A miR-centric view of head and neck cancers

Biochimica et Biophysica Acta 1816 (2011) 67–72

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a c a n

Review

A miR-centric view of head and neck cancers Janki Mohan Babu, R. Prathibha, V.S. Jijith, Ramkumar Hariharan ⁎, M. Radhakrishna Pillai Integrated Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India

a r t i c l e

i n f o

Article history: Received 25 February 2011 Received in revised form 15 April 2011 Accepted 19 April 2011 Available online 28 April 2011 Keywords: Head and neck squamous cell carcinoma MicroRNA Biomarker Oral cancer

a b s t r a c t Head and Neck Squamous Cell Carcinomas (HNSCCs) constitute the sixth most common cancer worldwide with an average 5-year survival rate of around 50%. Several microRNAs, small non-coding RNAs involved in post-transcriptional gene regulation, have been linked to HNSCC based on their differential expression in tumors. Here, we present a compilation of multiple types of information on each HNSCC linked miRNA including their expression status in tumors, their molecular targets relevant to cancer, results of gene manipulation studies and association with clinical outcome. Further, we use this information to devise a new scheme for classifying them into causal and non-causal miRNAs in HNSCC. We also discuss the possibility of using miRNAs as prognostic and diagnostic biomarkers for HNSCC, based on existing literature. Finally, we present available evidence that shows how altered expression of specific miRNAs can contribute to various “hallmarks of cancer” phenotypes such as limitless replicative potential owing to abnormal cell cycle regulation, evasion of apoptosis, reduced response to anti-growth signals, and Epithelial–Mesechymal transition (EMT). © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . MiRNAs as prognostic and diagnostic biomarkers in HNSCC . . . . . Defining causal microRNA genes in HNSCC . . . . . . . . . . . . . 3.1. Oncogenic microRNAs in HNSCC . . . . . . . . . . . . . . . 3.2. MicroRNAs with tumor suppressive roles in HNSCC . . . . . . 4. Epigenetic silencing of HNSCC miRNAs via promoter hypermethylation 5. Conclusions and therapeutic implications . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Head and Neck Squamous Cell Carcinoma (HNSCC), which includes epithelial cancers of the oral cavity, pharynx, nasal cavity, paranasal sinuses, salivary glands and larynx, represents the sixth most common cancer in the world, and accounts for more than half a million new cases annually [1]. The dismal average 5 year survival rates of around 50% for HNSCC warrants deeper understanding of its molecular carcinogenesis in order to accelerate discovery of new

⁎ Corresponding author at: Integrated Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram-695014, India. Fax: +91 471 2349303x234633. E-mail address: [email protected] (R. Hariharan). 0304-419X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2011.04.003

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markers for early detection and possible development of targeted therapeutics. To this end, an increasing number of powerful structural and functional genomics-based studies are beginning to uncover different types of genetic alterations associated with this tumor. The development of progressively better treatment regimens involving improved surgical procedures and radiation therapy, over the course of the past decade or two has resulted in a significantly enhanced quality of life for HNSCC patients [2,3]. However, this has not been complemented by any major improvements in the average 5 year survival for such patients. This is so because the implementation of new, organ-preserving treatment protocols has largely been unable to address the most common HNSCC associated problems of tumor recurrence (both second primaries and loco-regional recurrences) and distant metastases.

