Carlo M. Croce, MD, Ohio St. University Comprehensive Cancer Center, Columbus, OH
Gregory Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Victor Ambros, Ph.D., Dartmouth Medical School, Hanover, NH
David Baltimore, Ph.D., California Institute of Technology, Pasadena, CA
Michele A. Cleary, Ph.D., Rosetta Impharmatics, Pasadena, CA
Anindya Dutta, MD, Ph.D., University of Virginia School of Medicine, Charlottesville, VA
Scott M. Hammond, Ph.D., University of North Carolina, Chapel Hill, NC
Lin He, Ph.D., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Tyler Jacks, Ph.D., MIT Center for Cancer Research, Cambridge, MA
Sakari Kauppinen, Ph.D., University of Copenhagen, Copenhagen, Denmark
Joshua Mendell, MD, Ph.D., Johns Hopkins University, Baltimore, MD
Amy Pasquinelli, Ph.D., University of California, San Diego, La Jolla, CA
Tariq Rana, Ph.D., University of Massachusetts Medical School, Worcester, MA
Martine Roussel, Ph.D., St. Jude Children’s Research Hospital, Memphis, TN
Andrei Thomas-Tikhonenko, Ph.D., University of Pennsylvania, Philadelphia, PA
Layout and Goals
- The Basics of MicroRNAs
- Altered MicroRNA Expression in Cancer
- MicroRNA Pathways
- Moving MicroRNA toward the Clinic
The 2007 Forbeck Foundation Forum will focus on the topic of microRNA. MicroRNA, as the name implies, are small RNA that have been identified in human cells. Despite their small sizes, microRNAs have big roles in human biology. Recently, scientists have discovered that microRNAs are involved in cancer, and that microRNA will be useful in the prediction, the diagnosis and the treatment of cancer. The Forbeck Forum on MicroRNA and Cancer will be a catalyst for expanding this forefront of cancer research.
What are microRNAs and why are they important? To answer these questions, we need to recall the central dogma of biology established in the second half of 20th century based on the double-helical DNA structure and the studies of simple organisms such as bacteria. In this central dogma, DNA is the genetic repository. DNA is transcribed into three types of RNA- messenger (mRNA), transfer (tRNA) and ribosomal (rRNA). Together, these three types of RNA assemble proteins from amino acids and the proteins then carry out the biological functions. This central dogma has spun tremendous progress in biology, leading to the identification of mutated human genes that encode defective or dangerous proteins to cause various diseases including cancer. After the human genome sequence was completed at the beginning of this century, biologists were surprised to find that only 2% of the genome coded for proteins. Some of the genome sequences are used to build the infrastructure for gene expression and chromosome maintenance, but this infrastructure does not take up 98% of the genome.
Most recently, biologists have realized that our genome encodes RNA other than mRNA, tRNA and rRNA. Among these new types of RNA are the microRNAs. At the moment, computational methods have predicted that there are about 1000 microRNA genes in the human genome. The exact number will probably not be known for a while. So far, biologists have found microRNA to function as an inhibitor of mRNA. MicroRNAs bind to mRNA with two consequences, either causing mRNA to be degraded, or preventing mRNA from being translated into proteins. A microRNA can bind to several different mRNAs.
Conversely, more than one microRNA can target a single mRNA. In fact, two human microRNAs have been found to bind an mRNA that codes for a protein, which cancer cells depend on for their survival in the body. Patients with defects in those two microRNA genes end up with too much of this protein and therefore are at a higher risk for cancer. The Forbeck Forum will bring together leaders in the microRNA field. The organizers of this Forum will be Dr. Carlo Croce, who has discovered microRNA mutations in a large number of human cancers, and Dr. Greg Hannon, who has created microRNA to block genes that cause cancer. Because microRNAs are small, scientists believe that they can be efficiently delivered into cancer cells in the body; and microRNA holds the promise for being a brand new class of medicine.
