Cancer Initiation: March 2014

Sunday, March 2, 2014

The cells first line of defence against loss of control of the cell cycle depends on epigenetic repression of telomerase, a "molecular fossil"

Introduction
 As mentioned previously, the event most closely associated with cancer initiation is loss of control of the cell cycle. This very often is associated with loss of a "checkpoint" at one of the key cell cycle transitions. What natural defenses does the cell have against what may be a common occurrence? There are apparently multiple levels of defenses, but the first level seems to be a limitation on the number of times a cell can divide as a function of the length of the telomeres.
  The DNA of the cell is organized into units called chromosomes. Each chromosome is a single molecule.  On each end of the chromosome is a highly repetitive structure called the telomere. During mitosis, ( the M phase of the cell cycle), the chromosomal DNA is duplicated. The enzymes that mediate this process are called DNA polymerases. The function of  DNA polymerase is such that each time a duplication cycle takes place, a small amount of DNA on the telomere is lost.  Thus in somatic cells, or cells of the body, there is a limited number of replications that can take place. This number of generations is called the Hayflick limit after the scientist who determined that cell lines made from somatic cells could only divide a limited number of times.
   If there were not exceptions to the "Hayflick Limit" rule, then life would not be immortal, in the sense of perpetually maintaining a genome. The exceptions to the Hayflick Limit rule are cells we call "germ line" cells and "stem cells". These cells have an enyme called telomerase, whose function is to elongate telomeres that have been shortened by replication. The active subunit of the telomerase enzyme is called human telomere reverse transcriptase ( hTERT ). A reverse transcriptase is an enzyme that transcribes ( copies ) RNA to DNA, where the forward direction for a transcriptase (polymerase) is to copy DNA to RNA.
  Enzymes that are ribo-proteins, or consist of a portion of the enzyme that is a protein and a portion that is an RNA, are thought to be "molecular fossils" in the terms of White[1]. Typically, the RNA is the enzymatically active portion of the enzyme. We call these catalytically active nucleotides prosthetic groups, in that they help the function of the core protein. The molecular fossils hypothesis, or alternatively RNA World hypothesis holds that there was an earlier state in the evolution before the emergence of DNA and proteins, where RNA molecules performed both information storage and catalytic functions. A genome of these early progenitors is thought to consist of an RNA coding for an ensemble of early RNA enzymes. From these early progenitors, the catalytic machinery for proteins emerged, including ribosomal RNA. The subsequent appearance of proteins created a class of riboproteins in a manor described by White as follows[1].

The appearance of coded proteins would provide new opportunities for a metabolic system composed of nucleic acid enzymes. Proteins would bind to the nucleic acid enzymes and provide specific substrate binding sites not previously available. In time, proteins would completely displace nucleic acid enzymes in all but the catalytic core. Gene duplication and independent evolution would create families of homologous enzymes.[1]
  Thus, White, in 1976 described an archaic evolutionary system  which seems to accurately describe the emergence of telomerase while at the time, observing  different systems, nucleotides (co-enzymes) associated with the citric acid cycle and generation of ATP.
  One might understandably ask why we would take such a diversion into admittedly fringe topics when discussing regulation and deregulation of the cell cycle in cancer. The answer is that it always seems to be a good idea to keep the unending antics of RNA in mind. In particular, micro RNAs and their interactions with cellular components are now of primary interest in the study of cancer initiation. In taking the broad view, we hope to avoid overlooking significant clues.
   In other words, the reason to take a circumspect view is because there never seem to be any hard and fast laws in biology in the same sense as in physics or chemistry. Even the previously described mechanism for limiting the number of cell divisions seems to have exceptions. It seems to have been shown that even if the telomere lengthening catalytic activity is eliminated by introducing a mutation, ectopic (scientist mediated)  reintroduction if the disabled gene back into the cell is enough to transform some cell lines into a  cancer causing phenotype. First, we must recognize that there seems to be some other telomere maintenance system in addition to telomerase/hTERT. It is referred to as ALT by Stewart et. al. as follows: [2]
Telomerase activation is not the only mechanism by which cells can stabilize their telomeres. As many as 10% of human tumor-derived cell lines are telomerase-negative and rely on an alternative mechanism of telomere maintenance termed ALT [2]

