Applied Cancer Research. 2013;33(4):198-205. REVIEW

The role of in oral squamous cell carcinoma pathogenesis: a literature review

Anne Maria Guimarães Lessa1, Ludmila Faro Valverde2, Rosane Borges Dias2, Maria Cecília Mathias Machado1, Jean Nunes dos Santos3, Clarissa Araújo Gurgel Rocha3

Abstract In this review, we summarize the main aspects related to the involvement of microRNAs (miRNAs) in oral carcinogenesis. miRNAs are small non-protein-coding RNAs that function such as regulators of expression. They regulate various biological processes such as growth, differentiation and apoptosis and have been widely studied in carcinogenesis. miRNAs may exhibit oncogenic or tumor suppressor activity in cancer, depending on the biological context and the cell type. The altered expression patterns of miRNA in cancer could serve as molecular biomarkers for tumor diagnosis, prognosis and disease-specific prediction of therapeutic responses. The literature indicates that up-regulation of miR-21, miR-221, miR-184 and under expression of miR-133a, miR-375 and let-7b are the principal profile in oral squamous cell carcinomas. Keywords: gene expression; microRNAS; MIRN133 microRNA, human; MIRN184 microRNA, human; MIRN21 microRNA, human; MIRN221 microRNA, human; MIRN375 microRNA, human; mirnlet7 microRNA, human; mouth mucosa; mouth neoplasms; tumor markers, biological.

INTRODUCTION years later, Reinhart et al.12 observed that another C. elegans heterochronic gene, let-7, was also represented by a small Cells have developed several biological mechanisms to non-coding RNA capable of starting the temporal cascade of ensure that mitosis, differentiation and death occur in a coor- regulatory through an RNA-RNA interaction with the 3 dinated manner and disturbances in genes related with these untranslated region (UTR) of target genes. This discovery 1 cellular processes could result in tumor initiation and cancer . prompted scientists to investigate other small non-coding The mechanisms responsible for the human cancer patho- RNAs in various organisms and cellular systems. genesis are not fully identified, despite advances in molecular The identification of hundreds of miRNAs in worm, biology. However, researchers have identified small molecules animal and plant genomes has now attracted the attention of of non-coding RNA, called microRNAs (miRNAs) which can many researchers10,13-15. It is estimated that miRNAs comprise 2-5 function as tumor suppressor genes and/or oncogenes . 1-4% of all human genes, which makes miRNAs one of MiRNAs are small non-coding and single-stranded the largest regulators of gene expression16. Many miRNAs RNAs involved in numerous physiological and pathological are evolutionarily conserved from worms to humans, processes. They are a post-transcriptional regulators with implying that these miRNAs run essential processes during approximately 20 to 22 nucleotides that bind to comple- development and adult life, such as cellular development, mentary messenger RNAs sequences, usually resulting in proliferation migration, differentiation, and apoptosis1,9. 2,3,6-10 their silencing . In fact, miRNAs negatively regulate Another important property is the fact that only one 4,7 gene expression at a post-transcriptional leve . miRNA can target hundreds of different mRNAs, suggesting The first miRNA was identified in Caenorhabditis that miRNAs might act to regulate mRNA translation and elegans and named lin-4, in 1993. This miRNA controls the turnover7. To date, more than 2000 mature miRNAs have 11 timing and progression of the nematode life cycle . Seven been described in humans, and many of them have been identified to have an important role in disease progression4. 1 Undergraduate student - (Undergraduate student). It has also been estimated that more than 60% of human 2 Master Student - (Master Student). 2,17 3 PhD - (PhD). protein-coding genes are regulated by miRNAs . Previous studies have indicated that miRNAs may Send correspondence to: 1,18-21 Federal University of Bahia. participate in tumorigenesis and affect the etiology, Clarissa Araújo Gurgel Rocha. diagnosis, and prognosis of various cancers1,22-28. Moreover, Av. Araújo Pinho, nº 62. Salvador - BA. Brazil. CEP: 40110-150 the miRNAs may be involved in regulation of the immune Email: [email protected] and inflammatory response systems29,30, becoming Submitted: 09/15/2013 attractive molecules as markers as well as for prediction Aproved: 12/06/2013 of therapy response and surveillance of cancer31.

