miRNAs and Cancer: Some Thoughts

Micro RNAs are an interesting recent discovery which have been found to control cancers by making them stop or making them grow faster.

We first look at micro RNA and its process and its impact on prostate cancer (PCa). Micro RNAs, miRNA, are small single stranded RNAs which when in the cytoplasm may often bind to other RNA on complement binding sites and thus change or incapacitate the mRNA to which it binds from being translated into a protein. Craig Mello was awarded the Nobel Prize in 2006 for the discovery and his Nobel Lecture provides an excellent overview of the early stages of miRNA investigation.

We now briefly examine the miRNA production and action. This is graphically demonstrated below. From segments of the DNA, segments not containing genes, a long segment called a pri-miRNA is generated and it is then cut to a shorter segment called a pre-miRNA and transported to the cytoplasm outside of the nucleus. Then another protein called Dicer cuts up the pre miRNA into about 22 base single-stranded pair segments which are the miRNA,

Then as we show below the small miRNA can bind to mRNA at complement sites, and in fact the binding may allow for a loop which extends out from the binding sites composed of non-complement base pairs. This binding then inactivates the mRNA and prevents its translation to a protein.

The process continues as follows (See Garcia and Miska in Appasani):

 The process is described in some detail below. Here we describe the steps one at a time as is currently understood ( an alternative view of this is in the paper by He and Hannon, 2004).

It is also possible for the miRNA to target more than one mRNA since the miRNA may bind in its complement binding with many other such sites on other mRNAs. It is currently not clear what the affinity of binding is for an miRNA and any possible mRNA.

Also miRNA may be obtained from introns as well as exons. The former is called intronic and the latter called exonic. Now the exonic miRNA goes through the pri and preprocess whereas the intronic miRNA is cut directly to a pre miRNA segment (see Ying et al in Appasani).

miRNAs have been identified and currently there are well over 1,000. They are named in a simple numerical order such as miRNA 34.

 

miRNA is a single stranded product of the process above. An alternative double stranded product is called small-interfering RNA or siRNA. siRNA usually trigger mRNA degradation whereas miRNA may cause degradation or suppression of translation to proteins. For this section we shall not focus a great deal on the siRNA functions.

Now there may be some dynamics associated with this miRNA process as well. The model above assumes a simple one to one matching of miRNA and mRNA. However the generation of the two RNAs can be continuous and we should be looking at the concentrations. Thus is we define:

[miRNA] to be the concentration of the miRNA

and

[mRNA] the concentration of the targeted mRNA

then we have a dynamic process. Namely we can see a process such as follows:

If [miRNA] < [mRNA] then there will be excess mRNA and its product protein P will have a [P] >0. Otherwise the miRNA will bind to all mRNA and there will be no resultant protein.

One may view miRNA as a buffer agent which controls the [P] of its associated [mRNA]. One can see in dynamic form of a dynamical model. Now since the binding is not necessarily 1:1, namely the miRNA may bind to several mRNA, then we may want to expand the above as follows:

  

There has been a great amount of research regarding the impact of miRNA on cancer and especially on PCa. miRNAs may downregulate tumor suppressor genes such as PTEN. This has been seen in miRNA 21. Colin and Croce have provided several review article regarding miRNA and their influence on cancers. They argue that miRNA alterations are heavily involved in the initiation of many cancers. Their focus had been on CLL, chronic lymphocytic leukemia, and its initiating miRNAs, miR 15 and miR 16. Coppola et al (2010) provide a detailed summary of miRNAs and PCa.

The graphic from Coppola et al is shown below where it depicts a collection of miRNAs which impact various parts of the PCa process.

For example miR34 can cause the activation and recapitulate p53 which in turn induces cell cycle arrest and apoptosis. Loss of the miR34 can result in the impairment of the p53 control of apoptosis and permit the cells to proliferate. Coppola et al perform a detailed analysis of all of the above related miRNAs and their resultant impact on PCa. miR-21 up-regulation leads to PTEN loss and thus is an oncogene.

Recent work by Poliseno et al have shown that PTENmiR-106b. It had already been known that PTEN could be down-regulated by miR-22, miR-25 and miR-302. Their work demonstrated that miR-22 and miR-106b are overexpressed  in PCa miR-106b is an intronic miRNA. The work of Poliseno thus has demonstrated a proto-oncogenic miRNA dependent network that regulates PTEN and thus can have a significant role in initiating PCa.

Micro RNAs are regulators of mRNA, the post transcriptional result which is then used to generate via translation the operative protein. Currently there are nearly 1,000 identified miRNAs. They are generally 22 nucleotides long, short segments, and they usually target specific mRNA and silence it. Each one of the miRNA may act upon many mRNAs.