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The dismal average 5 year survival rate reflects some of the challenges of HNSCC management. First, the majority of HSNCC cases are detected with advanced disease which is typically associated with poorer prognosis compared to early-stage tumors. Second, the scarcity of good molecular targets, arising partly due to the considerable genetic heterogeneity of the disease, has dampened the development of targeted therapeutics in HNSCC. The biological heterogeneity of HNSCCs has also hampered the identification of accurate prognostic and diagnostic biomarkers of the disease. That increased molecular understanding of HNSCC is likely to lead to more efficient, targeted therapy for the disease that is supported by the recently reported success of EGFR-specific antibodies in the management of this cancer [4]. Thus, the identification of new biomarkers and therapeutic targets for HNSCC is warranted. Here, we review the evidence that supports the exploration of microRNAs as target molecules in the diagnosis and therapeutic development in HNSCC. MicroRNAs-single stranded, 18–23 nt, non-coding RNAs that repress target gene expression via either accelerated destruction of their transcripts or inhibition of translation, are known to play key roles in many physiological processes including cell division, development, cell death and cell migration [5–7]. Of the ~700 miRNAs identified so far in the human, more than a hundred have been implicated in various diseases including cancer [8,9]. Altered expression levels of cancer-associated miRNAs or ‘Oncomirs’ have been found in several types of cancer including carcinomas of the breast, prostate, lung, colon, head and neck, as well as tumors of the lymphatic system [10]. Both oncogenic and tumor suppressor miRNAs have been defined [11–13]. Moreover, over-expression of oncogenic miRNAs and down regulation of tumor suppressor miRNAs have been shown to make critical contributions to tumor formation, invasion and metastasis [14,15]. While it has been relatively straightforward to document miRNAs with aberrant expression status in different cancer types, efforts at elucidating the molecular pathogenesis pathways driven by or involving these RNAs have lagged behind considerably. Investigation and evaluation of aberrantly expressed miRNAs in the context of their target genes and pathways, link to chromosomal alterations, relationship with clinical outcome and functional significance as measured by in vitro knock-in or -out assays, are therefore warranted for driving translational research efforts. Here, we compiled a comprehensive list of aberrantly expressed miRNAs in Head and Neck carcinomas from recent literature and have annotated them with available information as outlined above. We present a new scheme with which we have classified HNSCC miRNAs into causal, non-causal and ‘not enough information available’ categories by taking advantage of the compiled information. In addition to describing molecular mechanisms that may at least partially explain how miRNA deregulations can contribute to HNSC carcinogenesis, we also discuss the possibility of using miRNAs as prognostic and diagnostic biomarkers for HNSCC. Finally, we also examine recent evidence for epigenetic silencing of miRNAs during oral carcinogenesis. Throughout the review, we suggest potentially useful future directions for further investigation. 2. MiRNAs as prognostic and diagnostic biomarkers in HNSCC Oral leukoplakia is a clinical entity defined as a white lesion in the oral mucosa [16]. A small proportion of leukoplakias (1 to 2%) turn malignant and they represent the most common premalignant oral lesion [16]. At present, it is not possible to distinguish a progressive leukoplakia from a non-progressive one with high confidence. Although several clinico-histological parameters and molecular genetic markers are currently used to identify leukoplakias with a high risk for malignant transformation, there is clearly a need for more accurate biomarkers [17]. In a recent study that looked to identify a miRNA expression signature associated with progressive leukoplakias using sequentially

progressive samples, the investigators built a multi-miRNA prognosis predictor [18]. The predictor is composed of eight miRNAs, whose high expression levels in progressive leukoplakias and invasive OSCC compared to non-progressive and normal tissues may hold some potential for further development into a miRNA based prognostic biomarker. Of the predictive set of eight miRNAs derived using a classical training–testing set of samples, three miRNAs, miR-345, -21 and 181b have highly significant associations with progressive oral cancer. It may also be worth investigating if the same results can be obtained if the expression levels are measured from exfoliated cells or saliva. A related problem is the identification of prognostic biomarkers in HNSCC. Tumor stage at the time of presentation, and more recently Human Papillomavirus (HPV) status of the tumor, in addition to tumor site and treatment regimen are widely used as predictors of survival in HNSCC [19]. The usefulness of using miRNAs as prognostic markers is only beginning to be explored. Childs et al. used real time PCR based profiling of a panel of 236 unique miRNAs to compare the miRNA expression levels between tumor and adjacent normal tissue in a cohort of 104 HNSCC patients. They found that low absolute expression levels of miR-205 and Let-7d are significantly associated with higher chances of loco-regional occurrence and shorter survival [20]. Furthermore, the expression levels of these two miRNAs predict prognosis independently of other, more classical prognostic markers. Another similar study identified lower expression levels of miR-451 in HNSCC tumors as a strong predictor for relapse [21]. There is some evidence to show that miRNAs may also hold potential as diagnostic biomarkers for HNSCC. It was recently shown that the expression ratio of miR-221 to miR-375 can be used to distinguish tumor from normal tissue with high specificity and sensitivity [22]. It was also reported that high expression levels of miR205 can be used to detect HNSCC positive lymph nodes [23]. This suggests that miR-205 can be considered as a marker for metastatic HNSCC, although the small sample size (n = 19) of that study warrants further validation of this marker in a larger sample. In a study that looked to identifying diagnostic miRNAs in the saliva, the authors reported significantly lower levels of miR-125a and miR-200a in the saliva of OSCC patients compared to control subjects [24]. Currently, non-invasive diagnostic biomarkers for OSCC do not exist and hence these results might be worth pursuing further. Another recent study reported high levels of miR-184 in the plasma of tongue squamous cell carcinoma patients [25]. Furthermore, the authors found that levels of miR-184 in the blood fell off following surgical resection of the primary tumor. The study also showed that miR-184 has anti-apoptotic functions in SCC cells and may act by altering levels of c-Myc. Inhibiting miR-184 in vitro reduced cell proliferation rates. 3. Defining causal microRNA genes in HNSCC Although about fifty miRNAs have been linked to HNSCC based on their altered expression in these tumors, sometimes reported by multiple, independent studies, an evaluation scheme to identify causal miRNAs in HNSCCs is needed. Here, we defined and used the following criteria to classify each HNSCC associated miRNA into either ‘causal’, ‘non-causal’ or ‘not enough information’ categories. a) Causal HNSCC miRNA. Satisfies all the following points. (1) Molecular– structural alteration leading to its altered expression is known in HNSCC (structural aberration or epigenetic modification at its loci). (2) Manipulation of the miRNA gene in an in vitro system leads to a verifiable phenotype. For example, knock-in of a TSG miRNA in a HNSCC cell line leading to suppression of proliferation or increased apoptosis and vice versa for oncogenic miRNA knock-in. (3) Involvement in other cancers and, (4) has at least one cellular cancer gene as its target.

