigens as the result of progressive loss of suppression on their promoters, and promoters that are responsive to multiple ubiquitous transcription factors such as sp1
  • initiation of tumor specific immune defenses triggered by recognition of CT antigens by cytotoxic T lymphocytes
Implications for cancer therapy
  Cancer therapies that seek to stimulate and take advantage of the immune system are now, as always relegated to "alternative" therapies, or second line, prospects. We can see from the implications of Van Der Bruggen and Boon [3] [4] [5] [6] there was never really any basis for this in research. As such there is much new interest in designing therapies that stimulate and take advantage of the immune systems natural targeting ability for cancer.

The hTERT ( telomerase ) promoter
  The responsiveness of the telomerase promoter has been characterized by Zhao et. al. [7]. Like CT antigens, the expression of hTERT is suppressed in normal somatic cells by promoter hypermethylation. The promoter itself has been shown to be responsive to ( the ubiquitous ) transcription factor sp1 as described by Zhao:
We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner.[7]
So we see that the ubiquitous promoter sp1 activates both telomerase and CT antigens, so that in both cases, expression is completely dependent upon the promoter methyation state. Thus, in the pathological case of global hypomethyation, both genes will become, at some point expressed, and presumably, the CT antigens are "warnings" or "rattles" that a clone of cells has defeated it's Hayflick limit defense, and presumably needs help from the immune system.

Therapeutic implications and applications
  Although the role of CT antigens has yet to be integrated into a "big picture" of cancer theory , their potential in relation to cancer detection ( biomarkers ), as well as implications for immune system based therapies has been recognized.[8] [11] [12].  A  logical framework for cancer defenses that actively included Cancer/Testis Antigens would therefore be of value in the therapeutic  and clinical  end of the cancer research spectrum because a logical and evidence based foundation for decision making is even more important in clinical practice than it is in education and basic research.

  There is a gene expression symmetry which exists between telomerase and CT antigens that is dictated by the evidence that both genes exist behind promoters that are responsive to ubiquitous transcription factors, and as such, they must be suppressed epigenetically by promoter hypermethylation in somatic cells.  Complementing this parallel expression profile, is the observation that one ( telomerase, hTERT ) is a significant danger to the host as a result of an immortalized clone of cells ( cancer ) and the other is a group of warning signals to the immune system, specifically cytolytic T cells. Underlying both these complementary functions is what has been called "global hypomethylation", or the observed progressive loss of promoter methylation occurs in a colony of cancer cells, presumably as a result of the loss of a cell cycle check point, and incomplete duplication of methylation patterns on the daughter strand of the duplicated DNA.

See Also:

Description of the "Rattlesnake Hypothesis" in developmental biology and cancer defense


[1] Costa F1, Le Blanc K, Brodin B. Concise review: cancer/testis antigens, stem cells, and cancer. Stem Cells. 2007 Mar;25(3):707-11. [PubMed] [Full Text]

[2]Yang F, Zhou X, Miao X, Zhang T, Hang X, Tie R, Liu N, Tian F, Wang F, Yuan J.
MAGEC2, an epithelial-mesenchymal transition inducer, is associated with breast cancer metastasis. Breast Cancer Res Treat. 2014 May;145(1):23-32. [PubMed Central]

[3] van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A,
 Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991;254(5038):1643–1647. [Abstract] [Full Text]

[4] Gaugler B, Van den Eynde B, van der Bruggen P, Romero P, Gaforio JJ, De Plaen E, Lethé B, Brasseur F, Boon T. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med. 1994 Mar 1;179(3):921-30. [PubMed Central]

[5]Van den Eynde B, Peeters O, De Backer O, Gaugler B, Lucas S, Boon T. A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma.
J Exp Med. 1995 Sep 1;182(3):689-98.[PubMed Central]

[6] Boon T, van der Bruggen P. Human tumor antigens recognized by T lymphocytes. J Exp Med. 1996 Mar 1;183(3):725-9.[PubMed Central]

[7] Zhao JQ1, Glasspool RM, Hoare SF, Bilsland A, Szatmari I, Keith WN. Activation of telomerase 8na gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3. Neoplasia. 2000 Nov-Dec;2(6):531-9.[PubMed Central]

[8]Caroline J. Voskens,Duane Sewell, MD, Ronna Hertzano,Jennifer DeSanto, Sandra Rollins,  Myounghee Lee,  Rodney Taylor,  Jeffrey Wolf,  Mohan Suntharalingam, Brian Gastman,  John C. Papadimitriou,  Changwan Lu,  Ming Tan,  Robert Morales,  Kevin Cullen, Esteban Celis,  Dean Mann, and Scott E. Strome,  Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma Head Neck. Dec 2012; 34(12): 1734–1746. [PubMed Central]

[9] Lim JH1, Kim SP, Gabrielson E, Park YB, Park JW, Kwon TK. Activation of human cancer/testis antigen gene, XAGE-1, in tumor cells is correlated with CpG island hypomethylation. Int J Cancer. 2005 Aug 20;116(2):200-6.[PubMed] [Full Text]

