Small RNA: A Large Contributor to Carcinogenesis?

J Gastrointest Surg (2009) 13:1379–1388 DOI 10.1007/s11605-009-0887-6

REVIEW ARTICLE

Small RNA: A Large Contributor to Carcinogenesis? Imran Bhatti & Andrew Lee & Jonathan Lund & Michael Larvin

Received: 21 October 2008 / Accepted: 24 March 2009 / Published online: 17 April 2009 # 2009 The Society for Surgery of the Alimentary Tract

Abstract Introduction Homeostasis in normal tissue includes balancing cell proliferation and apoptosis (programmed cell death). Mutations in proto-oncogenes or tumor suppressor genes may lead to disruption of normal cellular function, uncontrolled cell proliferation, and subsequent carcinogenesis. Discussion Micro-RNAs (miRNAs) are short (19–24 nucleotide) noncoding RNA sequences that inhibit protein translation and can cause the degradation of subsequent messenger RNA, thus playing an important role in the regulation of gene expression. Aberrant expression of miRNAs has been shown to inhibit tumor suppressor genes or inappropriately activate oncogenes initiating the cancer process. Unique miRNA expression profiles have been found in different cancer types at different stages, suggesting a possible diagnostic application. This review summarizes the current evidence supporting a link between aberrant miRNA expression and carcinogenesis and its possible role in improving diagnosis and treatment of cancers, particularly of gastrointestinal origin. Keywords Micro-RNA . miRNA . Cancer . Carcinogenesis

Introduction The genetic stability of normal tissue is maintained tightly by mechanisms which regulate cell proliferation and apoptosis (programmed cell death). Proto-oncogenes are genes that are susceptible to mutational activation to oncogenes, which may then promote cell growth and mitosis through cell signaling pathways, and tumor suppressor genes promote DNA damage responses to minimize the tumorigenic effect of mutations. Dysfunction in such genes may lead to disruption of normal cellular function, I. Bhatti (*) : A. Lee : J. Lund : M. Larvin Division of Surgery, School of Graduate Entry Medicine and Health, University of Nottingham Medical School at Derby, Derby City General Hospital, Uttoxeter Road, Derby DE22 3DT, UK e-mail: [email protected]

uncontrolled cell proliferation, and subsequent tumor formation or carcinogenesis. The precise mechanism for the initiation of cancer remains unclear. Micro-RNAs (miRNAs) are short (19–24 nucleotide) noncoding RNA sequences that are involved in the regulation of human gene expression (noncoding RNA is transcribed from a DNA sequence, but not translated into protein). miRNAs bind to messenger RNA and prevent gene expression by inhibiting protein translation.1,2 Newly discovered miRNAs are still poorly understood; however, studies performed on animal cells have shown them to be involved in key cellular, immune, and developmental processes.3 miRNA was first discovered in the nematode Caenorhabditis elegans in 1993 by Lee et al.4 The gene lin-4 was transcribed into a 22 nucleotide RNA molecule and found to inhibit protein synthesis.4 This molecule inhibited the expression of Lin-14 by directly binding with the 3′ untranslated region of its transcribed messenger RNA molecule.1 Lee et al.4 found that this process plays a crucial role in larval development. Furthermore, mutations in lin-14 miRNA caused abnormalities in the execution of a

1380

terminal differentiating program, preventing cells from reaching their fully differentiated state.4 Since 1993, over 5,000 miRNAs have been discovered in 58 different species (approximately 500 in humans). Each has multiple targets, which are thought to regulate 30% of protein coding genes.5 Aberrant expression of miRNAs has been shown to inhibit tumor suppressor genes or inappropriately activate proto-oncogenes initiating neoplastic transformation. Unique miRNA expression profiles have been found in different cancer types at different stages, suggesting a possible application in cancer diagnosis and perhaps future treatment strategies.6

Biogenesis and Function of Micro-RNA Formation of mature miRNA follows a three-step process: firstly, miRNA genes are transcribed into primary miRNA (pri-miRNA); secondly, the pri-miRNA is cleaved into premiRNA, which is then transported into the cytoplasm; and finally, the pre-miRNA is cleaved and unwound to form mature miRNA (Fig. 1). MiRNA genes are transcribed into double-stranded primiRNA by RNA polymerase II.7 Pri-miRNA can be found

