Cancer Initiation: Micro RNAs that regulate cell cycle check point components are implicated in loss of control of the cell cycle in glioblastoma

Thursday, January 23, 2014

Micro RNAs that regulate cell cycle check point components are implicated in loss of control of the cell cycle in glioblastoma


 Introduction
 When a cell is fully differentiated into a functional somatic cell of a particular tissue, it is thought to have permanently exited the cell cycle. The presumed normal fate of a cell that has differentiated and exited the cell, is to serve its physiologically relative functional life, and then die through a process of programmed cell death called apoptosis. When a cell has been transformed to a cancerous cell, it re-enters the cell cycle and begins to divide out of control.
   So, precisely where is the mechanism that fails and sends the cell back into the cell cycle?  This is a foundation of cancer research. There are multiple pathways by which cells exit and enter the cell cycle. These pathways most often must work in conjunction with molecular regulatory mechanisms called checkpoints .  When we use the term checkpoint, we most correctly are referring to these molecular mechanisms that work at the G1 to S transition and G2 to M transition, where (of course ) the four phases of the cell cycle are G1, S, G4 and Mitosis (M) .

Checkpoint implementation
  As a bit more review of cell cycle regulation, we must say that as the cell progresses through each phase of the cell cycle it produces proteins called cyclins, which are a bit like sands in an hourglass. When enough cyclins of a specific type have been produced, the cell reaches a threshold, or critical point where it is capable of moving thorough a checkpoint to the next phase.
   When this happens, it is because enough cyclins have been produced to activate a member of a family of proteins called cyclin dependent kinases. (CDK)   A kinase is an enzyme that phosphorylates ( adds a phosphate group ) to a substrate. In the case of cell cycle checkpoint implementation, the phosphorylation substrate of cyclin dependent kinase is retinoblastoma, or pRB when referring to the protein associated with the retinoblastoma ( rb ) gene.
  Retinoblastoma itself gets its name from a type of cancer with which it was originally associated. A retinoblast is a particular type of developmental cell in the retina of the eye, and cancer arising from these retinoblasts had been designated retinoblastoma before the discovery of molecular mechanisms underlying cancer. If fact retinoblastoma was predicted from genetics of afflicted patient families before the gene was discovered.  Retinoblastoma ( pRB) can be a cell cycle regulation component in all cells, not just retina cells.
   When a checkpoint is in the "blocked" state, pRB is bound to a protein called E2F. After pRB has been phosphorylated by a cyclin dependent kinase, it becomes unable to bind to E2F, and thus E2F is free to provide it's own signalling function.
  E2F itself is a transcription factor. When it is in its activated, it translocates ( moves ) to the nucleus of the cell and binds to the promoter region of appropriate genes on the cells DNA. In the case where the promoter has not been methylated,  and thus blocked, this is the initial step of gene expression for the genes that represent the next step in the cell cycle.
   In practice, specific ( numbered or lettered ) Cyclins, CDKs and E2Fs are associated with each checkpoint.  Below is a table:

Checkpoint Cyclin Cyclin Dependent Kinase
G1-S Cyclin A CDK4/6
G2-M Cyclin B CDK 1

Epigenetic factors controlling checkpoints

   More recently, epigenetic regulation of checkpoint genes has come under scrutiny of researchers. When we speak of epigenetics, we are referring to  micro RNAs that bind to and regulate specific messenger RNAs, promoter methylation, and reversible changes to the histones that package genes in the nucleus.
  Promoter methylation plays a large role in determining whether an activated gene ( transcription factor available ) actually becomes an expressed gene ( promoter unblocked by methylation and binding of MeCP2 ). Methylation patterns themselves are caused by an RNA/RNA interaction between the expressed gene and an associated micro RNA ( mi-RNA ). As such, most recent research has begun to focus on genes and their micro RNA regulators.
    Now we will transfer our general theory to actually observed cancer research. Much or most of the previously mentioned principles of cyclin/checkpoint regulation was actually done by Nobel Laureate Paul Nurse in fission yeast. The following quite is from the abstract of a recent paper [1] on the aggressive brain brain cancer glioblastoma multiforme (GBM).
Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138.  
After observing that gene expression of micro RNA 138 had been lost in lines of glioblastoma cells, expression was artificially induced (ectopic expression ) using a cloned version of the gene and a vector (  replication deficient viral transporter ). As a result of reinserting active miRNA-138 into cell lines from glioblastoma multoforme, the G1/S checkpoint was restored, and the cells exited their abnormal growth program.

In addition to the checkpoint proteins we previously discussed, We see an unfamiliar one called EZH2. EZH2 itself has been found to be implicated in many cancers, but its precise role was inknown. Its normal function relates to epigenetics, in the role of histone modifier as described in the following from Qui[1]:
 EZH2 possesses the histone methyltransferase activity and can lead to gene silence through methylating histones [15] and [16]. EZH2 is over-abundant in a broad range of human malignancies including GBM, and contributes to tumor proliferation and cell cycle control [12], [16] and [17]. The Cyclin-dependent kinase 4 and 6 (CDK4/6) are critical regulators of cell cycle G1/S transition and are specialized to phosphorylate and inactivate the cell cycle controller Retinoblastoma protein (Rb) [18].
 The new hypothesis is that EZH2 itself is a target of E2F, and as such a feedback loop can develop. What they go on to show is that model cell lines can be forced out of the cell cycle by introducing a micro rna which down-regulates members of the checkpoint pathway.
 Additionally, it is exhibited that the transcription factor E2F1 is able to drive EZH2 transcription [21]. Therefore, we come to the hypothesis that an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1 is involved in gliomagenesis, and this signal loop could be suppressed by the candidate miRNA.


Summary
   The addition of another, epigenetic level of understanding to neo-genic ( cancer causing ) pathways multiples the number of candidate therapies. As a result of new information, scientists can work on drug candidates that are more specific for a certain type of cancer. In this case a microRNA that is shown to inhibit EZH2 activity/expression is shown to rescue cancer cells, and force them out of the cell cycle by breaking a feedback loop associated with a checkpoint at the G1-S checkpoint.
   As a more general observation about the introduction of epigenetic models and hypothesis generation into cancer research, computational expertise, relating to RNA/RNA interaction is playing a greater role in research planning and interpretation. We are moving toward an era in which discovers will come from unique computational methods only.


References
[1 ] Qiu S, Huang D, Yin D, Li F, Li X, Kung HF, Peng Y.
Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme.
Biochim Biophys Acta. 2013 Oct;1832(10):1697-707. [Full Text]

[2] Chase A, Cross NC. Aberrations of EZH2 in cancer.Clin Cancer Res. 2011 May 1;17(9):2613-8. [Abstract]

[3] DeGregori J, Johnson DG. Distinct and Overlapping Roles for E2F Family Members in Transcription, Proliferation and Apoptosis. Curr Mol Med. 2006 Nov;6(7):739-48.  [PubMed]



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