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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.43 No.1 pp.27-40
DOI : https://doi.org/10.17779/KAOMP.2018.43.1.004

Drosophila melanogaster as a Potential Animal Model to Study the MicroRNA-related Pathophysiology of Human Oral Cancer

Ji Eun Jung1),2), Joo Young Lee5), In Ryoung Kim4), Hae Ryoun Park1),2),3),5), Ji Hye Lee1),2),3),5)*
1)Department of Life Science in Dentistry
2)BK21 PLUS Project
3)Department of Oral Pathology
4)Department of Oral Anatomy, School of Dentistry
5)Institute of Translational Dental Sciences, Pusan National University, Yangsan 50612, Korea
Correspondence: Ji-Hye Lee, Department of Oral Pathology and Life Science in Dentistry, 49 Pusandaehak-ro, Mulgeum-eup, Yangsan-si 50612, Republic of Korea Tel: +82-51-510-8259 E-mail: jihyelee@pusan.ac.kr
January 21, 2019 February 1, 2019 February 22, 2019

Abstract


MicroRNAs (miRNAs) are a group of small non-coding RNAs consisting of 18~24 nucleotides in length. Each miRNA is expected to bind a few hundreds of putative target mRNAs, thus inhibiting their translation into protein products mostly by degradation of targets. With its biogenesis extensively deciphered, miRNAs have been implicated in a variety of biological processes, including early development and cellular metabolism. In addition, dysregulation of miRNAs and subsequent alterations in the expression of its target molecules are thought to be linked to the pathophysiology of multiple human illnesses, including cancer. To establish the miRNA-target relationships important for developing a specific disease, it is critical to validate the putative targets of each miRNA suggested by computational methods in vivo. In this review, we will first discuss oncogenic and tumor-suppressive roles of miRNAs in human cancer and introduce computational methods to predict putative targets of miRNAs. Then, the value of Drosophila melanogaster as an alternative model system will be further discussed in studying human cancer and in validating the miRNA-target relationships in vivo. Finally, we will present a possibility of applying the mammals-to-Drosophila-to-mammals approach to study the roles of miRNAs and their targets in the pathophysiology of oral cancer, an intractable type of cancer with poor prognosis and survival rate.



동물 모델로서의 노랑초파리를 이용한 MicroRNA 관련 구강암 병태생리 연구

정 지은1),2), 이 주용5), 김 인령4), 박 혜련1),2),3),5), 이 지혜1),2),3),5)*
1)부산대학교 치의학전문대학원 치의생명과학과
2)BK21 PLUS 사업단
3)구강병리학교실
4)구강해부학교실
5)중개치의학연구소

초록


    Ⅰ. INTRODUCTION

    MicroRNAs (miRNAs) are defined as small RNA molecules consisting of 18~24 nucleotides in a single-stranded helix. Once binding to its specific targets, each miRNA functions to inhibit the translation of target mRNAs by inducing either endo-nucleolytic cleavage of its target or deadenylation followed by degradation [1]. Alternatively, translation of target mRNAs can be inhibited by miRNAs via either inhibition of translational initiation or elongation [1]. Rarely, miRNAs can act as stimulators of translation, as reported in miR-16- dependent activation of translation of myt1 kinase in Xenopus oocytes [2]. Since its first discovery in C. elegans [3, 4], the roles of miRNAs in various aspects of cell biology have been extensively studied, ranging from embryogenesis and developmental control to cellular metabolism and apoptosis [5, 6].