J Gastrointest Surg (2009) 13:1379–1388

as independent transcripts or incorporated into intronic regions of other genes 4. pri-miRNA is then cleaved by two distinct complexes: Drosha in the nucleus and Dicer in the cytoplasm, both members of RNase III enzyme family. Drosha cleaves the pri-miRNA into pre-miRNA which is transported out of the nucleus via a nuclear membrane transporter Exportin 5.8,9 Dicer cleaves the pre-miRNA which is then unwound by helicases to form two mature miRNAs. MiRNA inhibits messenger RNA (mRNA) translation10 by a number of mechanisms, including cleaving of the mRNA at the miRNA binding site and by translational repression of the target transcript.11 Cleavage of the mRNA involves the RNA-induced silencing complex (RISC), which has also been well documented in the shortinterfering RNA (siRNA) pathway. This complex associates with Argonaute 2 proteins Gemin 2 and Gemin 3 after it has been charged by the miRNA.12,13 Translational repression of the mRNA occurs in polyribosomes and involves an as yet unknown mechanism. Some studies have reported localization of mRNA and miRNA to cytoplasmic foci known as processing bodies (p-bodies).14,15 These p-bodies contain a decapping enzyme (hDcp 1/2), an exonuclease (hXrnl1), and a mRNA degradation protein (LSm 1–7).14,15 Kong et al.16 demonstrated that the mechanism involved in translational repression is determined by the promoter used to transcribe the target gene.16 They established that transcripts derived from the SV40 promoter are repressed at the initiation stage of translation, whereas mRNAs derived from the TK promoter are repressed at the post initiation stage.16 Although it was first thought that miRNAs only block translation, it has been suggested that they may also have a role in enhancing or even activating translation at certain points in the cell cycle.17

RNA Interference

Figure 1 Biogenesis and function of micro-RNA and siRNA. RISC RNA-induced silencing complex, AGO2 Argonaute 2 protein, siRNA short-interfering RNA.

The discovery of the gene-silencing phenomenon, also known as RNA interference (RNAi), has been further confirmed by exogenously administered or artificially expressed double-stranded RNAs which have been found to selectively inhibit target genes by analogous mechanisms to miRNA.18 Similar to miRNA, siRNAs undergo processing by Dicer endonuclease producing a single-stranded RNA for the RISC complex (Fig. 1). Similarities in processing of siRNA and miRNA suggest that functional pathways may cross over because the human genome contains a single specific gene encoding Dicer. The origin of endogenous siRNA is not fully understood. However, it is thought that transposons, viruses, and other repetitive elements in the genome may well play a crucial role in their formation.19

J Gastrointest Surg (2009) 13:1379–1388

1381

The silencing process is highly sequence specific and although it was a believed that effective RNA interference requires almost complete sequence homology, it now appears that as few as seven contiguous complimentary base pairs can mediate gene silencing.20 RNAi technology could be found useful in the treatment of cancer by knocking down the expression of dominant mutant oncogenes.

Role of Micro-RNA in Carcinogenesis Dysregulation of miRNAs may be viewed as a consequence rather than a cause of carcinogenesis. However, deletions, local amplifications, and chromosomal breakage in regions of miRNA genes suggest a more direct role of miRNAs in tumorigenesis. Over 50% of miRNA genes are localized in genomic regions known to be associated with cancer or in fragile sites (genomically unstable during replication).21 MiRNA profiling studies have revealed abnormal levels in various types of cancer cell lines and tumors.22 In these studies, multiple deregulated miRNAs have been found, which has assisted in classifying cancer types.23 The aberrant expression of specific miRNAs has also been associated with prognosis.24 Manipulating deregulated miRNAs by a process of degradation or miRNA inhibition with RNA interference methods may develop new opportunities for cancer treatment. miRNAs as Tumor Suppressors An association between miRNA was first reported by Calin et al.25 A deletion on chromosome 13 is recognized to be the most frequent abnormality associated with B cell chronic lymphocytic leukemia (B-CLL). It has been demonstrated that in the majority (68%) of B-CLL, the miR-15 and miR-16 gene located within the deletion on chromosome 13 was either unexpressed or down regulated.25 This study strongly suggests the role of these two miRNAs as tumor suppressors. Although their full target complement is unknown, they appear to mediate their effect by downregulating the anti-apoptotic protein Bcl2 (Fig. 2).26, 27 Further studies have revealed a seven-base pair mutation downstream from miR-16-1 hairpin in two of 75 CLL patients which correlated with diminished expression of miR-16. It was thought that this mutation caused a defect in the transcription of miR-16. Following on from this, significant progress has been made in specifying the function of miRNAs in a variety of cancers. The let-7 family was the first group of miRNAs shown to regulate the expression of Ras protein (protooncogene).28 Ras is a signal transduction protein that