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vivo evidence, these categories can serve to prioritize miRNA-based investigations in more complex in vivo model systems.

Table 1 Table of causal and non-causal HNSCC linked microRNAs. Sl. no

MicroRNA

Status

Type

1 2

hsa-miR-21 hsa-miR-106b-25 cluster (includes miR-106b, -93 & -25) hsa-miR-137 hsa-miR-125b hsa-miR-100 hsa-miR-423 hsa-miR-27a hsa-miR-10a

Causal Causal

Oncogenic Oncogenic

Causal Causal Causal Non-causal Non-causal Non-causal

TSG TSG TSG _ _ _

3 4 5 6 7 8

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This table provides the list of causal and non-causal HNSCC associated miRNAs, as defined in the manuscript. The ‘type’ assigned to each miRNA is based on a convergence of different kinds of evidence such as status of altered expression (up- or down regulated), effect on phenotype in in vitro knock-in/-out studies and type of chromosomal aberration (gain of copy number or loss of it) of its loci. TSG stands for Tumor Suppressor Gene. All the other HNSCC linked miRNAs (described in our HeNeCan miRs database) fall into the “not enough information” category.

b) Non-causal HNSCC miRNA. Manipulation of the miRNA in an in vitro system does not have any noticeable effect on the phenotype. c) Not enough information. When a miRNA fails to meet all four criteria defining a causal miRNA because of lack of experimental evidence or information (on at least one of the four parameters), we place it in this category. A list of causal and non-causal miRNAs in HNSCC, classified on the basis of our scheme is provided in Table 1. See our database HeNeCan miRs, available at http://tarmir.rgcb.res.in/henecan/ for a full list of HNSCC miRNAs, including those in the ‘not enough information’ category. It must be mentioned that while the criteria for classification of HNSCC-miRNAs given here is not based on any mechanistic in