    Recent studies have revealed miRNA-dependent regulation of biological processes implicated in development of human diseases, including cancers as well as cardiovascular and neurological disorders [7]. For instance, an array of miRNAs have been mapped to function as oncogenic or tumor-suppressive regulators [8], playing a critical role in tumorigenesis as well as in development of invasive and metastatic potentials of cancer cells [9]. Indeed, altered biogenesis and aberrant expression of miRNAs are significantly associated with in various types of cancers, including colon [10], lung [11], breast [12] and oral cancer [13]. In this review, we will summarize the recent findings in oncogenic and tumor- suppressive miRNAs and their putative targets implicated in tumorigenesis and progression of human malignant tumors. Furthermore, limitations of current computational approaches to predict putative targets of cancer-related miRNAs and an alternative approach using Drosophila melanogaster as an animal model to study the pathophysiology of human cancer will also be discussed for its potential.

    Oncogenic and Tumor-Suppressive MicroRNAs and Their Potential Contributions to Metastatic Behaviors of Cancer Cells

    After initial transcription of miRNA genes, the resulting pri-miRNAs are further processed to pre-miRNA in the nucleus, followed by its transport into the cytoplasm for DICER/ TRBP/AGO-dependent processing into mature miRNAs [6]. During this process, either accumulation of oncogenic miRNAs or loss of tumor-suppressive miRNAs can lead to development of malignant tumors [8]. For instance, miR-21 has been frequently documented as oncogenic miRNAs to target multiple tumor suppressor genes, including PTEN, thus implicated in development of various types of cancers (Table 1). In contrast, downregulation of let-7 family has been implicated in a number of human cancers, including lung, gastric and colon cancer as well as Burkitt’s lymphoma [8, 14] (Table 1). On the other hand, there are a few miRNAs such as miR-29 that may function as either oncogenic or tumor-suppressive regulators, depending upon their target genes or tissues, in which their expression profiles are altered [8] (Table 1).

    Some of these miRNAs play a crucial role not only in the initiation of tumor formation, but also in acquisition of aggressive behaviors of cancer cells, resulting in local invasion and distant metastasis. For instance, miR-21 appears to target MMP inhibitors, thus suppressing its expression and subsequently facilitating MMP activities that are essential for promoting invasive and metastatic events [9, 15] (Table 2A). In case of let-7, its targeting of HMGA2, HRAS, KRAS and NRAS is associated with inhibition of anchorage-independent growth and self-renewal of cancer stem cells, thus possessing a potential of metastatic suppressors [9, 15] (Table 2B). In line with these results, miRNAs have also been implicated in regulating a developmental process called epithelial-mesenchymal transition (EMT) that represents a series of cellular events occurring at the early phase of cancer progression. EMT is initiated with the loss of tight cell-cell contacts characteristic of epithelial cells, followed by acquisition of mesenchymal cell properties, leading to dedifferentiation of cancer cells with more invasive and migratory potentials [16]. In addition to some miRNAs that function both in tumorigenesis and cancer progression, including miR-21 and let-7 (Table 1 & Table 2), multiple miRNAs have been documented to specifically regulate the EMT process (Table 2). For instance, miR-205 and miR-200 family, with translation suppression of the ZEB family transcription factors for activating the EMT process, are consistently downregulated in cancer cells during EMT [17]. Similarly, other miRNAs such as miR-141 are known to suppress the expression of cytokines, including TGFβ that mediates the EMT process [18]. Downregulation of these miRNAs would free cancer cells from the molecular brakes that halt the EMT process, thus allowing them to acquire mesenchymal phenotypes [17, 18].

    Target prediction of cancer-related microRNAs: Limitation of its predictive potentials

    To fully understand the role of miRNAs in tumorigenesis and cancer progression described above, it is critical to identify mRNA targets of individual miRNAs. As the first step to accomplish this goal, a number of computational methods have been developed and employed over the past few decades [19, 20] (Table 3). Most of these tools require analyses of 3’-untranslated regions (UTR) of putative target mRNAs, with which computational algorithms are developed for finding seed matches of miRNAs, ranging from 6mer to 8mer sites [20]. In addition to the seed sequence of a miRNA matching to its putative targets, there are other aspects of prediction that needs to be incorporated into computational algorithms. For instance, the degree of conservation of seed sequences across different species needs to be carefully considered, together with free energy status of molecular binding between each miRNA and its specific target [21].