Applied Cancer Research, Volume 33, Number 4, 2013 199 Oncogenic miRNAs are found in malignant tumors and could act by stimulating cellular proliferation or inhibiting the action of tumor suppressor genes and genes that control apoptosis2,3,32,33. Some miRNAs have reduced expression in malignant tumors and are considered tumor suppressors. These miRNAs work against the development of cancer by inhibiting oncogenes3,32. In oral cancer, some studies demonstrated up and down-regulated miRNAs indicating that these molecules may participate in the molecular mechanisms involved in this tumor. Thus, this review aimed to provide an overview about miRNA and describe evidences of the potential participation of miRNAs in oral carcinogenesis. miRNA - Biogenesis Most miRNA-coding genes are located in intergenic Figure 1. Biogenesis of miRNAs. MicroRNA (miRNA) genes are generally regions and its biogenesis is controlled by two RNA-dependent transcribed by RNA Polymerase II (Pol II) in the nucleus to form large pri-miRNA enzymes, which convert a long primary transcript into a mature transcripts. These pri-miRNA transcripts are processed by the RNase III enzyme miRNA7. and its co-factor, Pasha, to release the ~70-nucleotide pre-miRNA miRNA biogenesis is a complex multi-step process that precursor product. RAN-GTP and exportin 5 transport the pre-miRNA into involves different cellular compartments. This process starts the cytoplasm. Subsequently, another RNase III enzyme, , processes the pre-miRNA to generate a transient ~22-nucleotide miRNA: miRNA* duplex. in the nucleus, where miRNAs are encoded by genomic DNA The mature miRNA strand is preferably retained in the RNA-induced silencing and transcribed by a RNA polymerase II into the long primary complex (miRISC) and the complementary strand undergoes destruction. transcript called pri-miRNAs5,9. The pri-miRNAs form secondary The mature miRNA then binds to complementary sites in the mRNA target structures named as “stem” regions2,6,34 and are processed in the to negatively regulate gene expression in one of two mechanisms: mRNA nucleus in pre-miRNAs containing 70 nucleotides by a complex degradation/cleavage or repression of translation of mRNA. formed by the RNase III enzyme (Drosha) and a double strand RNA binding protein (named Pasha or DGCR8)2,6,35. The characteristics, such as loss of cellular identity, increased pre-miRNAs are then exported to the cytoplasm by exportin capacity to grow and proliferate as well as alterations in 5 and submitted to an additional processing step in which a systems that control cellular death. Studies in a variety miRNA duplex is excised by another RNase III enzyme (Dicer). of organisms revealed that miRNAs have the capacity to Subsequently, the miRNA duplex is incorporated to the RNA- regulate these cellular processes, corroborating to cancer induced silencing complex (miRISC) and the complementary pathogenesis. miRNAs lin-4 and let-7, for example, have strand undergoes destruction5,6,34. The mature miRNA strand been related to control duration of the development of 36 is preferably retained in the miRISC complex and regulates its C. elegans , and mutations in these miRNAs results in target genes1 through two mechanisms: mRNA degradation abnormalities in cell cycle and cellular differentiation, or repression of translation of mRNA6,32, depending on the preventing cells from reaching a complete differentiated 33 degree of complementarity between the bases of the miRNA state . In addition, because each miRNA regulates and mRNA35. Figure 1 illustrates the biogenesis of miRNAs. the expression of hundreds of different genes, miRNAs can Studies on the biogenesis of the miRNA pathways function as master coordinators, efficiently regulating and 2 have shown that the miRNAs expression is regulated at coordinating multiple cellular pathways and processes . three different levels, including transcription, processing The role of miRNA in tumorigenesis was first 37 and subcellular localization. The regulation of pri-miRNA reported by Calin et al. in chronic lymphocytic transcription is one of the most relevant mechanisms leukemia (CLL). Ever since, there has been an explosion modulating miRNA abundance, by the action of several of publications showing miRNA deregulation in various factors including transcription factors, silencers, enhancers cancer types 2. Recent studies have shown that over and epigenetic modification in miRNA promoters. Moreover, 50% of miRNA codifying genes are located in DNA post-transcriptional factors can also modulate the miRNAs regions associated with cancer, corroborating to this 17,32,38 expression, such as p53, histone deacetylase I, and cytokines9. hypothesis . The mechanisms responsible for the alterations in miRNA and Cancer the expression of miRNAs involve epigenetic modifications It is not surprising that miRNA deregulation or mutations in the DNA sequences, such as deletions, 33 is a hallmark of several pathological conditions, substitutions, insertions, translocations and amplifications , including cancer7,8. Tumors in general share a number of resulting in deregulated miRNA expression. The oncogenic activity of miR-21, for example, has been reported in a