3.1. Oncogenic microRNAs in HNSCC We first discuss HNSCC linked miRNAs that can modulate cell cycle inhibitory proteins (Fig. 1). Over-expression of these oncogenic miRNAs contributes to deregulated cell cycle regulation thereby leading to initiation or promotion of carcinogenesis. MicroRNAs belonging to the miR-106b-25 cluster, miR-17-92 polycistron and miR-106a can all target the p21 transcript encoded by the CDKNIA gene which belongs to the CIP family of cyclin–CDK inhibitors [26]. Consistent with the oncogenic nature of these miRNAs, their over-expression has been observed in HNSCCs [21,27]. That miR-106b can promote tumor proliferation by deregulating key cell cycle check points has been shown previously [26]. Indeed, miR-106b cannot only target p21 directly, but also helps the cell go over a doxorubicin induced cellular DNA damage check [26]. Thus, knock down of these miRNAs in HNSCC tumor cells should result in a reduced proliferation phenotype — given the oncogenic status of these miRNAs in HSNCC and their effect on modulating cell cycle progression. Validating this hypothesis, Hui et al. found that knockdown of the miR106b-25 cluster in HNSCC cell lines reduces cell proliferation rates by inducing G1 phase cell cycle arrest [21]. MiRNA mediated deregulation of p21 is thus likely to play an important role in tobacco associated HNSC carcinogenesis because it can negatively regulate p53 mediated DNA damage induced by carcinogens in tobacco smoke. It is interesting to note that miR-221, known to suppress p27 and p57 transcripts, is overexpressed in HNSCC [22,28,29]. Moreover, reduced expression of p27 has been well documented in several HNSCCs and has even been suggested as a biomarker for cancer progression [30,31]. That genes belonging to the Rb family can be treated as established cancer genes, at least in HPV-positive HNSCC was concluded recently

miR – 137 DNA damage

CDK1 Cyclin A CDK6

Cyclin A

Cyclin D1

CDK1 p53

E2F

Rb

E2F

p p21

Cyclin E

Rb

CDK2 p

p

miR- 17- 92 cluster, miR- 106a miR – 106b- 25 cluster

Fig. 1. HNSCC miRNAs and the cell cycle. The Retinoblastoma (Rb) tumor suppressor inhibits the E2F transcription factors to control cell cycle progression. Transition across the G1 restriction point (maroon bar) begins when Rb is phosphorylated by active cyclin D1–CDK4/CDK6 complexes in response to mitogenic signals. Phosphorylation of Rb triggers the release and subsequent activation of E2Fs, which in turn induces cyclin E expression. Subsequent, additional phosphorylation of Rb by the cyclin E–CDK2 complexes promotes entry into S phase. MiR-137 targets CDK6 transcript for degradation and contributes to prevention of G1/S phase transition. Reduced levels of miR-137, reported in some HNSCCs, may lead to increased CDK6 levels resulting in accelerated cell proliferation. P53, which is involved in the response to DNA damage, works at the G1/S cell cycle check point. Detection of DNA damage by p53 activates a DNA repair system and results in cell cycle arrest at the G1 check point until the damage is repaired. This cell cycle halt is accomplished by activation of p21 which inhibits several cyclin–CDK complexes. Mir-106b-25 cluster members can target p21 mRNA while miRNAs belonging to the miR-17–92 polycistron and miR-106a have been shown to post-transcriptionally suppress both p21 and Rb. Over-expression of these miRNAs (reported in a subset of HNSCCs) can contribute to enhanced cellular proliferation and tumorigenesis.

