Review

Oncogene (2006) 25, 6220–6227. doi:10.1038/sj.onc.1209914

Implications of micro-RNA profiling for cancer diagnosis

J M Cummins1 and V E Velculescu1

1The Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins University Kimmel Cancer Center, Baltimore, MD, USA

Correspondence: Dr VE Velculescu, The Ludwig Center for Cancer Genetics and Therapeutics, Johns Hopkins University Kimmel Cancer Center, Baltimore, MD 21231 USA. E-mail: velculescu@jhmi.edu

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Abstract

Micro-RNAs (miRNAs) are a large class of small non-coding RNAs that regulate protein expression in eucaryotic cells. Initially believed to be unique to the nematode Caenorhabditis elegans, miRNAs are now recognized to be important gene regulatory elements in multicellular organisms and have been implicated in a variety of disease processes, including cancer. Advances in expression technologies have facilitated the high-throughput analysis of small RNAs, identifying novel miRNAs and showing that these genes may be aberrantly expressed in various human tumors. These studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer.

Keywords:

microRNAs, expression profiling, miRAGE, cancer, molecular diagnostics, biomarkers

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Introduction

Micro-RNAs (miRNAs) are single-stranded RNAs of 18–25 nucleotides found in both plant and animal cells. Mature miRNAs are generated from precursor molecules in a two-step process (Bartel, 2004). First, primary transcripts (pri-miRNAs), often several kilobases in length, are processed by RNase III enzyme Drosha into approx80 base hairpin structures called pre-miRNAs. Subsequently, pre-miRNAs are trimmed by the RNase III enzyme Dicer into approx22 base RNA duplexes. One strand of each duplex associates with the RNA-induced silencing complex (RISC), whereas the other is usually rapidly degraded. The miRNA–RISC complex targets mRNAs for translational repression or mRNA cleavage. miRNAs may bind mRNA targets with incomplete complementarily, resulting in a potentially large number of targets for each miRNA. Bioinformatic analyses have estimated that miRNAs may regulate as many as 30% of human genes (Lewis et al., 2005).

There has been considerable debate about the total number of miRNAs that are encoded in the human genome. Initial estimates, relying mostly on evolutionary conservation, suggested that there were up to 255 human miRNAs (Lim et al., 2003). More recent analyses using miRNA serial analysis of gene expression (miRAGE), microarray studies and bioinformatic approaches have identified hundreds of additional uncharacterized human miRNAs, many of which are non-conserved (Bentwich et al., 2005; Cummins et al., 2006). As such analyses have only examined a limited number of cell types, studies of additional human tissues will likely reveal many more miRNAs. Our estimate is that there are likely to be as many as 1000 miRNAs, comprising approx3% of the currently known genes in the human genome.

A current challenge is to elucidate the function of miRNAs in normal physiologic processes and in disease states. Several clues have recently pointed to the potential role of miRNAs in cancer and suggest that aberrations in miRNAs may be important in tumor progression (reviewed by McManus, 2003; Ambros, 2004; Xu et al., 2004; Caldas and Brenton, 2005; Chen, 2005; Croce and Calin, 2005; Gregory and Shiekhattar, 2005; Hall and Russell, 2005; Mendell, 2005; Miska, 2005; Morris and McManus, 2005; Esquela-Kerscher and Slack, 2006; Hammond, 2006; Hwang and Mendell, 2006; Slack and Weidhaas, 2006).

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miRNAs and cancer

Potential connections between miRNAs and cancer have been made at several levels. First, a variety of studies have suggested that miRNAs play important roles in cellular growth and differentiation. Functional investigation of miRNAs was originally explored in Caenorhabditis elegans with the discovery of lin-4 and let-7 and later in Drosophila with the identification of the Bantam miRNA (Ambros and Horvitz, 1984; Lee et al., 1993; Reinhart et al., 2000; Brennecke et al., 2003). From these studies, it became clear that miRNAs were critical players in cellular differentiation and developmental timing in these organisms. Indeed, when either lin-4 or let-7 were inactivated in C. elegans, the result was dysregulated cellular differentiation and the reiteration of embryonic cell types in later stages of development (Lee et al., 1993). Bantam overexpression in Drosophila resulted in increased cellular proliferation and prevention of apoptosis with resulting overgrowth of wing and eye tissue. More recent analyses of the RNase III enzyme Dicer in mice have shown that inactivation of this gene results in an inability to produce mature miRNAs and in defective cellular differentiation (Bernstein et al., 2003; Kanellopoulou et al., 2005; Muljo et al., 2005; Murchison et al., 2005). As two characteristics of tumor cells are abnormal cell proliferation and defects in differentiation, the potential dysregulation of miRNAs in human cancer has become an intense area of investigation.