The past few years have seen a veritable revolution in biology, with the discovery that small RNAs act in a broad range of biological processes and pathways. In the expanding world of small RNAs, microRNAs hold a special place in that they are evolutionarily conserved regulators of gene expression networks. These ~18-22 nucleotide, single stranded RNAs are derived from fairly conventional genes, with the exception being that the end-product of the gene is a small, non-coding RNA rather than an encoded protein. Standard transcriptional regulatory circuits govern the synthesis of the primary microRNA transcript, which is then processed through two steps to yield a mature small RNA. These small RNAs join RISC, the core effector complex of the RNA interference (RNAi) pathway, associating specifically with an Argonaute protein. The Argonaute protein uses the small RNA as a guide to select silencing targets based upon sequence complementarity between the small RNA and a target messenger RNA. Through such interactions each microRNA can regulate the expression of potentially hundreds of genes. While the human genome harbors 20- 30,000 protein-coding genes, microRNAs are numbered in the hundreds. However, this number, combined with broad target profiles, gives microRNAs potentially quite large roles in regulating the biology of an organism.
The Symposium brought together leaders in the field of small RNA biology with leaders in cancer biology with a special interest in small RNAs. This group was charged with addressing three questions, which can be broadly described as follows. Cancer is fundamentally a genetic disease, with alterations in oncogenes and tumor suppressors driving tumor initiation and progression. Historically, only the role of protein encoding genes has been examined in depth; however, mounting evidence has suggested that microRNAs may contribute to cancer at a disproportionately high rate, considering that there is only about 1% as many microRNAs as protein coding genes.
The first key question for Symposium participants was to examine the possibility that microRNAs play a special role in cancer, perhaps because of their ability to impact the expression of related networks of genes. A significant percentage of metastatic tumors present without an obvious primary origin. Understanding these lesions is critical, as that information is used to guide therapeutic decisions. MicroRNA expression patterns can often serve as a signature of cell type, and several studies have indicated that determining microRNA profiles may serve as a more effective record of cell fate decisions than the expression patterns of protein coding genes.
The Symposium was charged with discussing the issue of whether microRNA expression patterns may be used for diagnostic purposes, either to determine the cell of origin for a particular lesion or to reveal the underlying patterns of oncogenic mutations in a tumor. The prospect that small RNAs can be used directly for therapy has generated an enormous amount of excitement. Engaging the RNAi pathway directly, by delivery of artificial microRNAs, may allow us to directly target the expression of even mutant forms of protein coding genes selectively. Moreover, given the emerging roles of microRNAs in cancer, these naturally occurring small RNAs may serve as therapeutics or targets for inhibition. The final goal of the symposium was to critically examine the promise and progress in targeting of the microRNA pathway for clinical benefit.
The meeting began with a session devoted to the biology of microRNAs. Greg Hannon introduced the goals of the Symposium and provided an introduction to microRNA biogenesis and mechanisms of action. Key among the issues addressed was the lingering uncertainty over the precise manner through which microRNA repress the activity of protein coding mRNAs. Many hypotheses have been raised, each with some degree of supporting evidence. However, answers to such questions have stubbornly resisted being driven to the level of clarity that has been achieved for other small RNA-based regulatory events. Underlying mechanisms are critically relevant for two reasons. First, if one is to impact this pathway for therapeutic benefit, one must understand the mechanisms by which it acts. Second, understanding the mechanisms of microRNA-mediated control may allow the field to improve the ways that it uses to discover microRNA target mRNA relationships. Again, a confident sense of the regulatory networks that are touched by a given microRNA is critical for the use of these species for diagnosis or therapy. Greg presented data describing functional approaches to uncovering these regulatory networks. He also described emerging new classes of small RNAs, which are less conserved than microRNAs and whose biological roles are only now beginning to emerge.
Victor Ambros, who first discovered microRNAs, presented an overview of microRNAs in C. elegans. He pointed out that many microRNAs are deeply conserved through evolution; and described an example regulatory network in which one of the first microRNAs to be discovered, let-7, acts. Victor again touched upon the issue of defining microRNA targets, describing improved computational methods that take into account the accessibility of the potential microRNA binding site for predicting a regulatory interaction. He also described procedures to complement the computational predictions of microRNA-target pairs with a direct, biochemical method for target identification. Finally, he described a new component of microRNA pathways in C. elegans, which might form a conserved part of this regulatory mechanism.
Amy Pasquinelli continued the C. elegans theme discussing one of the potential mechanisms though which microRNA might regulate their targets. She discussed a specific translational regulator, EIF-6, and how it might act to prevent proper assembly of the ribosome on microRNA-bound mRNAs. She also discussed physiological regulation of the microRNA pathway under different metabolic conditions. Her studies highlight an emerging theme in microRNA biology, that stressing cells can change the output of microRNA mediated control. This is likely to be highly relevant in cancer cells, which encounter stress in many forms, ranging from hypoxia to metabolic stress.
