March 15, 2010 (Vol. 30, No. 6)
Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications
Scientists Scrutinize Promising Molecules as Potential Drug Targets and Biomarkers
MicroRNAs (miRNAs) finely regulate gene expression and play an important role in various cellular processes, including cell growth, differentiation, proliferation, and apoptosis. To date, more than 5,000 of these endogenous, noncoding single-stranded RNAs have been identified. miRNAs act through binding to complementary mRNA sequences, thereby preventing their translation into protein or accelerating mRNA breakdown. Investigators are working on exploiting these molecules for experimental and potential therapeutic applications.
As presenters will discuss at CHI’s “MicroRNA in Human Disease and Development” meeting later this month, miRNAs’ mechanistic reach extends well beyond suppression of gene expression and encompasses a complex system of post-transcriptional control. Investigators have found that miRNA regulatory processes involve miRNA activation of gene expression by interacting with complementary regions found in the promoter coding region, as well as the 3´ UTR of their mRNA targets.
Extensive regulation of miRNA itself occurs at the levels of miRNA promoter transcription, methylation, miRNA processing, RNA editing, and miRNA-target interactions.
Brian D. Brown, Ph.D., assistant professor of genetics and genomic sciences at the Mt. Sinai School of Medicine, will describe studies using artificial miRNA target sites to exploit or modulate endogenous miRNA regulation at the meeting. He says that several features of miRNA target sites on mRNA influence the results of miRNA binding to the site. These include complete or incomplete complementarity between miRNA and its mRNA target; the number of target sites on a transcript, which is directly proportional to miRNA-mediated suppression; and the sequence nucleotide sequence around an miRNA target site.
For example, in the nucleotide sequence surrounding a given target, secondary structures in the RNA can reduce target-site accessibility and reduce the chance of transcript regulation by the miRNA.
These observations suggest some guidelines, which will be discussed by Dr. Brown in his presentation, for leveraging endogenous miRNA regulation to improve the outcomes of basic functional studies and potential therapeutic applications including gene and cell therapy.
In the gene-therapy arena, the investigators observed that transgene expression from hepatocyte-specific promoters occurred in antigen-presenting cells, as well as liver cells, causing potential problems because gene therapies targeting liver cells can trigger an antitransgene product T-cell response.
Dr. Brown and his colleagues found that they could prevent this response by modifying the 3´ UTR of a vector to contain four tandem copies of a sequence that is perfectly complementary to miR-142, an miRNA highly expressed in APCs but not in hepatocytes. Studies of transgenic mice showed that miR-142-3p suppressed reporter gene expression more than 100-fold in all mouse cells of hematopoietic lineage, including apes.
The potential therapeutic value of designing vectors under the control of miRNA regulatory mechanisms was verified when hemophilia B mice were injected with the same miRNA-regulated vector and encoding factor IX. The mice were cured of factor IX deficiency, as well as immunological tolerance to factor IX, Dr. Brown explains.
When possible, “miRNA target sites for any type of study should be placed in transcript regions of high accessibility, for example the 3´ UTR A-rich regions, to optimize miRNA binding.”
RNAs have the potential to become a new class of biomarkers to detect cancer at its earliest stages, as well as to characterize specific cancers. Dirk Dittmer, Ph.D., and his colleagues at the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, are focused on understanding viral tumorigenesis—specifically, cancers that are caused by Kaposi’s sarcoma-associated herpes virus (KSHV/HHV-8) and the development of high-throughput tools for viral diagnostics.
Using Kaposi’s sarcoma (KS) as an example, Dr. Dittmer discussed the relative merits of pre-miRNAs vs. mature miRNAs as biomarkers. Dr. Dittmer’s laboratory has shown that tumor suppressor miRNAs (miR-222/221, let-7 family) are significantly downregulated in primary effusion lymphoma (PEL), a lymphoma caused by KSHV or human herpesvirus 8 (HHV-8). PEL occurs most commonly in patients with immunodeficiency diseases, including AIDS. The investigators also distinguished among miRNAs present in latently virus- infected nontumorogenic endothelial cells and Kaposi’s sarcoma cells, identifying 15 virally regulated miRNAs in the endothelial cells.
Two types of miRNAs—MiR-143 and -145—were elevated only in KS tumors, not virally infected endothelial cells, therefore, representing tumor-specific rather than virus-specific miRNAs. “Because many tumor-suppressor proteins are wildtype in KS and PEL, downregulation of multiple tumor suppressor miRNAs provides an alternative mechanism of transformation,” Dr. Dittmer says.
Few studies have simultaneously assessed all three levels of miRNA regulation. Dr. Dittmer says that his group was able to determine changes in gene copy number, pre-miRNA, and mature miRNA levels in a large set of PELs, detecting PEL-specific miRNA gene amplifications and concordant changes in pre-miRNA and mature miRNA. They also identified 68 PEL-specific miRNAs that, according to Dr. Dittmer, define the miRNA signature of PEL.