The most compelling evidence for a gene's involvement in human cancer is the observation of acquired genetic mutations in patient tumors. Such alterations can occur through point mutations or through larger chromosomal changes, including amplifications or deletions. Previous analyses have estimated that approximately 50% of miRNAs are located at fragile sites or regions known to be amplified or deleted in human cancer (Calin et al., 2004a). In particular, a cluster of six miRNAs (the mir-17–92 cluster) on human chromosome 13q31 has been shown to be frequently amplified in B-cell lymphomas (He et al., 2005b). Interestingly, these miRNAs are transcriptionally activated by a known oncogene (c-myc), and artificial expression of the mir-17–92 miRNA cluster in a lymphoma mouse model leads to accelerated and more aggressive disease (He et al., 2005b; O'Donnell et al., 2005). Recent analyses have identified amplification of the mir-17–92 cluster with concomitant miRNA overexpression in lung cancers, especially in tumors with small-cell histology (Hayashita et al., 2005). Additionally, a small number of germline or somatic mutations have been reported in a group of five miRNAs in chronic lymphocytic leukemia (CLL) (Calin et al., 2005). In at least two examples, such changes appeared to lead to reduced levels of the miRNAs, suggesting that these mutations may be functionally important. Further work will be required to evaluate systematically the compendium of miRNAs for genetic alterations in human cancers.

Changes in the expression of miRNAs have been observed in a variety of human tumors. Although expression differences may not necessarily reflect causal events of tumorigenesis, such changes may, nevertheless, regulate genes important in tumor pathogenesis and may be useful for classifying tumors and predicting their outcomes. Examples of such gene expression alterations in miRNAs have been detected in CLL (Calin et al., 2002), colorectal neoplasia (Michael et al., 2003; Cummins et al., 2006), pituitary adenomas (Bottoni et al., 2005), lung cancer (Takamizawa et al., 2004; Johnson et al., 2005), Burkitt's lymphoma (Metzler et al., 2004), B-cell lymphoma (Eis et al., 2005; He et al., 2005b; Kluiver et al., 2005), breast cancer (Iorio et al., 2005), glioblastoma (Chan et al., 2005; Ciafre et al., 2005) and papillary thyroid cancer (PTC) (He et al., 2005a).

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miRNA expression profiles

miRNA expression profiles derived from large-scale analyses of tumor samples have recently been shown to serve as phenotypic signatures of particular cancer types. For example, two independent studies have examined the miRNA profile of colorectal cancer compared to normal colonic epithelium (Michael et al., 2003; Cummins et al., 2006). Both studies described a general downregulation of miRNAs in tumor cells and were in agreement that levels of two miRNAs, miR-143 and miR-145, were significantly lower in colorectal tumor cells compared to normal colonic cells. Using the miRAGE approach, Cummins et al. (2006) uncovered over 50 differentially expressed miRNAs, of which 32 were downregulated in tumor cells (Michael et al., 2003; Cummins et al., 2006). Examples of miRNAs that are differentially expressed in human colorectal cancer are shown in Figure 1. Additionally, selected miRNAs that are aberrantly expressed in human cancer together with their clinical correlations are listed in Table 1.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Differentially expressed miRNAs in colorectal cancer. miRAGE was utilized to compare expression levels of miRNAs in two primary colorectal adenocarcinomas with matched normal colonic epithelia. The log10 ratio of expression levels in normal tissue to the average expression levels in tumor tissue is shown on the vertical axis.

