Tuesday, April 12, 2011

Striking the Heart of Hydra with miRNA

Cancer has long been depicted as a nasty crab.  Now Colin Barras describes it as a “distant animal ancestor, a ‘living fossil’ from over 600 million years ago.”  If cancer is an organism, it is an alien in our bodies.  An alien believed by many to be driven by cancer stem cells on an engine of endothelial-mesenchymal transformation (EMT) phenotype. 
By disrupting core signaling circuits, these alienic cancer cells (and mass) in our bodies are capable of resisting or evolving past the commonly used chemotherapeutic agents and even defeat the modern billion dollar kinase and growth receptor targeted MAbs or small molecule agents.  Current "magic bullet" therapies that cut one or more heads of Hydra have limits in this war against cancer.  With so many things going wrong at so many fronts, cancer is a system biology problem.  What is needed is identification and targeting of master regulators; microRNAs (miRs or miRNAs) are one class of such regulators.  MicroRNA-126 (miR-126) was the first miRNA to be described with a direct role in cancer development; forced expression (upregulation) of miR-126 leads to the development of acute myelogenous leukemia.  Now we have a long rap sheet (read here, here, here) implicating several families of miRNAs in various solid tumors and leukemia

MicroRNAs are key regulators in cell

A single miRNA family can bind an estimated 500 mRNAs, regulating gene expression of multiple genes, and potentially altering the course of a physiological or pathological process.  For instance, a single microRNA miR-181 can regulate T cell receptor binding and signaling, a process that requires over 40 kinases and phosphatases, by targeting post-transcriptional regulation of multiple phosphatases.  Thus, miRNA target is one of the most promising areas to apply a system biology or network approach to cancer.

MicroRNAs are 18-25 nucleotide evolutionarily conserved non-coding RNAs that act as master regulators of gene expression, influencing fundamental cellular processes, such as, proliferation, differentiation and death, and organ development.  MicroRNAs are transcribed from the intergeneic (e.g., introns) or intragenic regions by RNA polymerase II as 1-3 kb pri-miRNAs; pri-miRNAs are processed by ribonucleases Dosha and DGCR8 complex in the nucleus to 70-100 nucleotide long hairpin intermediates called pre-miRNAs, and are exported from nucleus to cytoplasm.  In the cytoplasm, pre-miRNAs are further processed by a ribonuclease Dicer into a 18-25 nucleotide double-stranded RNA; after strand separation, the mature or guide strand (aka miRNA) is incorporated into an RNA-induced silencing complex (RISC); the passenger strand is degraded.  RISC complex consists of miRNA, argonaute 1 – argonaute 4 proteins, and other proteins.  Besides miRNA, argonaute proteins are also potential targets for drug development in this area.

The binding of miRNA to the complementary sequences the 3’UTR of target mRNA triggers mRNA degradation; imperfect miRNA:mRNA bindings result in attenuation of translation and decreased protein concentration.  A single mRNA may have binding sites for multiple miRNAs.  Over 1000 miRNAs have been described, but only a handful have been correlated with certain cancers, for instance, miR-15a-miR-16-1 cluster is downregulated in CLL, whereas, miR-155 is upregulated in CLL; the former acts as tumor suppressor and latter as an oncogene.  MicroRNA genes are deregulated in cancer as a result of genomic alterations—chromosomal translocations, loss of hetrozygosity, etc.—or, extragenomic changes, such as, aberrant promoter methylations, loss of ribonuclease Dosha or Dicer activity, miRNA promoter modulation by oncoproteins, such as, MYC or p53.  Tumor suppressor miRNAs include miR-15a-miR-16-1 cluster, Let-7a-2 family, miR29b-1-miR-29a, miR-29b-2-miR-29c families,
miR-26a, miR-34a, miR-34b and miR-34c.  Onco-miRNAs include miR-17-92, miR-21, miR-155, mir-372 and miR-373  [see Table I in Garzon et al.] 

Therapeutics targeting miRNA pathways

Inhibitors targeting onco-miRNAs include antisense oligonucleotide (DNA or RNA) chains stabilized by various modifications.  Common oligonucleotide modifications include 2’O-methyl-group, phosphorothioiate linkage, and locked nucleic acid (LNA) constructs; LNA contain 2’-O and 4’O methylene linkages.  Antagomirs are 2’O-methyl-modified cholesterol conjugated, phosphorothioiate-linkage containing single-stranded RNA analogs.  Other strategies are: miRNA sponges (transcripts with multiple miRNA binding sites,) miR-mask (small oligonuecleotides that bind and 'mask' miRNA binding sites on target mRNAs) and screening for small molecule inhibitors targeting miRNA transcription.  Newer nanoparticle delivery methods are key to bringing these strategies to clinic.  The approaches considered for tumor suppressor miRNAs are to increase or mimic expression by gene delivery or other methods. 

Turning miRNA discoveries into products 
*The list is becoming longer by the day (read here); although, I don't expect all to be oncology focused.
  • Mirna Therapeutics (Austin, TX) pipeline has two lead candidates, tumor suppressors Let-7 for non-small cell lung cancer, and miR-34a for metastatic prostate cancer.  The second lead candidate miR-34a has a role in lung, prostate and skin cancers and T-cell leukemias, and is also expressed in cancer stem cells. [...]  The company expects to file INDs late this year or early 2012.