    These computational approaches certainly reduce our burden to identify miRNA-target relationships. However, they often report a few hundreds to thousands of putative targets for each miRNA, albeit recent efforts in improving the accuracy of their predictive ability, including combinatorial approaches based upon multiple prediction platforms. Even so, modified approaches can only maximize the power of prediction by reducing the number of false positive results, while still missing known true targets in some cases [22]. Therefore, it holds true that translational repressive activities of individual miRNAs against putative targets needs to be experimentally verified to rule out false positive targets. Furthermore, target genes may not be sensitive to subtle repression of translation provided by miRNAs, corresponding to a number of false positive target prediction results that failed to be reproduced in cells [23], hence further emphasizing the importance of experimental validation of putative targets of each miRNA predicted by computational methods.

    Drosophila melanogaster as a versatile in vivo model system to study human cancer

    Another important checkpoint of an experimental validation step for individual miRNA-target relationships implicated in human cancer includes whether miRNA-target interactions validated in in vitro settings would hold true in vivo. Investigation on this matter relies upon utilization of appropriate animal cancer models. Although a number of mammalian model systems such as rodents have been employed to study human cancer in vivo, they often require significant time and effort in genetic manipulations and analysis of disease phenotypes. Here, we first discuss the promise of Drosophila melanogaster as an effective and alternative model for investigating the pathogenesis of human cancer.

    For the last 100 years or so, Drosophila melanogaster, also known as fruit flies, has been providing indispensable knowledge to understand diverse molecular mechanisms that underlie cellular processes essential for all stages of development across species. Among them lies an array of important molecules that are linked to the pathogenesis of human malignant tumors. For instance, the genes important for maintaining cellular and tissue polarity, including discs large, scribble and Notch, were first cloned in Drosophila, of which abnormal regulation would often lead to development of malignant tumors [24, 25].

    Besides such initial contributions, Drosophila holds a few advantages over other animal model systems for studying human cancer. First of all, relatively simple husbandry and short generation time of Drosophila with highly prolific nature makes it suitable for investigating the effects of genetic or chemical manipulations on tumorigenesis and cancer progression. A single mating between parental flies often produces a few hundreds of progenies in about 10 ~ 11 days, when cultured at 22°C, thus well suited for experimental designs such as drug screens that require a large number of animals exposed to genetic or chemical manipulations. Second, with a relatively low redundancy in Drosophila, the significant extent of homology between the human and Drosophila genomes provides an excellent opportunity to reveal the novel functions of human cancerrelated genes with little to no previous characterization and significant redundancy. Indeed, it is estimated that Drosophila has orthologues to nearly 70% of the human genes linked to diseases, including cancer [26]. In many cases, the loss of Drosophila genes can be rescued by inducing the corresponding human genes, further emphasizing the power of Drosophila genetics in biomedical research. In addition, there are wealth of experimental resources that are mostly accessible to the research community, including thousands of mutant and double-stranded RNA collections, enhancer and/or protein traps, cell lines and gene constructs established by the colleagues and distributed by the public stock centers and collections. To go along with such openness in the atmosphere of Drosophila research community, Flybase, a well-organized and maintained public database for the Drosophila genome and research, has been the core of vibrant exchange of knowledge among the community and more.