Figure 2 miRNA role in regulating cell cycle and apoptosis. Clear arrows—inhibit pathway. Black arrows—activate pathway.

control cellular processes such as proliferation, differentiation, and apoptosis. Mutations in RAS occur in 15–30% of human cancers, and overexpression of RAS is commonly found in lung cancer.29 Johnson et al. confirmed that let-7 inhibits RAS expression in human cancer cell lines (Fig. 2).28 Reduction of let-7 in lung cancer led to RAS overexpression, resulting in cellular overgrowth and contributing to carcinogenesis.28 Conditions of stress activate both p53-induced transcription of several miRNAs including transcription of the miR34a gene.30,31. Overexpression of miR-34a was associated with an arrest of the cell cycle and apoptosis and reduced expression of multiple genes responsible for cellular proliferation and angiogenesis. Most of these genes were predicted targets for miR-34a which included the apoptosis inhibitor Bcl-2 (Fig. 2).31,32 The genetic region of containing the miR-34a is often deleted in many types of cancer. Furthermore, the inhibition of miR-34a by antisense oligonucleotides inhibited the p53-dependent apoptosis during DNA damage.31 Therefore, miR-34a may be considered as another important tumor suppressor. miRNAs as Oncogenes miRNAs may act as oncogenes, either by inhibiting tumor suppressor genes or by inhibiting genes that restrict the activity of oncogenes. A recent study suggested that miR155 (required for functioning of B and T cells) exhibits strong oncogenic properties by interacting with MYC oncogene.33 Its expression is increased in many cancers including Burkitt’s lymphoma, Hodgkin’s disease, lung cancer, breast cancer, and pancreatic cancer.34–36 Bioinformatics predict that miR-155 target cytokines, chemokines, and transcription factors.37 In pancreatic cancer cells, miR155 inhibits Tp53inp1 (pro-apoptotic protein) causing cellular overgrowth.36

1382

Overexpression of miR-372 and miR-373 induced proliferation and malignization of human primary cell culture by increasing the RAS oncogene.38 These miRNAs target LATS2 to cause suppression and thereby activating CDK2 (cyclin-dependent kinase) and the cell cycle (Fig. 2).38 Cluster/Family miRNAs A group or cluster of miRNAs with oncogenic or tumor suppressor properties may be located close together. Two independent studies described the relationship between the miRNA cluster, mir-17-92, and the MYC oncogenic pathway. The mir-17-92 cluster was found to be located within a region on chromosome 13 (13q31–32) which is commonly amplified in human B cell lymphomas, follicular lymphomas and brain cortex lymphomas.39 It was further demonstrated that the miRNAs from mir-17-92 cluster were overexpressed in lymphoma cell lines that carried this amplification, and expression levels correlated with the gene copy number of the mir-17-92 locus.40 To test whether mir-17-92 actively contributed to lymphogenesis, experiments were undertaken on mice that had developed lymphomas due to an overexpression of MYC oncogene. He et al.40 proved that additional expression of the mir-1792 cluster accelerated C-Myc-induced tumorigenesis in mice. It was therefore suggested that mir-17-92 was the first noncoding oncogene, recognized as oncomir-1 (Fig. 2).40 Transformation of mouse hematopoietic stem cells into B cell lymphoma caused by oncogene C-Myc was accelerated by transfection of overexpressed cluster fragment lacking the mir-92-1 gene.40 Expression of miR-92-1 cluster also increased the rate of proliferation of lung cancer cells in cell culture.41 The predicted targets of such cluster miRNA include tumor suppressors: PTEN-inducing apoptosis42 and Rb12inhibiting E2F (transcription factor that plays a crucial role in the cell cycle and tumor suppression).43 Interestingly, the miR-17-92 cluster can also be activated by the protooncogene C-Myc (see Fig. 2).44 Cluster miRNAs such as miR-17-5p may also act as tumor suppressors. miR-17-5p can suppress the translation of E2F1 mRNA, resulting in an oncogenic effect.44 Such observations in miRNA clusters demonstrate the involvement of complex regulatory mechanisms. miRNA as Tumor Suppressors and Oncogenes Depending on the type of tumor cell, miR-21 and miR-24 may act as an oncogene or tumor suppressor gene. In HeLa cells (immortal cervical cancer cells), inhibition of miR-21 or miR-24 by the use of anti-miR oligonucleotides resulted in accelerated proliferation.45 Inhibition of miR-24 in A549