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[32]. MicroRNAs belonging to the miR-17-92 cluster and miR-106a, both of which are up-regulated in HNSCC, have been shown to target Rb1 transcript for degradation in solid tumors [21,33]. In fact, the role of miR-17-92 and miR-106a in tumor progression is well known [33] Gene manipulation studies of these miRNAs have not been carried out in HNSCC and therefore their effects on cell proliferation remain to be validated experimentally. Over-expression of miRNA targeting pathways involved in growth or tumor suppression has also been reported. Aberrant signaling in the Transforming Growth Factor beta (TGF-beta) pathway in HNSCC has been demonstrated repeatedly [32]. A role for the miR-106b-25 and miR-17-92 clusters in controlling TGF-beta signaling in the context of tumor progression is emerging [34]. It has been strongly suggested that when over-expressed in tumors, the miR-106b-92/ miR-17-92 cluster can interfere with the cross talk between TGF-beta and Myc pathways, leading to cell cycle deregulation and evasion of apoptosis [34]. It seems likely that this mechanism, demonstrated explicitly in gastrointestinal tumors, contributes to enhanced proliferation of miR-106b-92 over-expressing HNSCC. This hypothesis is supported by in vitro experiments [21,27]. One of the signaling pathways contributing to apoptosis evasion that is relevant to HNSCC is the PI-3K/PTEN/AKT pathway [32]. The oncogenicity of over expressed miR-21 is believed to stem at least partly, from its ability to target tumor suppressive/apoptosis promoting genes [35–39]. It is interesting to note that at least half a dozen studies have documented miR-21 over-expression in primary HNSCCs and in HNSCC derived cell lines, suggesting that deregulation of this miR is common in HNSCC [18,20–22,40,41]. The importance of miR-21 involvement in head and neck cancers is evidenced by multiple levels of information. First, in vitro studies have shown the functional significance of miR-21 up regulation. Specifically, transfection of miR-21 into HNSCC cells results in significantly increased growth rates whereas inhibitor driven knock-down of miR-21 in these cells reduces cell proliferation [41]. Further, it has been demonstrated that inhibition of miR-21 enhances cytochrome-c release, thereby triggering apoptosis [41]. Second, there is experimental evidence to show that miR-21 affects multiple tumor suppressive pathways by directly targeting key genes such as PTEN, TPM1, PDCD4 and SERPINB5 [35–39]. However, the exact mechanisms responsible for the oncogenic effects of miR-21 are debated since miR-21 is also thought to deregulate apoptosis-related genes [41]. It will be interesting to examine the status and role of TPM1, PDCD4 and SERPINB5 in HNSCC. Third, over-expression of miR21 is part of a miRNA expression signature that is known to strongly correlate with progression of premalignant leukoplakia to invasive oral carcinoma [18]. Lastly, miR-21 is known to be frequently overexpressed in several solid tumors and hematological malignancies [33,42]. MiR-155 shows increased levels of expression in HNSC tumors and its possible role in oral carcinogenesis has been investigated in some detail [21,27,41]. There is evidence to show that it can target tumor suppressors such as APC, and thereby exhibit oncogenic properties [27]. MiR-155 has also been linked to various other solid tumors [33]. Interestingly, it was demonstrated in invasive breast cancer that miR155 can play a role in TGF-beta induced Epithelial–Mesenchymal transition or EMT via targeting of the RhoA transcript [43]. MiR-155 can be evaluated further as a possible therapeutic target in HNSCC, specifically in the context of metastatic disease. 3.2. MicroRNAs with tumor suppressive roles in HNSCC Reduced expression of almost all members of the Let7 family with the exception of let-7i (which is up-regulated) has been noted in HNSCC [21,27,41]. KRAS suppression by Let-7 family members has been relatively well studied [44]. It seems possible that enhanced KRas activity combined with or contributed by, loss of miRNA-mediated

K-Ras suppression can promote oncogenesis. This apart, HMGA2, the other characterized molecular target of Let-7 miRNAs, is also known to be associated with several forms of cancer [45]. Moreover, reduced Let-7d expression is associated with poor prognosis in HNSCC [20]. Down regulation of miR-125a/b in HNSCC has been documented by independent studies [20,21,24,27]. Not only has reduced levels of 125a been suggested to be a predictive biomarker for OSCC, but also functional studies that involve introduction of miR-125b into OSCC derived cell lines result in reduced cell proliferation [24,46]. A possible molecular mechanism contributing to this effect might be ERBB2 targeting by miR-125a/b which has been experimentally determined [47]. Indeed, high levels of expression of ERBB2 have been observed in oral squamous cell carcinoma suggesting disruption of miRNA suppression of this gene [47]. Suppressed miR-200a expression has also been observed in primary OSCC [24,40]. It is also known that ZEB1 and ZEB2, two important zinc finger transcription factors, are cellular targets of miR-200a [48,49]. Importantly, members of the miR-200 family can inhibit EMT and tumor cell migration by directly targeting ZEB1/ZEB2 which acts as transcriptional repressors for E-cadherins [48]. It can be postulated that suppressed levels of miR-200a may promote EMT by contributing to reduced E-cadherin expression. Thus, down-regulation of miR-200a together with over-expression of miR-155 can favor EMT (Fig. 2). Interestingly, ZEB1/ZEB2 has also been shown to be central to regulation of latency maintenance of Epstein–Barr virus (EBV) in epithelial cells [50]. Given the association between latent EBV and nasopharyngeal carcinoma, the EBV-miR-200a-HNSCC connection needs to be investigated in more detail. Down regulation of miR-133a/b in primary HNSCC was reported by two recent studies [20,51]. Importantly, knocking-in miR-133a/b in HNSCC cells resulted in reduced cell proliferation or increased apoptosis [52]. Increased levels of PKM2, the validated cellular target of miR-133a/b, have also been associated with cancer progression [52]. At least 4 independent studies have shown suppressed miR-100 levels in both primary HNSCC and in derived cell lines [21,40,46]. Additionally, transfecting cells with miR-100 dampen cell proliferation rates [46]. Although a few of its target genes such as FGFR1, MMP13 and ID1, are known, the exact role of this miRNA in HNSCC carcinogenesis remains to be elucidated. Several other miRNAs, in addition to the ones discussed here, are known to show altered expression in HNSCCs. However, their functional