Full figure and legend (168K)


It has been postulated that tumor miRNA profiles may resemble those that of their antecedent stem cells and thus reflect developmental lineage (Lu et al., 2005; Meltzer, 2005; Hwang and Mendell, 2006). In accord with the hypothesis that expressed miRNAs may serve as markers of the differentiated state, most studies of miRNAs in cancer have demonstrated downregulation of miRNAs (Lu et al., 2005). For example, of the six miRNAs that were most dramatically differentially expressed in colorectal cancers (>10-fold), all but one were downregulated in tumor cells (Cummins et al., 2006). However, upregulation of specific miRNAs, such as the mir-17–92 cluster described above, appears to be associated with oncogenesis (He et al., 2005b; Volinia et al., 2006). In addition, other miRNAs, including miR-125b and miR-143, have been directly associated with the differentiation and proliferation of certain cell types (Esau et al., 2004; Lee et al., 2005). Finally, miRNA expression patterns may change when cells are treated with differentiation-promoting agents (Kasashima et al., 2004; Stegmaier et al., 2004), and recent studies have shown that undifferentiated human embryonic stem cells express certain miRNAs (Suh et al., 2004). Therefore, although it seems likely that downregulation of certain miRNAs may be a feature of tumor cells and de-differentiation, it remains to be seen whether miRNA expression changes are a cause or consequence of neoplasia. One approach to address this issue would be through systematic disruption of miRNA genes in human cancer cells, using somatic gene targeting approaches (Kohli et al., 2004). Alternatively, recent methods may permit knockdown of miRNAs using modified antisense oligonucleotides (Hutvagner et al., 2004; Meister et al., 2004) or siRNAs that target miRNA precursors (Lee et al., 2005) to evaluate their function.

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Are miRNA expression profiles more useful than those of mRNAs?

Regardless of the functional effect of expression changes of miRNAs, RNA expression profiles may serve as useful diagnostic and prognostic markers of human cancer. With the advent of microarray and serial analysis of gene expression data, there have been a multitude of efforts to correlate mRNA expression signatures with tumor pathology and disease prognosis. However, one of the difficulties with these approaches has been the observation of significant numbers of samples, usually poorly differentiated tumors, that can be difficult to classify using mRNA expression signatures (Ramaswamy et al., 2001). Such results suggested that there may be no robust mRNA markers that show consistent differential expression between tumors and normal tissues of different lineage.

Surprisingly, Lu et al. (2005) have recently shown that miRNA expression signatures can be extremely informative for cancer diagnosis. For example, miRNA profiles of marrow samples from patients with acute lymphocytic leukemia (ALL) were able to accurately distinguish between subsets of patients with different molecular pathologies (e.g. BCR/ABL-positive samples, T-cell ALL samples and samples with mixed lineage leukemia gene rearrangements). These experiments indicate that even within individual malignancies, miRNAs can be used to distinguish among different mechanisms of tumorigenesis. Additionally, a miRNA classifier comprising 217 miRNAs was developed to evaluate tumors of unknown origin. This miRNA classifier was tested (without modification) on 17 poorly differentiated tumor samples with non-diagnostic histological appearance (Ramaswamy et al., 2001; Lu et al., 2005). The miRNA-based classifier established the correct diagnosis of the poorly differentiated samples with substantially greater accuracy (12 of 17 correct) than the mRNA-based classifier (one of 17 correct), further demonstrating the advantage of miRNA profiles to mRNA profiles for diagnostic classification. These observations were all the more unexpected because of the small number of miRNAs evaluated (approx200) as compared to the number of mRNA transcripts normally examined in such studies (>15 000). Perhaps, because of their central role in development and their direct effects on global gene regulation, miRNAs are among the best markers for human cancer. In contrast, it has been speculated that mRNA expression may be less informative as only a small percentage of the protein-coding transcripts are regulatory molecules (Chen, 2005).

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From differential expression to informative markers