Michele Cleary began the move from the basic biology of microRNA pathways to their biological roles in mammals, with a discussion of her comprehensive efforts to identify the targets of many microRNAs. Her colleagues at Rosetta were among the first to report a robust method for understanding microRNA regulatory pathways. Michele hammered home the notion that if we are to consider microRNAs as therapeutics, we must understand the full spectrum of their targets, not just the one or two microRNA-target pairs that generally emerge in a typical academic report. She presented the roadmap that she and her colleagues are developing for that purpose and showed examples, such as miR-106b, where regulatory circuits could be uncovered and validated by this integrated approach.
Additional specific examples of the biological impacts of microRNAs were provided by Jun Lu, one of the Forbeck Scholars. Jun Lu was among the first to show the value of microRNA expression profiles for diagnosis of human cancers. At the Symposium he showed a beautiful example of microRNAs acting in hematopoiesis, specifically with miR- 150 acting at critical decision points in the erythrocyte differentiation pathway, probably through is regulation of a critical developmental transcription factor, c-myb. This provided an interesting example of a very linear microRNA-target interaction, as a contrast to the view that microRNAs exert their biological effects by regulating broad target networks. David Baltimore continued the theme of microRNA biology with his description of the roles of microRNAs during inflammatory responses. Specifically, he discussed the response to LPS in macrophage, and the roles of microRNAs in regulating gene expression networks in this system. The focus was on miR-155, a known oncogenic microRNA and the product of the BIC non-coding RNA gene and on miR-146 family members.
The meeting continued to move toward a cancer focus with observations from Anindya Dutta on the role of let-7 in regulating the HMGA2 oncogene. Anindya also discussed the role of growth regulatory microRNAs in myogenic differentiation. He raised important concerns about methods for identifying new microRNAs and for quantifying changes in microRNA expression relevant to different physiological states. He noted potential anomalies in the output of microRNA profiling methods that could point to significant regulation of microRNA biogenesis or metabolism.
This theme was followed by Scott Hammond and Carl Novina. Scott had been among the first to report global changes in microRNA pathways in cancer, with the majority of microRNAs that were linked to differentiation decisions being expressed at lower levels in tumors and with microRNAs characteristic of stem/progenitor cells being expressed more prominently. Scott pursued the underlying mechanism that led to these observations and showed that microRNA biogenesis was regulated at the first processing step. He revealed that LIN-28 controlled the processing of the let-7 family of microRNAs, and perhaps by extension differentiation promoting microRNAs more broadly. This discovery may have fundamental implications for the adaptation of cancer cells to maintenance of a less differentiated state and for resistance to differentiation inducing signals.
Carl Novina, another of the Forbeck Scholars, described the coordinate regulation of miR-211 and TRPM1 in melanoma. While TRPM1 is considered the host gene for miR-211, with the microRNA being processed from a TRPM1 intron, Carl noted a divergence in the expression of these genes in tumors. He proposed a complex relationship between splicing of the host intron and the microRNA machinery that may begin to reveal another underlying complexity in the pathways that lead to small RNA production.
Andrei Thomas- Tikhonenko discussed the roles of microRNA in B-cell lymphomas. He started with the curious observation that Pax-5 expression increased the levels of oncogenic transcription factors, Myb and Ets1 without changes in Myb or Ets1 mRNAs, suggesting some form of post-transcriptional control. He found that Pax-5, itself a transcriptional regulator, acted by repressing the expression of miR-15 and miR-16, two known tumor suppressive microRNAs. These in turn normally repress Myb and Ets1 expression, allowing Pax-5 to drive the growth of CLL cells via its control of the mir-15-16 microRNA cluster. Broad changes in the epigenetic state of the genome often accompany tumor formation.