miR- 200a/b

ZEB1/ZEB2

E- cadherin gene

E- cadherin expression

Other Regulatory signals

RhoA

Epithelial- mesenchymal transition (EMT)

miR-155

Fig. 2. HNSCC linked miRNAs and the ZEB1/ZEB2 network. The E-box transcription factors, ZEB1 and ZEB2 act as transcriptional repressors of E-cadherin, a key cellular adhesion protein. ZEB1/ZEB2 expression levels in epithelial cells are kept under check by members of the miR-200 family. In HNSCC, suppressed miR-200 levels cause upregulation of ZEB1 and ZEB2. This results in reduced levels of E-cadherin, which in turn contributes to induction of Epithelial–Mesenchymal transition (EMT). Independently, over-expression of miR-155, observed in some HNSCCs, causes increased posttranscriptional suppression of RhoA, a molecule involved in the maintenance of the epithelial phenotype. Loss of RhoA promotes EMT.

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significance in the context of HNSCC-linked cellular signaling pathways has not been investigated in any detail. We have developed a database which holds the entire catalog of HNSCC-linked miRNAs that we compiled in this study. This annotated catalog can be accessed freely at http://tarmir.rgcb.res.in/henecan/. There is sufficient literature to show that microRNAs tend to be located in chromosome fragile sites and many have been mapped to regions of genetic aberration [51,53,54]. Indeed, a number of chromosomal abnormalities have been repeatedly characterized in HNSCC [55]. At least ~25 HNSCC-linked miRNAs are associated with chromosomal regions known to be altered in this form of cancer. Although it is tempting to associate up- and down regulations of specific miRNAs with gain or loss of chromosomal arms or individual bands, a formal mechanistic relationship has not been demonstrated in any case so far. 4. Epigenetic silencing of HNSCC miRNAs via promoter hypermethylation The role of promoter hypermethylation in the silencing of cellular genes, especially TSGs, has been well documented. A similar search for miRNAs that could be silenced via hypermethylation yielded two miR137 and miR-193a, which are down-regulated in OSCC [56]. Moreover, a screen for the targets of these miRNAs identified CDK6 and E2F6 as major targets. The role of both CDK6 and E2F6 in cell cycle regulation is well-known. This suggests a possible mechanism whereby epigenetic silencing of miRNAs can lead to deregulated cell proliferation. Importantly, in a later study, it was shown that miR-137 promoter hypermethylation is significantly associated with poorer average survival in a set of 67 HNSCC patients [57]. 5. Conclusions and therapeutic implications Our review highlights how miRNA deregulations can contribute to carcinogenesis by affecting many of the important phenotypes associated with cancer. We have presented available evidence that demonstrate how altered expression of select miRNAs may contribute to “hallmarks of cancer” such as limitless replicative potential owing to abnormal cell cycle regulation, evasion of apoptosis, reduced response to anti-growth signals, and Epithelial–Mesenchymal transition. Elucidation of the molecular mechanisms for miRNAs that are strong predictors of clinical outcome and conversely, investigating the clinical association of miRNAs known to target important tumor suppressors/ oncogenes can lead to a more complete picture of the role of miRNAs in HNSCC. Given the causal nature of some miRNAs in HNSCC, it would be worth investigating if these miRNAs can be exploited as therapeutic targets in HNSCC. Antisense targeting of miRNAs for therapeutic purposes is emerging as a promising approach. Conflict of interest The authors declare no conflict of interest. Acknowledgements We would like to acknowledge the Department of Science and Technology (DST), Government of India for supporting the microRNA– oral cancer work of RH in the form of a DST-SERC FAST TRACK Young Scientist Award (SR/FT/LS-064/2008). We are also grateful to Mr. Anand Krishnan, Senior Research Fellow, Integrated Cancer Research Program, RGCB, for his critical comments and suggestions. References [1] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics, 2008, CA Cancer J. Clin. 58 (2008) 71–96.

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