Telomerase Shown to have function outside of telomere extension
   The Stewart group designed a disabled hTERT designated HA, and combined it with fluorescent marker protein GFP. When the cell cycle was stimulated with oncogene Ras,  the presumed  Hayflick limit was defeated  with the ectopic addition of the HA, or catalytically deficient hTERT. It was described as follows:[2]
 As expected, the hTERTHA-GFP expressing cells did not form tumors. In contrast, the hTERTHA-Ras expressing cells formed tumors at the same efficiency as the hTERT-Ras-expressing cells (Table 2 ), indicating that telomere elongation was not critical for tumor formation.
So Stewarts 2002 paper is something that many scientists would be tempted to overlook because it does not fit into some ideological "dogma" associated with the most commonly interpreted function of hTERT.  In fact, Stewart may have set the stage so to speak for later observations about the role of telomerase in tumor transformation.
  Just as the process of differentiation can be regarded as the transformation of cells from germ line cells to mesenchymal cells, or stem like cells, to fully differentiated cells ( epithelial cells in the case of organs ), cancer is the reverse of that process. Progression of cancer through its stages consists of an epithelial to mesenchymal ( EMT ) transition.

More about the epithelial mesenchyme transition
   The role of telomerase in the  EMT is well known [3], and has been observed in multiple types of cancer. Below is a quote from a paper related to gastric cancer as opposed to breast cancer. It is apparent that the role of telomerase in forcing a cell back to a "stem cell" like phenotype ( mesenchymal tissue like ) is a generally observed progression of neoplasms ( cancer ) .
    Because epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are key factors in cancer metastasis and relapse, and hTERT has been shown to exhibit multiple biological activities independently of its telomere-lengthening function, we address a potential role of hTERT in EMT and CSCs using gastric cancer (GC) as a model.[3]

   We additionally  learn that the mechanism related to hTERTS function in elongation and maintenance of telomeres, is related that JAK2/STAT3 pathway.[4]
  Here we demonstrate that STAT3 physically interacts with CD44 and NF-kB and activates the catalytic subunit of telomerase (hTERT) in human breast cancer stem cells. STAT3 plays a role as a signal transducing molecule between CD44 and NF-kB. In addition to functioning as a catalytic subunit of telomerase, hTERT has been reported to function as a transcription co-factor which drives EMT and cancer stem cell phenotype in human cancer.[4]
   As a matter of reference, and for better understanding of the histology, Singh and Settleman [6] provide a nice reference figure if the EMT transition.  The EMT is a natural occurring process in the case of wound repair, where de-differentiation and re-differentiation is a naturally regulated process, that seems to get "hi-jacked" in the case of cancerous cell transformation.

EMT cell surface markers
   Two of the most potentially lethal properties of the acquired mesenchymal phenotype ( often referred to grade ) are that that they tend to dis-associate as a function of loss of cell adhesion, and they tend to "home" for bone. It seems that these two lethal results  of the EMT transition can be attributed to markers stimulated by activation of hTERT.
  As mentioned in the above quite hTERT is a key factor in the transition to the more aggressive "mesenchymal" cell type, or phenotype. The markers for this more "stem like" grade of cancer are cd44+ and cd24-  where ( of course ) cd stands for cluster of determination, and usually means cell surface marker by which cells can be labelled immunologically or sorted in a cell sorter ( flow cytometry).

 EMT and loss of cell-cell adhesion 
   If this section  seems overly general, it is because all the details of loss of cell adhesion have not yet been decoded, and loss of cell adhesion in the EMT is still somewhat of a "paradox". [5] In general, normal (mammary) glandular tissue consists of differentiated epithelial cells that enclose lumenal caviities guareded by tight junctions between cells. In addition, the basal portion of these glandular cells recognize and bind to extra-cellular signalling molecules in the basement membrane. When an EMT is initiated by a cancer causing event, these cell surface proteins and associated cellular infrastructure disintegrate. The resulting sells loose their (histologically) visible structure, and become free to migrate through the blood stream to other parts of the body. Thus, the most dangerous phase of breast cancer has been enabled.