They further showed that transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used for the classification of human tumors. “Both pre- and mature miRNA profiling have their place in biomarker research. Pre-miRNAs are ideally suited to investigate signaling events that change miRNA expression, for example, in response to drugs. Mature miRNA profiles integrate miRNA expression, nuclear export, and stability and thus, are more useful in longer-term experiments or if there is a suggestion that a particular tumor changes miRNA processing.”
Aurora Esquela-Kerscher, Ph.D., of the department of microbiology and molecular cell biology at Eastern Virginia Medical School, will present recent findings on the role of the let-7 miRNA family in processes related to cellular growth and differentiation. Since the early phases of carcinogenesis resemble embryonic development, often involving the re-expression of embryonic mesenchymal genes, Dr. Esquela-Kerscher explains, many of the same cellular processes used to direct appropriate embryonic development are the same mechanisms that go awry in cancerous tissues.
Her lab focuses on analyzing how miRNAs control developmental events and how their dysfunction contributes to cancer progression using a combination of Caenorhabditis elegans and mammalian model systems to functionally characterize miRNAs.
In particular, she and her colleagues have focused on the role of the let-7 family during cancer progression. Let-7 miRNA controls the timing of cell-cycle exit and terminal differentiation in C. elegans, she says, and is poorly expressed or deleted in human lung tumors.
At the meeting, Dr. Esquela-Kerscher will present findings using the nematode to understand how let-7 functionally overlaps with other miRNAs, such as those belonging to the lin-4 family, to direct common developmental events associated with cancer-related pathways. For instance, animals that carry a triple deletion for the lin-4 homologue, mir-237, and certain let-7 homologues have morphological defects and fertility problems related to abnormal mitotic and meiotic abnormalities in the germline that were not found in the single- or double-deletion combinations.
Dr. Esquela-Kerscher and colleagues have also begun to determine how the let-7 miRNA family functions as tumor suppressor genes in mammals by negatively regulating the RAS, MYC, and HMGA2 oncogenes, as well as several cell-cycle progression genes. The scientists showed that let-7 overexpression in cancer cell lines alters cell-cycle progression and reduces cell division, and reciprocally inhibiting let-7 function leads to increased cell division in A549 lung cancer cells, providing evidence that let-7 functions as a tumor suppressor in the lung.
“Our work now shows that multiple genes involved in cell-cycle and cell-division functions are also directly or indirectly repressed by let-7. This work reveals let-7 miRNA to be a master regulator of cell-proliferation pathways.”
Dr. Esquela-Kerscher and colleagues at Yale University further showed that let-7 miRNA directly inhibits the growth of lung cancer cell xenografts in immunodeficient mice. Using an established orthotopic mouse lung cancer model, the investigators showed that intranasal let-7 administration reduced tumor formation in vivo in the lungs of animals expressing a G12D-activating mutation for the K-Ras oncogene.
Dr. Esquela-Kerscher is also studying the role of let-7 outside of the lung, particularly in tissues of urothelial origin. Her lab found that let-7 is dysregulated in prostate cancer cells differing in their metastatic status, indicating a novel role for this miRNA in prostate cancer progression.
Furthermore, she is interested in determining if let-7 and other miRNAs can be useful diagnostic and prognostic biomarkers for prostate cancer. Due to her access to a vast repository of human prostate specimens from the Virginia Prostate Center at Eastern Virginia Medical School, her lab has begun to analyze the miRNA expression profiles in FFPE specimens taken from the primary prostate tumor, normal adjacent tissue, and the lymph-node metastasis of individual prostate cancer patients, as well as in tumors from patients presenting insignificant and organ-confined cancer.
They are also determining the “miRNA signatures” in blood, urine, and expressed prostatic secretions that were obtained from both normal and cancer patients. Dr. Esquela-Kerscher is optimistic in the future use of miRNAs for the treatment and diagnosis of a wide range of human cancers.
“The challenge will be to effectively deliver small RNAs to the tumor and to develop reliable and affordable miRNA-based biomarkers that can be measured in the clinic using noninvasive means.”
miRagen Therapeutics develops miRNA-based therapeutics for cardiovascular and muscle disease. William S. Marshall, Ph.D., miRagen’s president and CEO, will talk about identification of miRNAs associated with cardiovascular disease at the meeting. miRagen is developing miRNA inhibitors targeting miRNAs 15 and 208 to treat post-myocardial infarction remodeling and chronic heart failure, respectively. These miRNA targets were identified through extensive profiling efforts, and the company has performed validation experiments to show that these miRNAs control significant disease drivers thought to contribute to the development of pathological hypertrophy.
The company recently showed that inhibition of miR-15, an miRNA that regulates several pathways implicated in the control of apoptosis, reduces the area of cardiac tissue death after a myocardial infarction. miRagen scientists have consistently observed upregulation of the miR-15 family (miR-195 and miR-16) in diseased versus normal heart tissue from multiple animal models and human samples, suggesting that upregulation of these miRNAs may inhibit the expression of pro-survival factors, thereby contributing to cardiomyocyte death.
Subsequent studies have shown the benefits of inhibiting the targeted miRNA’s in rodent disease models. The company has demonstrated the benefits of antimiR-15 treatment after myocardial infarction in limiting the size of infarct, as well as reducing pathological hypertrophy in longer term studies. AntimiR-208 treatment in the transaortic banding model of hemodynamic overload showed a reduction in pathological hypertrophy, improvement of cardiac function, and inhibition in muscle form switch to the slower α-myosin form.