J Gastrointest Surg (2009) 13:1379–1388

cells (carcinomic human alveolar epithelial cells) caused effective suppression of cell growth, whereas inhibition of miR-21 had no effect. Many tumors (colon cancer, pancreatic cancer, glioblastomas, or breast cancer) are associated with a high expression of miR-21.46,47 miR-21 also acts through different mechanism depending on the cancer type. In glioblastoma cell culture, miR-21 causes apoptosis through the activation of caspases (cysteine proteases that play important role in apoptosis),46 whereas in hepatomas, miR21 may act as an oncogene by suppressing PTEN (tumor suppressor; see Fig. 2).47 Bioinformatic analysis show that many other miRNAs indicate both proto-oncogenic and tumor suppressive activity. However, the false positive prediction rate of these targets is high (approximately 30%).48 Global Loss of miRNA Expression Dysregulation in miRNA expression associated with carcinogenesis is not only caused by chromosomal defects but also due to problems in miRNA processing machinery. Kumar et al.49 reported for the first time that widespread reduction of miRNA expression was associated with carcinogenesis. By inhibiting the miRNA-processing enzymes Drosha and Dicer in cell lines, the authors were able to produce a global state of miRNA suppression. As a result, these cells demonstrated enhanced cellular growth and proliferation. When injected into nude mice in whom cancer was established, the tumors grew faster and became more invasive.49 It was also proven that loss of miRNAs led to upregulation of proto-oncogenes such as RAS and CMyc. Tumor invasion and metastasis have also been demonstrated to be associated with deregulated miRNAs.50

miRNAs in Tumor Diagnostics and Prognosis The uniqueness of miRNA profiling in particular tumor types could be helpful in cancer diagnostics (Table 1). Several studies have demonstrated that aberrant expression of miRNA exists at the early stage of cancer pathogenesis and that the expression changes as the tumor develops.22–24,51–53. This suggests that miRNAs have an important function in the development and differentiation of tested tumors. Expression analysis of miRNAs in solid tumors resulted in successful classification into subtypes by their origin and stage of differentiation.23,51. These results suggest that miRNA profiles may be useful for diagnosis but also for providing prognostic information.52 However, reproducibility needs to be improved before this technique can be generally applicable. Further studies are required to ascertain the value of miRNAs as diagnostic tools, and

J Gastrointest Surg (2009) 13:1379–1388

their potential value as prognostic markers by correlating with parameters such as metastatic potential and response to current treatments. Esophageal Cancer Feber et al.53 was able to demonstrate that miRNA profiles distinguish different esophageal tissue types (adenocarcinoma vs. squamous cell carcinoma (SCC)) and also discriminate malignant from normal tissue. miRNA profiles of normal squamous epithelium were more similar to squamous cell cancer than to adenocarcinoma samples. Similarly, miRNA profiles of Barrett’s esophagus were more similar to adenocarcinoma than squamous cell cancer,53 supporting the hypothesis that miRNA is involved in the pathogenesis of esophageal cancer. In the same study, miR-21 was found to be upregulated in both esophageal adenocarcinoma and SCC. This upregulation of miR-21 has also been observed in breast, lung, prostate, colon, and stomach cancer.22 MiR-192 showed higher expression in esophageal adenocarcinoma but lower expression of miR-203 was found relative to normal squamous epithelium.53 Different miRNA profiles were observed between cancer types. A further study demonstrated that prognosis was improved in esophageal cancer expressing lower levels of miR-103/107.54

1383

In another study, the most consistent highly expressed miRNA found in pancreatic cancer was miR-221. MiR-221 expression is also important in thyroid cancer and has a suggested role in angiogenesis.58 It has also been found to target KIT a cytokine receptor found on stem cells. KIT plays an important role in cell survival, proliferation, and differentiation. MiR-21 has also been shown to have increased expression in pancreatic cancer. It has been suggested that it plays an important role in preventing apoptosis, therefore functioning in an analogous way to a proto-oncogene.46 In one study, miR-21 was overexpressed in 79% of pancreatic cancers compared with only 8% (p