In addition to global views of miRNA expression landscapes in cancer, there have been several individual miRNAs which appear to have diagnostic significance in certain malignancies. One such example occurs in CLL, which is the most common leukemia of adults in the Western world and was one of the first in which miRNA expression dysregulation was demonstrated. Croce and Calin (2005) had extensively analysed a common deletion in chromosome 13q13.4 present in approximately 50% of CLL cases, but could not find a protein-encoding gene that was genetically altered in the minimally mapped deleted region. In 2002, however, they reported that two miRNAs, miR-15a and miR-16-1, were located in the minimally deleted region and were downregulated or absent in 68% of CLL samples (Calin et al., 2002). It was later shown that miR-15a and miR-16-1 expression silenced the antiapoptotic factor Bcl-2, suggesting that their low or absent levels in CLLs inhibited apoptosis by reactivation of Bcl-2 (Cimmino et al., 2005). As further proof that miRNAs were players in the pathogenesis of CLL, Croce and co-workers reported mutations in small group of miRNAs in 11 of 75 patients with CLL and a miRNA expression signature composed of 13 mature miRNAs that was associated with prognostic factors and disease progression (Calin et al., 2005; Calin and Croce, 2006). Interestingly, the expression signature exhibited high correlation with already proven prognostic markers, Zap-70 expression levels and the mutational status of the rearranged immunoglobulin heavy-chain variable region (IgVH). Patients with low levels of Zap-70 and mutated IgVH have a more indolent disease course, whereas those with high levels of Zap-70 and wild-type IgVH usually have an aggressive course. Additionally, the miRNA expression signature differentiated patients with a short interval from diagnosis to initial therapy from those patients with a significantly longer interval. Whether these CLL miRNA signatures have prognostic significance independent of Zap-70 expression and IgVH mutation status has yet to be determined.

Examination of miRNA expression patterns in lung cancers by Takamizawa et al. (2004) identified a reduction in tumors of the let-7 miRNA, a homolog of one of the original miRNAs described in C. elegans. The authors were able to classify 143 cases of lung cancer into two major groups according to the let-7 expression. Those showing reduced let-7 expression had significantly shorter survival after surgical resections (P=0.0003). Impressively, let-7 levels were more powerful predictors of patient survival than age, tumor histology and smoking history. The only factor that more significantly impacted survival was disease stage. Overexpression of let-7 was shown to inhibit cancer cell growth in vitro, although the mechanism in human cancer remains unclear. One clue may come from the recent observation that in C. elegans the let-7 family of miRNAs negatively regulates let-60/RAS, the C. elegans homolog of the human RAS oncogene, which is frequently mutated in lung tumors (Johnson et al., 2005). The correlation of let-7 levels with disease outcomes in lung cancer has recently been independently confirmed by a study which also implicated miR-155 as a prognostic marker in lung tumors (Yanaihara et al., 2006).

Diagnostic potential has also been recognized for miRNA profiling in PTC, breast cancer and B-cell lymphoma. miRNA profiling of PTCs by de la Chapelle and co-workers showed upregulation of numerous miRNAs, including a set of five miRNAs that clearly distinguish between PTC and normal thyroid tissue (He et al., 2005a). The authors reported that unaffected tissue surrounding the cancers in several patients also showed upregulation of the most highly expressed miRNA in PTC, miR-221, perhaps representing an early event in the pathogenesis of PTC. For breast cancers, miRNA profiles have been reported to distinguish breast tumors from normal breast epithelium, and have been correlated with specific breast cancer pathologic features such as estrogen and progesterone receptor status, tumor stage, vascular invasion and proliferation index (Iorio et al., 2005). A single miRNA, B-cell integration cluster (BIC)/miR-155, appears to be dramatically overexpressed in several types of B-cell lymphomas, including a subset of diffuse large B-cell lymphomas (DLBCLs) (Eis et al., 2005; Kluiver et al., 2005). DLBCL samples with an activated B-cell phenotype had high levels of BIC/miR-155, whereas those with the germinal center phenotype did not. As the activated B-cell subtype of DLBCLs has a worse clinical prognosis, determination of miR-155 overexpression in DLBCL may be useful in the management of these patients.

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Future applications and potential limitations

Given the rapid progress over the past several years, it is likely that miRNAs have a promising future in the realm of cancer diagnostics. Golub and co-workers have already demonstrated the potential for miRNA profiles to classify metastases of unknown origins. This discovery is significant as metastatic carcinomas from an unknown primary site account for approximately 5% of all newly diagnosed malignant lesions and carry an 85% 1-year mortality rate (Brigden and Murray, 1999). Early determination of tumor origin provides a potential opportunity for targeted therapy and eliminates the use of empiric chemotherapy, which may have limited efficacy and significant morbidity and mortality.

The utility of miRNA profiling in CLL, lung cancer, thyroid cancer, breast cancer and B-cell lymphoma is now apparent. In CLL and lung cancer, specific expression signatures are associated with either favorable or poor prognoses. Patients falling in the category with unfavorable outcomes may be placed into appropriate clinical trials, treated more aggressively, or receive palliative care depending on the particular case. Likewise, patients with favorable prognoses can benefit by having a better indication of their outcome and avoiding potentially harmful treatments. An exciting future prospect is that the miRNA patterns associated with particular outcomes may ultimately provide insights into the underlying etiologies of disease and uncover therapeutic targets.