Emerging reports suggest a potential role for miRNAs in both neuronal survival and the accumulation of toxic proteins associated with neurodegeneration, and how these proteins may themselves influence miRNA expression.
Kai-Christian Sonntag, M.D., Ph.D., assistant professor of psychiatry at Harvard Medical School, will discuss studies under way to determine whether dysregulation of signaling pathways in Parkinson disease (PD) pathogenesis are associated with deregulated miRNAs. He and his colleagues, through miRNA profiling on laser-microdissected dopaminergic (DA) neurons from normal individuals and PD patients, found distinct miRNA expression patterns.
According to Dr. Sonntag, “these studies showed dysregulation and, in particular, profound downregulation of gene expression relevant to PD pathogenesis. Additionally, using quantitative real-time (qRT)-PCR, we validated some of the microarray results and could confirm the observed downregulation of gene expression in PD. We also demonstrated that genes were differentially expressed within the DA neurons up to 200-fold, independent of gender or disease. Although this was consistent within the sample populations, we also observed high variation between individuals.”
miRNA profiling was performed on the same sample population as the microarrays using laser microdissected DA neurons and high-throughput megaplex TaqMan® miRNA qRT-PCR assays (Applied Biosystems, part of Life Technologies) for 382 miRNAs including three conserved nuclear miRNAs.
“Regardless of disease or gender we found that in all samples the SN DA neurons exhibited a distinct pattern (fingerprint) of miRNA expression. From the 379 miRNAs about 161 were expressed above detection threshold and the majority were higher expressed in PD.” However, Dr. Sonntag says, as in the microarrays, differences were often subtle.
The scientists also did computational analysis to determine PD-specific miRNAs based on target predictions. “When we did negative correlations, we could identify about 20 miRNAs that had significant target correlations. We then analyzed the pool of all targets for these 20 miRNAs using the same methodology as for the microarrays, for example, clustering the genes according to signaling pathways relevant to PD. This revealed that about 10 PD-specific miRNAs were prominent and four to six of them were overrepresented.”
It appears that predicted targets for these miRNAs don’t seem to be key factors in PD. Rather the PD-specific miRNAs were associated with all signaling pathways related to PD such as growth factors, neurotransmitters, ion channels, protein degradation, synaptic dysfunction, and cytoskeleton, but surprisingly little mitochondrial dysfunction and programmed cell death. In addition, miRNAs seem to preferentially target members of gene subfamilies that are deregulated in our microarrays.”
Cancer Cell Profiling
Stephanie Fulmer-Smentek, Ph.D., R&D group manager for biological systems at Agilent Technologies, has been working with the National Cancer Institute (NCI) to characterize miRNA and mRNAs for the NCI-60, a set of cancerous cell lines derived from nine different tissues. Dr. Fulmer-Smentek will present new miRNA and mRNA profiling data obtained using Agilent’s gene-expression and miRNA microarray platforms, providing what she and her team hopes will be a model system for such analyses.
Dr. Fulmer-Smentek and her group worked with isolated total RNAs from the NCI-60, performing hybridization assays using whole-genome expression arrays to detect mRNA expression, they then analyzed the samples for miRNA using the company’s miRNA microarray. This approach allowed comparison between gene-expression profiles and miRNA profiles, and they reported that the data demonstrated high producibility across replicates for both platforms.
The researchers found a strong correlation between the two profiles and the tumor cell tissue of origin. But, Dr. Fulmer-Smentek adds, “we found that the diversity within and between tissues of origin is much greater for miRNA than mRNA. This data might be useful in terms of providing a framework to which other data can be compared. But, whether data from the NCI-60 will allow definitive identification of the tissue of origin for other samples remains to be seen. There is, however, strong evidence in the field that miRNA profiles can be used to define the tissue of origin for cancer samples.
“In connecting the mRNA data with the miRNA we do find evidence of some of the same interactions in terms of genes that are known to be related to deletion-specific miRNAs, for example, the miRNA17-92 cluster on chromosome 13.” This suggests that the NCI-60 can indeed serve a role in helping to define interactions between different miRNA and their mRNA targets.
“One of the interesting things we found was that, in general, for many different tissues, there is a strong correlation between cell lines for both the miRNA and mRNA profiles, but the level of correlation is quite different for different tissues of origin.” For example, she found that the leukemia cell lines were quite distinct from the other cancer cell lines according to their miRNA and mRNA profiles, but show “a lot of diversity within the group.”
The data that was developed during these studies “provides a huge wealth of information for researchers,” says Dr. Fulmer-Smentek. “It extends the present knowledge of the cell lines, and allows for more extensive analysis, not just within these two new datasets, but also with all of the other available biological data about these cell lines and extending to other similar types of data on other cancer samples.” The take-home point is that there is a tremendous “opportunity to extend the analysis to many other types of data, putting miRNA and mRNA in context of the larger biological picture.”
Patricia F. Dimond, Ph.D. ([email protected]), is a life science consultant.