[10] James SR, Cedeno CD, Sharma A, Zhang W, Mohler JL, Odunsi K, Wilson EM, Karpf AR.
DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene MAGEA11.  Epigenetics. 2013 Aug;8(8):849-63. [PubMed Central]

[11] Stacey S Willard and Shahriar Koochekpour  Regulators of gene expression as biomarkers for prostate cancer Am J Cancer Res. 2012; 2(6): 620–657. [PubMed Central]

[12]  Comber JD1, Philip R2. MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.  Ther Adv Vaccines. 2014 May;2(3):77-89. [PubMed Central]

Tuesday, May 20, 2014

The paradox of global hypomethylation and local hypermethylation in terms of the sea-saw molecular logic of epigenetics

The observation of global DNA hypo-methylation is relatively easy to understand given our simplistic model of loss of a checkpoint in the cell cycle coupled incomplete duplication of methylation patterns during the DNA "maintenance" operations of the synthesis and growth phase phase of the cell cycle. The actual duplication if the cells methylation imprint is mediated by DNA Methyl - Transferase 1, (DNMT1) the catalytic portion of a replication complex. For a review and brief description of the DNA Methyl Transferases, we provide a link to Kim[1]. From Kim we have the following description of DNMT1:

 In proliferating cells, DNMT1 is found to be associated with replication foci (Leonhardt et al., 1992), ensuring methylation of the daughter strand during DNA replication. Knockout of Dnmt1 in the mouse genome resulted in global demethylation and embryonic lethality (Li et al., 1992).    (Kim[1] )
  Here, demethylation refers to complete loss of DNA CpG Island methylation, where as the word hypo-methylation refers to under, or incomplete methylation.
As further evidence that the loss of complete methylation is sufficient to induce loss of control of the cell cycle, Pacaud et. al. [3] disrupted the DNMT1 DNA maintenance complex ( DNMT1/PCNA/UHRF1) induces tumorigenesis.
 But  loss of methylation is only half the story, the other half is that tumor suppressors, such as our now familiar mediators of checkpoints ( pRB ) and apoptosis "guardian" genes, ( p53, BRCA1/2 ) become hyper- methylated, and as a result are suppressed.
 Lets look at an actual example related to Non Hodgkins Lymphoma. In Yim[4] We are introduced to our familiar sea/saw regulation of cancer related genes by micro-RNA's:

MicroRNAs (miRNAs) are short, non-coding RNA sequences of 18–25 nucleotides, which can repress the translational of multiple protein-coding mRNAs by sequence-specific binding to the 3′untranslated region. Depending on the genes targeted, miRNA can be tumor suppressive if an oncogene is repressed, or it can be oncogenic when a tumor suppressive gene is repressed. Recently, aberrant methylation of tumor suppressive miRNAs has been reported in different types of cancers including lymphomas. (Yim[4])
So we see, that if a micro-RNA that should be suppressed by promoter methylation is in fact expressed due to the progression of "global hypomethylation", then it may become expressed. If the molecular target gene ( transcript ) of the micro-RNA  is a "tumor supressor", then that target gene will become promoter hyper-methylated and its expression will be blocked.. Yim[4] also provides a slightly more technical description of the expression of a micro RNA, and the details of how it blocks a target.
These intermediates pre-miRNAs are exported via Ran-GTP-dependent exportin-5 (XPO-5) into the cytoplasm, where these pre-miRNA stem-loops are further processed into mature miRNA duplex (Yi et al., 2003). Eventually a single-stranded mature miRNA is produced, ready to function when it is loaded onto the DICER1-TAR RNA-binding protein-containing RNA-induced silencing complex (RISC; Liu et al., 2004). The biosynthesis and processing of miRNA is summarized in Figure Figure11. (Yim[4])
In general terms, the micro-RNA is processed by Dicer into a short single stranded piece of RNA that is incorporated into a silencing complex ( RISC ), which will mediate the hyper-methylation of the promoter of the target gene. By this process, we complete the pathway from loss of checkpoint, global hypomethylation and hyper-methylation of tumor suppressors, thus describing the "paradox".

 In conclusion, we note that micro- RNAs are relatively new addition to the molecular biology soup. Here we see that they provide an important logical component in describing a mechanism behind the often observed phenomenon of tumor suppressor hyper-methylation, while in fact, the majority of the cells DNA is undergoing hypo-methylation as a result of the loss of checkpoints  and/or DNMT1 integrity.