Homing to Bone
  The other major result of the EMT transition is that resulting disassociated "stem like" cells begin to "home for bone". This is one of the (again) most lethal aspects of breast cancer. Once cancer cells have metastasized to bone, they become impervious to immune defenses and surgery. The tendency to home to bone is now presumed to be due to the inappropriate expression of cd44 / HCELL. [7][8]. Sackstein provides the best description of the roll of the HCELL "glycoform" of cd44:

The molecular effectors that mediate cancer metastasis are best known for their role(s) in directing normal leukocyte trafficking, such as to lymphoid organs and to sites of inflammation and tissue injury. The braking adhesive interactions of flowing cells onto the vascular endothelium consist of shear-resistant tethering and rolling of circulating cells onto the endothelial surface. This primary step is mediated principally by the selectin class of adhesion molecules. The selectins comprise a family of three lectins that bind sialofucosylated glycans on their respective ligands. One of these selectin ligands, known as HCELL (Hematopoietic Cell E/L-selectin Ligand), is a specialized sialofucosylated glycoform of CD44 that is characteristically expressed on human hematopoietic stem cells.[8]
In essence, what Sackstein is saying is that HCELL is a normal marker and binding surface protein that has a normal role in the trafficking of white blood cells ( leukocytes). When it is mis-expressed on free circulating cancer cells, it causes them to leave the circulation system in a manor similar to leukocytes, and take residence in bone marrow. The result is new colonies of cancerous cells in bone marrow. They recognize bone marrow by "binding" or tethering to the suface of the blood vessel. The blood vessel signals that initiate rolling are carbohydrate decorations on proteins. Proteins that bind to carbohydrates are called "lectins".  The HCELL marker is a member of a special subset of lectins called selectins.

Summary
   Epigenetic repression of telomerase/hTERT in somatic cells provides a first line of defense against loss of control of the cell cycle. When telomerase is expressed, it aids in transforming the cell to a more "stem like" phenotype. It contributes in many ways, some unknown, to what is called the epithelial mesenchymal transition. As a result of this transition, cells change bio-markers to become cd24- cd44+ . This transition creates a new population of cells which loses its tendency to form regular cuboidal/columnar epithelium as a result of loss of cell to cell adhesion markers. The loss of cell adhesion leads to cell disassociation. Likewise, the cd44 marker when expressed, causes the disassociated "stem like" cells to home for bone. Those cells which defeat immune defenses and lodge in bone marrow stroma are likely to be protected from the defenses of the immune system by mechanisms that have not been completely described. It is these bone resident cells which begin new untreatable colonies ( metastasis ), and initiate the final stage of the cancer.



References

 [1] White, H.B. Coenzymes as fossils of an earlier metabolic state J. Mol. Bio. 7 101-104 (1976)

 [2]Sheila A. Stewart, William C. Hahn, Benjamin F. O'Connor, Elisa N. Banner, Ante S. Lundberg,Poonam Modha, Hana Mizuno, Mary W. Brooks, Mark Fleming, Drazen B. Zimonjic, Nicholas C. Popescu, and Robert A. Weinberg Telomerase contributes to tumorigenesis by a telomere length-independent mechanism  Proc Natl Acad Sci U S A. 2002 October 1; 99(20): [PubMed Central]

[3]Liu Z, Li Q, Li K, Chen L, Li W, Hou M, Liu T, Yang J, Lindvall C, Björkholm M, Jia J, Xu D. Telomerase reverse transcriptase promotes epithelial-mesenchymal transition and stem cell-like traits in cancer cells.Oncogene. 2013 Sep 5;32(36):4203-13. [PubMed]
 
[4] Noga Bloushtain-Qimron, et. al. The JAK2/STAT3 signaling pathway is required for growth of CD44+CD24– stem cell–like breast cancer cells in human tumors J Clin Invest. 2011 July 1; 121(7): 2723–2735.[PubMed Central]

[5] Mei Chung Moh and Shali Shen  The roles of cell adhesion molecules in tumor suppression and cell migration A new paradox Cell Adh Migr. 2009 Oct-Dec; 3(4): 334–336. [PubMed Central]

[6] Singh A, Settleman J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene. 2010 Aug 26;29(34):4741-51. [PubMed Central]

[7] Chung SS1, Aroh C1, Vadgama JV2. Constitutive Activation of STAT3 Signaling Regulates hTERT and Promotes Stem Cell-Like Traits in Human Breast Cancer Cells. PLoS One. 2013 Dec 30;8(12) [PubMed Central]

[8] Sackstein R, Merzaban JS, Cain DW, Dagia NM, Spencer JA,
Lin CP, Wohlgemuth R (February 2008). Ex vivo glycan engineering of CD44 programs
human multipotent mesenchymal stromal cell trafficking to bone.
Nat. Med. 14 (2): 181–7. [PubMed]

[9] Jacobs PP, Sackstein R.  CD44 and HCELL: preventing hematogenous metastasis at step 1. FEBS Lett. 2011 Oct 20;585(20):3148-58 [PubMed]