    Taking advantage of these characteristic features, Drosophila has been successfully implemented to model multiple types of human cancer in vivo, including oncogenic RAS-related epithelial and glial tumors (glioblastoma), rhabdomyosarcoma, acute myeloid leukemia and colon cancer, as well as the multiple endocrine neoplasia syndrome type 2 (MEN2) [24, 27]. When Drosophila is adopted as a model for human cancer study, either one of the following approaches can be employed; Drosophila-to-Mammals and Mammals-to-Drosophilato- Mammals (Fig. 1A). As an example of the Drosophila-to- Mammals scenario, many of junctional tumor suppressors, including the Scribble-Dlg-Lgl proteins, were first cloned or identified in Drosophila as a key module to control cell polarity and found in later studies to control tumorigenesis in mammals [28]. On the other hand, a successful implementation of the Mammals-to-Drosophila-to-Mammals approach was demonstrated in the case of MEN2, in which deregulation of the Ret signaling pathway was first mapped in human patients and later found to be also tumorigenic in Drosophila. Subsequent chemical screens for MEN2 were performed based on morphological changes in Drosophila eyes, leading to a discovery of ZD6474/Vandetanib, a novel receptor tyrosine kinase (RTK) inhibitor, to inhibit tumorous tissue growth in Drosophila [29] and to successfully employed in clinical trials for MEN2 [30]. Taken together, it is evident that Drosophila studies have made significant contributions to the field of oncology, spanning from initial discoveries of oncogenic or tumor-suppressive molecules to integration of drug screens to identify effective therapeutic agents. With continuing advances in technology and better understanding of cancer pathology, implementation of Drosophila in studying human cancer holds considerable promises for development of novel therapeutic remedies.

    Drosophila melanogaster as a model system to validate the putative targets of cancer-related microRNAs

    In addition to protein-coding genes involved in tumorigenesis, such as Ras and Ret described above, Drosophila can also serve as a model to study the role of non-coding RNAs, including miRNAs, in the pathophysiology of human cancer, more specifically miRNA-dependent regulation of oncogenic or tumor-suppressive target genes. According to miRbase (www.mirbase.org), the online data resource assembled for available miRNAs in different species, there are 1,917 human and 258 Drosophila (melanogaster) precursor miRNAs mapped in the genomes, from which 2,654 and 469 mature miRNAs are processed, respectively.

    The functions of miRNAs in Drosophila have been extensively studied in recent years, revealing the contributions of miRNAs to a variety of biological processes that are dysregulated in human cancer cells or tissues. For instance, miRNAs such as dmiR-7 and dmiR-278 may play a role in maintaining the stem cell fate and regulating subsequent cellular differentiation, while others, including dmiR-8 and dmiR-14, are sufficient to promote or inhibit tissue growth via regulation of cell proliferation and apoptosis in vivo [31]. Recent study has revealed that 136 human disease-related miRNAs are orthologous to about 83 Drosophila miRNAs when compared for the seed sequence, many of which are involved in human cancer [32]. Direct molecular targets and interacting protein networks of these cancer-related miRNAs also include known human cancer-related genes, such as Jak/STAT, Hedgehog, Hippo, EGFR and Yorkie [31] (Table 4), thus further confirming the validity of Drosophila as an in vivo model to study miRNA-dependent regulation of oncogenic or tumor suppressive target genes detected in humans.

    Potential application of the Mammals-to-Drosophilato- Mammals approach to study microRNA-related target gene regulation in oral cancer

    As stated earlier, miRNA-dependent alterations in cellular signaling pathways have been extensively studied in a variety of human cancers in order to further improve our understanding of their pathophysiology and eventually to develop novel therapeutic options. However, whether such significant contributions of miRNAs can also be mapped to oral cancer remains elusive, with relatively recent advances in clinical and experimental findings [13]. Oral cancer is the sixth most prevalent type of malignant tumors worldwide, most of which belongs to the category of oral squamous cell carcinoma (OSCC). Despite technical improvements in the last few decades, the survival rate of OSCC patients remains relatively poor, mostly due to the lack of effective treatment options other than a surgical intervention.

    Earlier experimental approaches have been mostly focused on miRNA profiling analyses either with in vitro cultures or with clinical samples obtained from patients with oral cancer. These studies have yielded a spectrum of miRNAs, of which expression or activity is altered in cultured cancer cell lysates as well as in tissue biopsy or secreted saliva specimens obtained from control groups and patients [13, 33-38] (Table 5). Several attempts have been made to examine the potential of aberrant miRNA expression profiles as cancer biomarkers with diagnostic and/or prognostic value [13]. However, the caveat of these studies lies in the fact that there is no experimental evidence beyond simple correlations to prove direct miRNA-dependent regulation of any oncogenic or tumor-suppressive genes occurring in development or progression of oral cancer.