Applied Cancer Research, Volume 33, Number 4, 2013 200 number of different malignancies including glioblastomas, In the past years, the increase of the OSCC cases in acute myeloid leukemia, breast, colon, pancreas, lung, non-smokers and in nonusers of alcohol, suggests that prostate, liver and stomach cancer17,39,40. other immunological, environmental or genetic factors also MiRNAs have become attractive molecules as markers contribute to the pathogenesis of this cancer31,38. for the diagnosis, prognosis, as well as for prediction of Recent results of molecular and epidemiological therapy response and surveillance of cancer once they studies have suggested that the Human Papilloma Virus have proven to be stable in clinical specimens41. These (HPV) is also an important etiologic factor in a subset of molecules can function as both tumor suppressor genes this tumor, particularly those that develop in pharynx, or oncogenes1,2,4, by stimulating cellular proliferation and tonsil and oropharyngeal cancer31,49. Of the more than inhibiting the action of tumor suppressor genes and genes that 130 types of HPV, the high-risk HPV type 16 is the control apoptosis2,32,33. On the other hand, some miRNAs have most commonly associated with head and neck cancers, reduced expression in malignant tumors and are considered accounting for about 90% of HPV-associated OSCCs31,50. tumor suppressors33. The same miRNA can act as an oncogene Qian et al.51 identified miRNAs that are expressed in in one type of cells and as a tumor suppressor in another, persistent infection and may thus be associated with the due to different targets and mechanisms of action2. development or maintenance of epithelial lesions. Thus, MiRNAs could affect all hallmarks of malignant cells miRNAs may be involved in regulation of the immune including self-sufficiency in growth signals, insensitivity and inflammatory response systems which may control to growth inhibitors signals, evasion of apoptosis, HPV clearance and escape from immune surveillance. limitless replicative potential, angiogenesis, invasion Therefore, functional genetic polymorphisms of miRNAs and metastasis2,3. In addition, miRNAs participate as may lead to individual variations in immune function, master regulators of epithelial-mesenchymal transition3 inflammation, and apoptosis that modify viral immune and are also critical in the deregulation of tumor escape, antiviral defense, and evasion of apoptosis, leading microenvironment, acting as managers of heterotypic to modification of the risk of HPV-associated OSCC31,52. signaling in cancer-associated fibroblasts (CAFs) and The development and progression of OSCC involve extracellular matrix (ECM)42. At this point, several studies a complex process resulting in a progressive acquisition of have indicated that certain miRNAs found in CAFs are genetic and epigenetic alterations47. Genetic damage may capable of regulating the process of tumor metastasis occur mainly by activation or amplification of oncogenes based on the secretion of growth factors as well as ECM related with cell survival and proliferation53. In this process, degrading metalloproteinases3,42. mutated DNA sequences are transcribed into mRNA MiRNAs have a very restricted tissue-specific and then translated into functionally aberrant proteins. expression and the apparent modulation of miRNA However, RNA is not an intermediate passive product. Gene in cancer tissues may represent the manifestation expression is also dependent on RNA based mechanisms of a different cell population in the tumor2. Using such as alternative splicing, miRNA and RNA editing54. high-throughput techniques including miRNA microarrays, Researchers have focused on epigenetic alterations related unique miRNAs expression profiles are being confirmed with OSCC pathogenesis24,55,56 and advances in cancer to mediate critical pathogenic processes in human epigenetics and miRNA have increased the complexity of disease. To translate these discoveries to viable therapies, OSCC tumorigenesis. Recently, many molecular studies on appropriate and stable chemical molecules must be head and neck cancer - specifically OSCC - have highlighted designed6. Recently, miRNA expression was found to the deregulation of miRNA25,38,57-59. The involvement of correlate with the response and resistance to specific miRNA in oral carcinogenesis is poorly known and there drugs and scientists have studied these small molecules are few studies focusing on their functional roles in OSCC57. as potential clinical targets8. Heimberger et al.43 highlight Downregulation of miR-133a has been reported in that one of the major limitations of using miRNAs as tongue squamous cell carcinoma24,60. In a follow up study, therapeutic approach against cancer is to obtain an Wong et al.61 demonstrated the tumor-suppressor functions adequate delivery to neoplastic lesions. of this miRNA that inhibits cellular proliferation, migration and invasion and induce apoptosis in OSCC. Oral Cancer and miRNA The oncogenic activity of miR-21 and miR-221 and Oral cancer is an important type of malignant the decreased expression of the let-7 miRNA family have tumor associated with a high morbidity and mortality. been frequently observed in OSCC38,55,56,58,62. The expression Approximately 90% of oral cancers are oral squamous cell of miR-21 was negatively associated with decreased levels carcinomas (OSCC)44,45 and the incidence of this tumor is of PTEN and TPM1, proteins that regulate apoptosis and rising in developing countries45,46. Tobacco and alcohol cell cycle events, respectively. A study revealed that miR-21 abuse are major risk factors47,48, and tumor development is facilitates the growth of tumor cells in OSCC, particularly correlated with the intensity and duration of the habit44,45. through negative regulation of TPM156. This finding has

