Journal ClubCancer Biology

MicroRNAs Add an Additional Layer to the Complexity of Cell Signaling

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Science Signaling  02 Aug 2011:
Vol. 4, Issue 184, pp. jc5
DOI: 10.1126/scisignal.2002182

Abstract

MicroRNAs (miRNAs), key posttranscriptional regulators of many biological processes, have been implicated in many diseases, including cancer. In a recent paper, Avraham and colleagues take a systems biology approach to determine whether and how miRNAs are involved in the regulation of oncogenic signaling networks downstream of epidermal growth factor (EGF). The authors showed that EGF stimulation orchestrated the transcription of both miRNAs and transcription factors. An early decrease in the abundance of a subset of miRNAs allowed for the induction of messenger RNAs of immediate early genes. Expression of this group of miRNAs was also decreased in tumors that showed deregulated signaling through the EGF receptor (EGFR) or the related receptor HER2. Their biological properties of redundancy, multiplicity, and rapid responsiveness make these small noncoding RNAs important regulators of cell signaling.

Signal transduction networks are triggered when an extracellular ligand activates a receptor that, in turn, drives alterations of intracellular molecules, thus inducing a cellular response. This often results in a cascade of signals within the cell, with each step amplifying the signal. Therefore, a limited stimulus can result in a large response relayed to the nucleus as a transcriptional program, for example. To prevent uncontrolled amplified signaling, the cell requires safeguard mechanisms to attenuate cascades in a rapid, widespread, and coordinated manner. Perturbations of receptor tyrosine kinases (RTKs) that lead to uncontrolled signaling are often associated with tumor initiation and progression. Two of these RTKs, epidermal growth factor (EGF) receptor (EGFR or HER1) and the related receptor HER2, affect many breast and brain tumors by relaying unrestrained signaling due to gene amplifications or activating mutations (1). A central path from RTK activation to cytoplasmic signaling and nuclear transcriptional programming is well established in growth factor–stimulated tumor initiation and progression. This complex network contains multiple feedback and feedforward mechanisms and is further regulated by microRNAs (miRNAs) (2, 3).

miRNAs are small, highly conserved, single-stranded RNAs that act by binding to complementary sequences, often in the 3′ untranslated regions (UTRs) of messenger RNA (mRNAs), which leads to translational inhibition or degradation of the message (4). In the past decade, roles for these modulators of gene expression have emerged in many biological processes, including cell cycle regulation, differentiation, migration, and metabolism (5, 6). Aberrant expression of miRNAs is common in cancers, and these small RNAs can function as oncogenes or tumor suppressors (7).

Avraham and colleagues used a systems biology approach to elucidate how miRNAs are involved in the regulation of oncogenic signaling networks (2). The authors measured the coordinated regulation of miRNAs and mRNAs over time following EGF stimulation of MCF10A human mammary epithelial cells. The authors focused on a subset of 23 immediate down-regulated miRNAs (ID-miRs) that were repressed within 60 min after treatment with EGF. This same group of ID-miRs was repressed in brain or breast tumors driven by constitutive EGFR signaling or genomic alterations in the genes encoding EGFR or HER2, suggesting that suppression of these miRNAs could play an important role in tumorigenesis.

Concurrent alterations in the abundance of miRNAs and mRNAs following EGF stimulation were then compared computationally. The seed sequence of each miRNA usually includes bases 2 to 7 from the 5′ end of the miRNA, and a perfect match to a target sequence is required to down-regulate expression. Based on the complementarity between the sequences carried by mRNAs in their 3′ UTRs and seed sequences of the ID-miRs, the authors found that mRNAs of immediate early genes (IEGs) were more likely to contain sequences recognized by ID-miRs. In addition, the expression pattern of ID-miRs negatively correlated with IEGs after exposure to EGF. Therefore, the authors hypothesized that loss of expression of this group of ID-miRs permitted the rapid induction of oncogenic transcription factors (TFs) encoded by IEGs, such as c-FOS and EGR1 (Fig. 1).

Fig. 1

Schematic representation of EGF-regulated microRNAs and transcription factors that converge on target genes. In parallel to the transcription factors (TFs) that are activated by EGF signaling and mediate transcriptional activation of corresponding immediate early genes (IEGs), immediate down-regulated microRNAs (ID-miRs) that act to repress IEG translation are down-regulated by an unknown mechanism. The protein products of the IEGs induced by EGF signaling collectively stimulate transcription of delayed early genes (DEGs) to complete the response cycle.