As He and Hannon state:

Non-coding RNAs participate in a surprisingly diverse collection of regulatory events, ranging from copynumber control in bacteria1 to X-chromosome inactivation in mammals2.MicroRNAs (miRNAs) are a family of 21–25-nucleotide small RNAs that, at least for those few that have characterized targets, negatively regulate gene expression at the post-transcriptional leve.

Members of the miRNA family were initially discovered as small temporal RNAs (stRNAs) that regulate developmental transitions in Caenorhabditis elegans6. Over the past few years, it has become clear that stRNAs were the prototypes of a large family of small RNAs, miRNAs, that now claim hundreds of members in worms, flies, plants and mammals.

The functions of miRNAs are not limited to the regulation of developmentally timed events. Instead, they have diverse expression patterns and probably regulate many aspects of development and physiology. Although the mechanisms through which miRNAs regulate their target genes are largely unknown, the finding that at least some miRNAs feed into the RNA INTERFERENCE (RNAi) pathway has provided a starting point in our journey to understand the biological roles of miRNAs.

miRNAs are simple yet complex entities and key players in the epigenetics which control gene expression.

It is clear from the above that miRNAs can positively and negatively impact many elements in the pathways we have considered in HGPIN and PCa. Coppola et al review several of the key ones. For example:

·       miR-146: Down regulates the AR.

·       miR-34: Can recapitulate p53 resulting in apoptosis and arrest.

·       miR-23: can result in c-myc overexpression and cell proliferation.

In a recent paper by Poliseno et al they have identified several others:

·       miR-106b: Down-regulates PTEN and triggers PIN in murine models.

·       miR-22, miR-25, miR-302: Down-regulating of PTEN.

Similarly the papers by Petrocca et al and that by Calin and Croce detail many of the miRNAs and their impacts on many cancers. As seen in the above graphic these are but a few in the overall targeting of PCa control genes. As Coppola et al state:

The hypothesis that miRs can be regarded as new broad-spectrum oncogenes or tumor suppressor genes has opened a revolutionary field of research with exciting diagnostic and therapeutic perspectives.

The compelling hint of a widespread miR deregulation in cancer pathogenesis came from the analysis of the genomic distribution of 186 miR. In this study, it was demonstrated that more than half of them mapped in cancer-associated genomic regions, namely in chromosomal sites prone to deletions, amplifications or recombinations. These aberrations can result in miR down- or up-regulation, conferring selective advantages to mutated cells.

Additional mechanisms of miR deregulation include altered expression of miRs as a consequence of excessive or deficient processing; aberrant transcription of the precursors by epigenetic silencing of miR promoters or as a result of the activity of oncogenic transcription factors; and more rarely, point mutations in mature miRs or in target sequences that can interfere with normal target recruitment

The problem that we will have in any modeling of HGPIN and PCa is not only do we have issues regarding the somewhat well-known genes but the impact of the epigenetic factors is unknown, complex, and possibly random.

Furthermore miRNAs can act in a positive or negative manner depending upon the cell and the activated networks in the cell. From Croce (2009) we have:

Importantly, miRNAs should not be described as oncogenes or tumor suppressor genes, unless the tissue or cell type involved in their action is specified. For example, miR-221 and miR-222 target an oncogene, KIT, and inhibit the growth of erythroblastic leukaemia30, and therefore function as tumor suppressors in erythroblastic cells. but they also target at least four important tumor suppressors — phosphatase and tensin homologue (PTEN), p27, p57 and tissue inhibitor of metalloproteinases 3 (TIMP3) — and function as oncogenic miRNAs by suppressing these tumor suppressors in various human solid tumours31 (TABLE 1). Therefore, before describing an miRNA as a tumor suppressor or an oncogene, it is necessary to specify in which cell or tissue, as cellular context is crucial for the function of miRNAs….

Recent work on miR-34 has demonstrated its impact on p53 (Rokhlin et al) and the fact that miR-34 significantly mediates the role of p53 in apoptosis in AR dependent PCa.

As we have indicated elsewhere, the concept of the cancer stem cell has received significant attention. There has also been a great deal of work on the area of linking miRNAs and the stem cell model for PCa. In a recent work by Liu et al (2011) the authors demonstrate the nexus between miR-34a and its ability to inhibit PCa stem cells by directly repressing CD44. They observe that cancer stem cells have been observed in many solid cancers by using the fact that CD44 adheres to the cell surface. PCa stem cells with enhance clonogenic and tumor initiating and metastatic capacities are often enriched with CD44+ cell population. The work of Liu et al demonstrated that the administration of miR-34a to PCa cells inhibited PCa metastasis and inhibited PCa regeneration. This is one of the first uses of miRNA as a tumor suppressor.