Applied Cancer Research, Volume 33, Number 4, 2013 201 been validated in other diseases, in which high levels of expression was significantly down-regulated, suggest- miR-21 expression contributes to neoplastic phenotypes55. ing that miR-145 is a candidate tumor suppressor in the According to Reis et al.63, elevated expression of miR-21 was pathogenesis of OSCC72,73. strongly correlated with the progression of pre-malignant Drug resistance is a major obstacle to successful leucoplakia to invasive oral carcinoma. The same was cancer treatment8,74,75. The identification of new found by Gombos et al.62, in which the miR-21 was most approaches that can circumvent this problem will markedly up-regulated in the OSCC samples. lead to improve clinical strategies and outcomes for MiRNA expression profile has a potential to separate cancer patients. miRNAs, key post-transcriptional progressive from the non-progressive leukoplakia. A gene regulators, are known to be master regulators superexpression of miR-21, miR-181 and miR-345 was of many cellular processes, including drug resistance. identified by Cervigne et al.58 and correlated with the Drug resistance related miRNAs can be used to stratify severity and progression of the pre-malignant lesions or patients and select those treatment options most likely the biological characteristics of OSCC, such as metastatic to prove efficacious for a particular individual8,75. For potential, recurrence possibilities and chemosensitivity or example, Cai et al.76 linked the epigenetic-silencing chemoresistance54. of let-7e to the development of cisplatin resistance in The identification of miRNA in blood plasma is , which inspires potential therapeutic another potential role for these molecules. For example, strategies targeting let-7 e to improve the treatment of overexpression of miR-184 in plasma from patients with cisplatin-resistant. tongue squamous cell carcinoma tissues was described by Dai et al.76 evaluated miRNA expression profiles Wong et al.60 These authors also observed the reduction of of head and neck squamous cell carcinoma with this miRNA in plasma after surgery of tumors60. Liu et al.64 docetaxel-induced multidrug resistance. They found that also revealed that plasma miR-31 was significantly elevated the resistant cells showed significant downregulation in OSCC patients, and it was also reduced after surgery. of miR-100, miR-130a, and miR-197 and upregulation These data indicate that both miR-184 and miR-31 might in miR-101, miR-181b, miR-181d, and miR-195 be an oncogenic miRNA in OSCC, and that its detection expression when compared with their parent cells. in plasma could be a clinically useful approach65. According to Sun et al.77, miR-200b and miR-15b may Silencing of miR-137 and miR-193 a may have a key regulate chemotherapy-induced epithelial-mesenchymal function in the progression of oral cancer by tumor-specific transition in human tongue cancer cells by targeting hypermethylation in OSCC, suggesting that the epigenetic BMI1. Chang et al.78 believe that ectopic expression silencing of these miRNAs plays a pivotal role during oral of miR-200b and miR-15b with miRNA mimics can carcinogenesis24. In addition, underexpression of miR-375 effectively sensitize human tongue cancer cells to may represent a significant alteration in the biological chemotherapy, but inhibition of miR-200b and miR-15b profile of OSCC25,38,59,66 and the proportion of miR-221 and can confer chemoresistance. Also, let-7d plays a role miR-375 could delineate cancerous and non-cancerous in regulating chemoresistant ability in oral cancer. In tissues with a high sensitivity and specificity38, involving addition, upregulation of miR-302 expression leads to miRNA in the molecular diagnosis of OSCC. Harris et al.67 self renewal, clonal formation, and cisplatin resistance found that the miR-375 represents a potential prognostic in cancer stem cells from head and neck squamous cell marker of poor outcome and metastasis in head and neck carcinoma79. squamous cell carcinoma, which include oral cancer, and After critical analysis of the selected articles, we that it may function by suppressing the tumor’s invasive observed that miR-21, miR-221, miR-184, miR-133a, properties. miR-375 and let-7b were the most cited in literature as Disturbances in the p53 pathway are frequently possible involved in the pathogenesis OSCC, as shown in observed in human OSCC and miR-504 plays as a negative Table 1. However, studies are necessary in order to obtain regulator of endogenous wild type p5368. In fact, the additional information on tumor suppressor miRNAs and overexpression of miR-504 demonstrated that it plays an oncogenic miRNAs involved in the pathogenesis of OSCC, important role during levels of the tumor suppressor69. including pre-malignant lesion. Demonstration that miRNA The potential role of miR-504 as new diagnostic, prog- expression is associated with tumor phase in some types nostic and therapeutic tools has been recently discussed of cancer may also represent an important instrument by Wu et al.69 and by Gorenchtein et al.70, both hypoth- for prognostic analysis and can be used for OSCC tumor esizing a clinic advantage in OSCC patient management. staging. In recent years, researchers have concentrated In addition, in OSCC, miR-504 expression is related with on genetic and epigenetic alterations in OSCC cells. The cellular invasion and lymph node metastasis71. In different understanding of miRNAs and their potential interactive types of cancers, the miR-145 has been downregulated action with such changes create new perspectives for and it also seems to be true for OSCC, whereas miR-145 understanding cellular transformation59.