    Here we propose to employ the Mammals-to-Drosophilato- Mammals approach (Fig. 1B) to validate the miRNA- target gene relationships relevant to oral cancer in vivo. Considering the number of miRNAs with altered expression in oral cancer and their putative targets, Drosophila can be the platform of choice for conducting a genetic screen to investigate tumorigenic or suppressive potentials of individual miRNAs and their actions on regulating target genes relevant to oral cancer. Indeed, the majority of miRNAs with abnormal regulation in oral cancer cells or tumor tissues is conserved in humans and Drosophila, together with their putative targets in common (Table 5, bold). Genetic manipulation of single or multiple miRNAs in Drosophila will allow us to dissect their roles in tissue growth and development of oral cancer and potential molecular networks that involve specific targets of each miRNA. Meanwhile, genetic modifier screens can also be performed in Drosophila to discover novel molecular candidates with the ability of modulating the pathophysiology of oral cancer. Results from this initial Drosophila platform will then be translated into either in vitro or in vivo systems using cell-based assays or biopsy samples to confirm the contributions of each miRNA-target network to tumorigenesis in mammals. In addition, the compatibility of novel molecular mechanisms discovered in Drosophila will be thoroughly examined with mammalian systems as potential targets to treat oral cancer. Ultimately, these targets will be subject to pre-clinical trials in the hope of developing effective treatment remedies for curing oral cancer in future.

    Ⅱ. CONCLUSION

    miRNAs in human malignant tumors have been a focus of recent investigations to elucidate their functions in cancerrelated molecular networks and potentials as biomarkers to aid early diagnosis of various types of cancer. Despite such efforts in deciphering the contribution of miRNAs to the pathophysiology of human cancer, our understanding of miRNA-centered molecular networks encompassing their putative targets is often limited due to the failure of experimental validation of miRNA-dependent target regulation projected by computational methods. Here we have introduced Drosophila melanogaster as a potentially powerful animal platform to model human cancer in vivo. The previous studies using Drosophila have clearly documented successful applications of the Mammals-to-Drosophila-to-Mammals approach to study multiple types of human cancer. With this knowledge, we finally propose the possibility of implementing such approach in order to improve our understanding of oral cancer, a highly intractable type of malignancy, of which treatment options have been barely modified over the past decades. The novel findings obtained from this approach will likely provide a valuable insight into the molecular networks that play crucial roles in tumorigenesis and progression of oral cancer and lead to development of more effective treatment remedies, thus improving the quality of life and survival rate of patients suffering from this rather refractory and fatal disease.

    ACKNOWLEDGMENTS

    This work was supported by a 2-Year Research Grant of Pusan National University to Ji-Hye Lee.

    Figure

    KAOMP-43-1-27_F1.gif
    Mammals-to-Drosophila-to-Mammals approach to study human diseases, including miRNA-dependent regulation of oral cancer.

    (A) The workflow is portraited to explain the research design of the mammals-to-Drosophila-to-mammals approach in studying human diseases. Experimental settings established in the mammalian and Drosophila systems are indicated in white and gray boxes, respectively. (B) A similar approach can be applied to study miRNAs involved in tumorigenesis and progression of oral cancer and their interaction with molecular targets that function as either oncogenes or tumor suppressors.

    Table

    MicroRNAs as oncogenes and tumor suppressors

    MicroRNAs as important regulators of cancer metastasis

    Computational methods for predicting targets of miRNAs

    Examples of Drosophila microRNAs with validated targets and their functions

    Oral and head and neck cancer-related microRNAs with their putative targets and functions

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