(Let-7 regulates multiple cancer-associated pathways; Fig from Mirna Therapeutic website)

  • Regulus Therapeutics in San Diego was created in 2007 as a partnership between Isis Pharmaceuticals (Carlsbad, Calif.) and Alnylam Pharmaceuticals (Boston, Mass.)  Their oncology program contains onco-miRNA miR-21 and a tumor suppressor miR-34; miR-34 expression is lost in melanomas and neuroblastomas. 
  • MiReven (Australia), a company dedicated to commercialize miR-7.  MicroRNA-7 regulates EGFR signaling (read here).
  • SPC3649 of Santaris Pharma (Denmark) is the first miRNA therapeutic to enter clinical trail.   Last September, it entered Phase II trails.  SPC3649, an antagomir and LNA, targets miR-122 for treatment of HCV infection to prevent liver cirrhosis and liver cancer.

 Others are developing diagnostics based on miRNA signatures
  • Rosetta Genomics (Rehovot, Israel) launched three microRNA-based tests, miRview™ squamous, miRview™ mets and miRview™ meso, in 2009 for cancer
  • Exiqon (Copenhagen) uses proprietary miRCURY LNA™ Universal RT microRNA PCR platform to detect colorectal cancer biomarkers in less than 0.2 ml of blood.[..|..]
  • Asuragen (Austin, TX) - none listed yet on website; they do provide miRNA profiling services in collaboration with Affymetrix.  Many others also provide profiling services or tools: WaferGen Biosystems has SmartChip Human microRNA Panel V2 containing 1200 human miRNA types from the miRBase database.[..]
  • Compendia Bioscience (Ann Anbor, Mich.) received an SIBR grant last August to develop miRNA-based diagnostics [..]

And, quite a few are making tools to allow all the discoveries happen

Ambion provides miRNA mimics, antagomirs; System Biosciences sells miRZip, an anti-miRNA lentiviral systems; SwitchGear Genomics (Menlo Park, Calif.) provides Synthetic miRNA Target GoClone™ reporters to study miRNA activity [..]; antagomirs are obtained from Alnylam; miRNA microarrays and related services are available from Exiqon (LNA arrays), L.C. Sciences and ABI; GeneTools provides morpholinos.


  • Targeting microRNAs in cancer: rationale, strategies and challenges. Garzon et al. (Oct. 01, 2010) Nature Reviews Drug Discovery 9:775-789| DOI | Scholar | PubMed
  • The role of microRNAs in acute myeloid leukemia. Hyde and Liu  (Nov. 24,  2010) F1000 Biol Reports 2010, 2:81 |DOI | FullText | ; also see, Interfering with Cancer: MicroRNAs may drive the development of leukemia. Hyde and Liu (Jan. 01, 2011) The Scientist 25(1):47 | FullText
  • Current prospects for RNA interference-based therapies. Beverly L. Davidson & Paul B. McCray. Nature Reviews Genetics 12, 329-340 (May 2011) | doi:10.1038/nrg2968 | Abstract |  ; TABLE 1: Clinical trials for RNAi therapy [Table link
  • [miRNA POSTER] Regulation of microRNA biogenesis, function and degradation. Jacek Krol, Inga Loedige and Witold Filipowicz.  Nature reviews Genetics 11(10), October 2010 [Free Link]
  • .
  • Tumours could be the ancestors of animals by Colin Barras (March 11, 2011) New Scientist issue 2803 p.12  |FullText
  • Cancer tumors as Metazoa 1.0: tapping genes of ancient ancestors. Davies and Lineweaver (20110 Physical Biology Vol.8, Article. 015001 | DOI | FullText |
  •  .
  • RNA interference | Wikipedia |
  • miRNA Blog by Christoph  http://mirnablog.com/
  •  .
  •  Santaris Pharma A/S advances miravirsen, the first microRNA-targeted drug to enter clinical trials, into Phase 2 to treat patients infected with Hepatitis C virus, September 22, 2010 — [press release]
  • miRNA and biotech world - [in news]
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  1. What a comprehensive lay of the land when it comes to miRNA. I came across a neat way to sum up the importance of microRNA in an article this morning: "How can you understand disease if you don’t know the control? It’s like playing music but you don’t know where the volume control is."

    You mention in that list of growing products/discoveries that you don't expect all to be oncology focused. What other areas of disease do expect this therapeutic technology to be more readily applicable to in the near future?

    Thanks again for the informative post.


  2. Kelly,

    Other clinical areas where miRNA discoveries and RNAi are having an impact are metabolic diseases, diabetes, viral diseases, cardiovascular, neurodegenerative diseases and regeneration. See: TABLE 1 (Clinical trials for RNAi therapy) in "Current prospects for RNA interference-based therapies" by Beverly L. Davidson & Paul B. McCray, Jr. Nature Reviews Genetics 12, 329-340 (May 2011) http://www.nature.com/nrg/journal/v12/n5/fig_tab/nrg2968_T1.html

    *I liked the music metaphor. Thanks.


  3. There are many articles that suggest that miRNA can be regulated by small molecules. But I did not find any reference that can specifically say how the small molecule can be so specific that can bind to desired miRNA (out of 1200 known human miRNAs) not others. Please share your views or let me know if there is any study on this aspect.
    Thanks in advance