Carlo Croce highlighted a potential role for microRNAs in control of this process. He found that the miR-29 family of microRNAs controls DNA methylation patterns through its regulation of de novo methyltransferases DNMT-3a and DNMT-3b. These microRNAs often show reduced expression in lung tumors, perhaps leading to deregulation of DNA methyltransferases and aberrant tumor suppressor methylation. In fact, Carlo showed that artificially increasing the expression of miR-29 in lung cancer cells restored methylation patterns to normal and allowed re-expression of tumor suppressor genes that had been silenced inappropriately by DNA methylation. This suggests that miR-29s may join the expanding family of tumor suppressive miRNAs.
Tyler Jacks continued the tumor suppressor theme with his discussion of the role of let-7 in a mouse model of lung cancer driven by k-ras activation. He showed that regulated expression of let-7 in this tumor type could potently inhibit the growth of these tumors in animals. This correlated with the suppression of proposed let-7 targets including ras itself and HMGA2. Tyler had previously shown that partial loss of key components of the microRNA biogenesis pathway promoted tumor formation, indicating a broad impact of microRNAs on tumor suppression. His work with let-7 not only reinforced that notion but also provided one of the first examples of a tumor suppressive impact of a single microRNA an animal model of human cancer.
Lin He discussed the roles of microRNAs in known tumor suppressor networks, focusing on miR-34 as a known transcriptional target of p53. miR-34 can mediate the downstream effects of p53 activation on cell cycle arrest, senescence and growth arrest, depending upon the precise cellular context. While miR-34 likely forms a key component of a tumor suppressor pathway, it may also be a tumor suppressor in its own right.
Studies presented by Kristina Cole, a Forbeck Scholar, focused on genomic rearrangements that might impact miR- 34a. This gene is located on at 1p36, a region of common loss in many different tumor types. Kristina showed that 1p36 deletions in neuroblastoma often affect the miR-34 gene, and that these impacts often correlated with lowered miR34 expression. MicroRNAs were also placed in other well known oncogenic pathways by Josh Mendell. A surprisingly large number of microRNAs are regulated by c-myc, with repression of microRNAs being a common consequence of c-myc binding at those sites. Indeed, Josh presented evidence that one of the c-myc repressed microRNAs, miR-195, could antagonize myc-mediated increases in cell proliferation, if the microRNA were released from c-myc control. Myc can also activate the expression of microRNAs, with the oncogenic mir-17-92 cluster being a classic example.
Andrea Ventura, the fourth Forbeck Scholar, described his studies on this cluster and its relatives. Loss of function of these microRNAs, specifically in hematopoietic compartments, caused a defect in B-cell development, with prevalent death of B220-positive pre- B cells. This phenotype was entirely consistent with the demonstrated role of mir-17-92 in B-cell lymphoma, where is inappropriate expression led to increased survival of pre-B cells, thus contributing to tumorigenesis.
Martine Roussel provided a paradigm for efforts to examine microRNA function in other tumor types, with her studies of medulloblastoma. This tumor, which likely arises as a result of a failure of granule neuron progenitor cells (GNPs) to differentiate, showed numerous alterations in microRNA expression. One hypothesis, based upon the general role of microRNAs in regulating cell fate decisions, is that restoring some element of the microRNA expression profile in these cells might induce differentiation even of the transformed derivatives of GNPs. Martine has developed powerful mouse models that not only permit her to address these hypotheses but that also allow the testing of other more conventional therapeutic strategies.
The meeting concluded appropriately with two reports on the possible use of small RNAs as therapeutics. Tariq Rana discussed the use of nanoparticles to deliver both natural and artificial microRNAs to a variety of tissues. He also showed enticing data on the role of microRNAs in regulating the replication and spread of HIV. Sakari Kauppinen provided the flip-side of Tariq’s work, specifically the delivery of modified nucleic acids (LNAs) as specific inhibitors of microRNA function. He showed impressive data demonstrating inhibition of miR-122 in the liver in primates, which gave therapeutically relevant biological effects, in this case on serum cholesterol levels. Given the many demonstrations of the impact of microRNAs both as oncogenes and tumor suppressors during the Symposium, the final two talks sparked optimism that the biological insights that had been discussed over the preceding two days could ultimately lead to impact on the lives of patients.
As with the best of meetings, perhaps the most significant outcomes were not the presentations themselves but were the informal interactions and discussions that arose from bringing together a group of leading investigators with diverse experience and expertise but with a common interest in this emerging and very important area of cancer biology. It is these interactions that will continue to resonate through the field in the form of new ideas and collaborations.