An especially interesting prospect is the potential for miRNAs to serve as early warning markers for cancer initiation or progression. In the case of PTCs, the accumulation of miR-221 in peritumoral tissue has been postulated to be an early event in PTC carcinogenesis. It remains a tantalizing possibility that other pre-invasive neoplastic lesions may have dysregulation of miRNA levels that could serve as sentinels of tumor initiation. Unfortunately, the reproducible detection of minute quantities of such RNA species in the blood or other easily accessible bodily fluids may prove challenging and will likely necessitate further technical advances.

On the horizon, miRNA profiles may ultimately inform the targeted treatment of cancer. For example, in lung and breast cancers, patients with epidermal growth factor receptor mutation and HER2/NEU amplification, respectively, have shown benefit from targeted antibody therapies against these receptors (Pao and Miller, 2005; Piccart-Gebhart et al., 2005; Romond et al., 2005). In these cases, the tumor cells are thought to be dependent on dysregulated intracellular signaling, and are therefore more sensitive to inhibition of mutant proteins driving these pathways (Weinstein, 2002; Varmus, 2005). It will be important to determine if mutant or dysregulated miRNAs control pathways that are essential for tumor growth, as such miRNA-regulated proteins might be useful therapeutic targets. At least two examples already suggest that miRNA dysregulation may affect major oncogenes such as c-myc and RAS (Johnson et al., 2005; O'Donnell et al., 2005). Additionally, the recent successes of inhibition of miRNAs at the cellular level (Hutvagner et al., 2004; Meister et al., 2004; Lee et al., 2005) suggest the possibility of direct targeting of miRNAs that are amplified or upregulated in patient tumors.

Most of these clinical applications will depend on accurate assessment of miRNA profiles in human samples. Major hurdles in such analyses will be convenience, cost and the acceptance of a standard profiling platform. Fortunately, miRNAs, unlike larger RNAs, remain largely intact in routinely collected formalin-fixed paraffin-embedded clinical samples (Meltzer, 2005), enhancing their potential utility and suggesting that their overall levels may be less likely to be affected by storage or collection procedures. A variety of platforms have recently been developed for miRNA expression analyses (Table 2). Higher throughput expression approaches can in general be classified as hybridization-based methods using microarrays, or cloning and sequencing approaches (including miRAGE). While the latter have the advantage of being open systems that permit identification of novel miRNAs and accurate miRNA quantitation, the former are cost effective and more amenable to a large number of routine analyses. Examination of individual miRNAs can be performed by Northern hybridization and specialized real-time PCR, and can be assessed in cellular contexts through in situ hybridization. At present, no standard methodology exists for hybridization-based profiling of miRNAs, and as a consequence comparison of expression data from different experiments can be difficult. A set of commercially available standard miRNAs or samples would be helpful in comparing results among analyses of miRNA profiles for both research and clinical use. In addition, the current processes for microarray analyses of miRNAs involve technically challenging enrichment, ligation and/or amplification steps, which make the prospect of miRNA profiling more expensive and labor intensive than mRNA profiling. However, technology development in the field, particularly in the realm of bead-based flow cytometry, single molecule detection and massively parallel sequencing coupled with the miRAGE approach, may help establish an automatable, high-speed process for miRNA profiling in the near future (Lu et al., 2005; Neely et al., 2006; Service, 2006)


In summary, we are entering an era where analyses of miRNAs appear poised to play a role in clinical management of human cancer. Although still in their infancy, miRNA analyses offer possibilities in tumor classification, disease prognosis, early cancer detection and therapeutic decision making. Technical hurdles for such analyses remain, but these are likely to be surmountable with new technologies designed to make miRNA profiling fast, reliable and cost effective. Although clinically relevant miRNA studies move forward, it should be remembered that this field is relatively young and many questions remain. The total number of human miRNAs has yet to be determined, the targets of miRNAs and their roles in cellular pathways are unexplored and the function of dysregulated miRNAs in human cancer remains largely a mystery. Given the important functions that these small RNAs have already been show to play in normal biology, it would be a safe bet that they will have a similarly large role in human cancer.

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