In a recent paper by Xia (2008) the author states:

The key characteristics of stem cells are that they are capable of self-renewal and differentiation. The mechanisms by which stem cells maintain self-renewal and differentiation are complicated. In the past years, protein-coding genes had been broadly investigated in stem cell self-renewal and differentiation. Recent studies indicate miRNAs as one of the most abundant classes of post-transcriptional regulators proved to be crucial in a wide range of biological processes, which suggest that miRNAs may also play essential roles in stem cell self-renewal and differentiation. Disruption of Dicer function in murine ESs influences miRNA processing and greatly impairs their ability to differentiate …

Cancer stem cells (CSCs) are the cells within a tumor that possess the capacity to self-renew and to produce the heterogeneous lineages of cancer cells that comprise the tumor. CSCs can thus only be defined experimentally by their ability of self-renewal and tumor propagation.

The implementation of this approach explains the use of alternative terms in the literature, such as “tumor-initiating cells” to describe putative CSCs. …

The identification of growth and differentiation pathways responsible for CSC proliferation and survival will help in the discovery of novel therapeutic targets. Previous studies have shown that many signal pathways may participate in regulating CSC functions, including Wnt/β-catenin, Notch, and Sonic hedgehog homolog (SHH). The canonical Wnt cascade has emerged as a critical regulator of stem cells and activation of Wnt signalling has also been associated with various cancers …

CSC maintenance is dependent on β catenin signaling. Moreover, because Wnt/β-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas. It is therefore hypothesized that inhibition of Wnt signaling may provide an effective way to reduce the unwanted stem cell renewal which results in cancers.

Inhibition of Wnt signalling may prove to be an effective road to inhibit the uncontrolled cell renewal that drives cancer. Acting as novel and pivotal regulators of protein-encoding genes, miRNAs will have great potential in regulating CSCs’ biological functions by targeting CSCs-related signal pathway molecules.

 

References:

1. Araud, T., Overview of the miRNA Pathways, Thesis, U Geneva, 2008.2.     
2. Bartel, D., MicroRNAs: Genomics, Biogenesis, Mechanism, and Function, Cell 2004 pp 281-297.
3. Calin, G., C. Croce, MicroRNA Signatures in Human Cancers, Nat Rev 2006 pp 857-866.
4. Calin, G., C. Croce, Chrmosomal Rearrangements and microRNAs, Jrl Clin Inves 2007 pp 2059-2066.
5. Coppola, V., et al, MicroRNAs and Prostate Cancer, Endo Rel Canc 2010 pp F1-F17.
6. Croce, C., Causes and Consequences of microRNA Dysregulation in Cancer, Nature 2009 pp 704-714.
7. Gottwein, E., B. Cullen, Viral and Cellular MicroRNAs as Determinants of Viral Pathogenesis and Immunity, Cell Host 2008, pp 375-387.
8. 7.      He, L., G. Hannon, MicroRNAs: Small RNAs with a Big Role in Gene Regulation, Nature, 2004 pp 522-532.
9. Huang, C., et al, A Study of Cancer Related MicroRNAs through Expression Data and Literature Search, World Acad Sci & Engr, 2009 254-256.
10. Jones, P., S. Baylin, The Epigenomics of Cancer, Cell, 2007 pp 683-692.
11. Mello, C., Return of the RNAi World: Rethinking Gene Expression and Evolution, Nobel Laureate Lecture 2006.
12. Petrocca, F., et al, Emerging Role of miR-106b-25/miR-17-92 Clusters in the Control of Transforming Growth Factor β Signalling, Can Res 2008, pp 8191-8194.
13. Poliseno, L., et al, Identification of the miR-106b-25 MicroRNA Cluster as a Proto-oncogenic PTEN-targeting Intron That Cooperates with Host Gene MCM7 in Transformation, Sci Sig 2010.
14. Rakhlin, O. et al, MicroRNA-34 Mediates AR-dependent p53-indiced Apoptosis in Prostate Cancer, Can Bio & Therapy 2008 pp 1288-1296.
15.  Sevigani, C., et al, Mammalian microRNAs: A Small World for Fine-tuning Gene Expression, Mam Gene V 17 2006, pp 189-201.
16. Xia, H., Great Potential of MicroRNA in Cancer Stem Cell, Jrl Can Mol 2008 pp 79-89.