[1]  Gun-Do Kim, Jingwei Ni, Nicole Kelesoglu, Richard J. Roberts, and Sriharsa Pradhan Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases EMBO J. Aug 1, 2002; 21(15): 4183–4195. [PubMed Central]

[2] Sceusi EL1, Loose DS, Wray CJ.
Clinical implications of DNA methylation in hepatocellular carcinoma. HPB (Oxford). 2011 Jun;13(6):369-76.  [PubMed Central]

[3] Pacaud R, Brocard E, Lalier L, Hervouet E, Vallette FM, Cartron PF.
The DNMT1/PCNA/UHRF1 disruption induces tumorigenesis characterized by similar genetic and epigenetic signatures. Sci Rep. 2014 Mar 18;4:4230.  [PubMed Central]

[4] Yim RL1, Kwong YL, Wong KY, Chim CS.  DNA Methylation of Tumor Suppressive miRNAs in Non-Hodgkin's Lymphomas.   Front Genet. 2012 Nov 8;3:233.[PubMed Central]

Sunday, May 11, 2014

Description of the "Rattlesnake Hypothesis" in developmental biology and cancer defence


The Rattlesnake Hypothesis  (RsH)  is an organizing principle which describes organization of gene expression in the developmental biology of higher organisms, and presents an underlying principle which describes natural cancer defenses.
   First must make some notes about the word "hypothesis" in the title and in the concept Rattlesnake hypothesis. In the epistemology of science, hypothesis means something close to "testable conjecture", where testable refers to a controlled laboratory study.  This is not quite what the rattlesnake hypothesis is. In science, it would be more closely related to the word theory which translates to explanation. such as in Darwins's theory of natural selection as a foundation of evolution.
 By way of note, this is not a blog that adheres to the epistemology of science. This is a blog that hopes to contribute to science education. Education has it's own epistemology which is a branch of cognitive science. In the field of cognitive science, the Rattlesnake Hypothesis is more akin to what might be called a  graphic organizer. As is often the case, this description rather awkwardly straddles the epistemologies of science and education, which is often the case in the field of science education.
  The role of the RsH in cancer defense
  We have previously described the suppression of telomerase and the concept of the Hayflick Limit in a previous post. The epigenetic suppression of telomerase can be thought of as a laboratory observable definition of the difference between what might be classified as "germ line" and "stem" cells and what might be described as "somatic" cells, where somatic means "of the body". In broad terms, germ line cells  and stem cells must be "immortal", where as somatic cells are terminally differentiated to their physiologically relative function, and have exited the cell cycle. The fate of somatic cells is presumed to be apoptosis, or programmed cell death at the end of their functional life.
 So, in general terms, two things can go wrong, stem cells can loose control of their cell cycle, and somatic cells can loose suppression of telomerase. In this sense, telomerase is one end of the "Rattlesnake", that is, it is the fangs. If a cell does not have "stem" cell markers and is expressing telomerase, it is a threat to the whole biological system. There must be a marker for the system to throw up warning signs to activate the immune system to destroy the threat. (cancer)
  The role of progressive hypomethylation in cancer progression.
Once checkpoints in the regulation of the cell cycle have been defeated, the process of DNA synthesis becomes compromised. As such, the cell enters into mitosis before the machinery of the S ( synthesis ) phase has completed duplicating DNA and copying promoter methylation markers. Incomplete promoter methylation during duplication can now be thought of as an underlying explanation of the commonly observed phenomenon of "global hypomethylation" associated with cancer stage progression. As a result of global hypomethylation, epigenetically suppressed antigens called cancer testis antigens, (C/T antigens) become expressed. C/T antigens get their name because they are (were originally ) found in two places, cancer and testes. That poses the question "What do cancer and testis have in common?" The RsH proposes an answer to the C/T question, which is that both of these undifferentiated immortal cells pose a threat to the organism since they can be carcinogenic if not properly defended.
  As a note, it has long been noted that cells of the testes (sperm) initiate an autoimmune reaction if their protective encapsulation within the body is breached. The conspicuous location of the testes may be a result of their potential danger in the case of loss of cell cycle control. Also, it is worth while to note that testicular cancer is one of the more common forms of neoplasm.
   The Rattle of the RsH
We have described telomerase as the "fangs" of the rattlesnake, now we are going to go ahead and denote C/T antigens as the "rattle" of the rattlesnake. The purpose of C/T antigens has not been previously proposed, but here we are going to go ahead and say that C/T antigens form a second line of defense against cancer after the Hayflick limit. In other words, we are going to say that if a clone of mitotic cells have breached the Hayflick limit due to loss of suppression of telomerase, global hypomethylation will begin to occur. As it progresses, somaticly suppressed C/T antigens will loose their epigenetic suppression. Since the promoters for C/T antigens are responsive to ubiquitous transcription factors, cells will express these C/T antigens, and as such, such cells will be targeted by the immune system.
 Cells that are potentially cancerous must be, in some way immortal. In about 90% of fully progressed cancers, telomerase is expressed. Likewise the immune system has always been known to target cancer cells. Here we propose that there is an "dead mans switch" in each cell such that if cancer/testes antigens are not continually suppressed at each mitotic generation, an immunological attack will be mounted against the cell. In most cases, potential neoplasms are destroyed by the immune system. In some cases, cancerous cells develop ways to suppress immunological attack, or merely overwhelm the immune system by brute force. In any case, the rattlesnake hypothesis provides an organizing principle by which each form of cancer can be further described in terms of it's mitotic and immunologic state.
  That is each clone of cells can be described, and possibly quantified in terms of it's immortality potential  ( telomerase, fangs ) , and it's immunological potential ( Cancer testis antigens, rattle ). Together they describe both  ends of the cancer rattlesnake.