CREDIT: X. FU/BAYLOR COLLEGE OF MEDICINE

In silico seed sequence analysis of ID-miRs and their target IEGs revealed features of redundancy (the same IEG being targeted by several miRNAs) and multiplicity (several IEGs targeted by the same miRNA) in this network (2). Because the target sequences to which miRNAs bind are often as short as six or seven nucleotides within the 3′ UTRs of target mRNAs, a single miRNA can recognize multiple targets. This multiplicity inherent in miRNA function is important biologically for coordinate regulation of mRNAs that may function in the same or similar cellular processes. For example, miR-122 has been shown to coordinately regulate the expression of mRNAs that encode proteins with related functions in lipid metabolism (8). Yet, genetic analysis in Caenorhabiditis elegans showed that only 10% of individual miRNA knockouts led to clear developmental or morphological defects (9). This illustrated the existence of functional redundancy among miRNAs, or among miRNA-regulated pathways. This redundancy between miRNAs suggests that miRNAs may have evolved into functionally related families that control or refine specific aspects of terminal differentiation programs of specific cell types. These features of multiplicity and redundancy are highlighted by the observation that miRNAs tend to exhibit distinctive expression profiles both during normal development and in specific disease states, such as cancer (10, 11).

Evidence of miRNA multiplicity was shown by Avraham and colleagues in cause-and-effect experiments in which individual ID-miRs were overexpressed in MCF10A cells (2). Whereas miR-630 was the only miRNA found to reduce cell viability on its own, most were able to inhibit EGF-induced migration and decrease the expression of multiple predicted target IEGs when individually overexpressed. Given the redundancy of some miRNAs, the authors knocked down the endonuclease Dicer, which is required for processing pre-microRNAs into their mature forms, by RNA interference rather than targeting individual ID-miRs. Serum-starved cells showed increased expression of many of the IEGs following Dicer knockdown, consistent with the findings that these IEGs are attenuated by miRNA overexpression.

Rapid response is another distinguishing feature of miRNA-mediated gene regulation that contributes to the efficient and specialized regulatory network of cellular responses to extrinsic stimuli. Transcriptional repressors must interact with sophisticated machinery localized in nuclei—not in the cytoplasm where translation occurs—to repress expression of specific target genes. This response of gene repression at the transcriptional level is delayed further by the stability of existing mRNAs. In contrast, miRNAs can rapidly repress protein production at the site of translation—the ribosome—or by sequestering mRNAs in P bodies (12), which are distinct foci within the cytoplasm that contain enzymes that mediate mRNA decay. In addition, owing to their small size and noncoding nature, miRNAs may be produced de novo more rapidly than transcription factors, thus shortening response time. The authors found that the IEGs were induced upon Dicer knockdown in growth factor–deprived cells (2), raising the potential of abundant ID-miRs in resting cells to act as restraint factors to prevent preexisting IEG mRNAs from being translated for cell proliferation and migration. This mode of regulation of IEGs by miRNAs improves the cellular response time as compared to de novo synthesis of transcription factors and possibly also has energy-saving benefits (13).

Avraham et al. show that down-regulation of ID-miRs may be a consequence of EGF signaling and often occurs in cancers associated with EGF pathway activation (2). It is unknown whether down-regulation of these miRNAs is a consequence of uncontrolled signaling or is one of the factors that contributes to it. The expression of some individual ID-miRs is lost in human cancers, suggesting that they may be causally involved. In addition, Dicer and other components of the miRNA biogenesis pathway are haploinsufficient tumor suppressors, indicating that an overall depletion of miRNAs may promote cancer development and progression (14, 15). Further evidence for these particular miRNAs playing tumor suppressive roles comes from Avraham and colleagues, who found that the viral oncogene v-Fos contains a shorter 3′ UTR than the endogenous c-Fos gene, which contains binding sites for miR-101 and miR-155. The 3′ UTR of v-Fos lacks the miR-101 binding site and contains a single base replacement within the miR-155 site, thereby escaping regulation by these two ID-miRs (2).

These findings may have important therapeutic implications. Because there are 23 ID-miRs, it is unlikely that restoring all of them would be a viable cancer treatment option. However, because these miRNAs show redundancy and multiplicity in their target specificities, treatment with a small number of exogenously synthesized miRNAs may be effective. In addition, as we learn more about the mechanism by which this group of miRNAs is coordinately regulated, perhaps an agent could be developed to restore ID-miR expression, or more traditional agents that target the protein products of miRNA-regulated genes could be used. Additionally, EGFR- and HER2-targeted therapies are currently being used clinically, and it may be important to test whether these 23 ID-miRs have an impact on sensitivity to drugs targeting these receptors.

Avraham and colleagues’ systematic analysis of miRNA and mRNA expression following EGF stimulation has revealed a mechanism whereby miRNAs act as a safeguard to prevent uncontrolled oncogenic signaling. This highlights a previously underappreciated facet of growth factor receptor signaling regulation and expands on the pivotal role of miRNAs in tumorigenesis. The authors focused on an early network, the ID-miR-IEG axis, because it appears to be a general target in breast and brain tumors. Further studies of global transcriptional response to EGF—including the so-called delayed early responses that occur after the immediate early phase—will undoubtedly reveal other important subnetworks regulated by this pathway in cancer and other diseases.

References

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