Applied Cancer Research, Volume 33, Number 4, 2013 202 Table 1: Biological profile of MicroRNAs in OSCC. miRNA Detection Expression profile Significance in OSCC Reference miR-21 Cell lines in OSCC tissue Overexpressed Facilitates anchorage-independent growth; Avissar et al., 2009; promotes cell proliferation, up-regulation Cervigne et al., 2009; associated with decrease Zheng et al, 2011; Gombos et al, 2013 miR-221 OSCC tissue Overexpresscd Up-regulation delineates cancerous tissue Avissar et al., 2009; Cervigne et al., 2009; Gombos et al., 2013 miR-184 Plasma of OSCC cancer patients Overexpressed Promotes cell proliferation and inhibits Wong et al., 2008; apoptosis Kozaki et al., 2008 miR-375 OCSS tissue Underexpressed Reduces cell proliferation and clonogenicity Avissar et al., 2009; and mediates cell cycle arrest, Hui et al., 2010; down-regulation, outlines cancerous tissue and Lajer et al., 2011; associated with alcohol use Harris et al., 2012 miR-133a OSCC tissue cell Underexpressed Inhibits cellular proliferation, migration and Wong et al., 2008; invasion; induces apoptosis Tran et al., 2007 let-7b OSCC cell lines Underexpressed Suppression of cell proliferation Avissar et al., 2009; Zheng et al., 2011 miRNA: MicroRNA; OSCC: Oral

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