Recently identified miRNAs that undergo deregulation in HNC.
Head and neck cancer (HNC) is referred to the cancers of aerodigestive tract covering number of structures viz, oral and nasal cavity, paranasal sinuses, lips, salivary glands, oropharynx, hypopharynx, pharynx, larynx, and local lymph nodes. It is the sixth most common cancer in the world. MicroRNAs (miRNAs) are small single‐stranded noncoding RNAs (ncRNAs) of about 19–25 nucleotides. These miRNAs have been reported to influence number of biological activities, i.e., gene regulation, differentiation, organ formation, cell death, cell proliferation, and stress responses. The first ever study involving miRNAs in HNC was published in 2005. Since then, association between dysregulation of miRNAs and head and neck tumorigenesis has been documented by a number of researchers. This chapter has covered a comprehensive state of the art literature review of the recent studies about the role of miRNAs in HNC including oral squamous cell carcinoma (OSCC) and human nasopharyngeal carcinoma. Despite significant improvement in multimodal treatment, the prognosis of advanced HNC is quite poor. Recent studies are promising regarding the potential role of miRNAs as prognostic indicators. Recently, some miRNAs have been discovered as important diagnostic biomarkers. In fact, miRNAs are found circulated stably in different body fluids, i.e., urine, blood, saliva, as well as in breath. Hence, these miRNAs can be assessed easily with noninvasive methods. miRNAs are the key therapeutic targets in addition to their prognostic and diagnostic value. Use of synthetically designed “miRNAs sponges,” miR mimics (agomiRs), miR antagonists (“antagomiRs”), and miR inhibitors (antimiRNAs oligonucleotides) is an innovative strategy to modulate oncogenic and tumor‐suppressive pathways. Our understanding of miRNAs involvement in HNC is in its infancy. The discovery of miRNAs heralds a complete new paradigm in the understanding of exact molecular pathways involved in HNC development. More detailed studies are required for better understanding and therapeutic targets to treat HNC.
- gene regulation
- head and neck cancer
- nasopharyngeal carcinoma
- noncoding RNAs
- oral squamous cell carcinoma (OSCC)
- therapeutic targets
1. Head and neck cancer epidemiology
Head and neck cancer (HNC) is referred to the cancers of aerodigestive tract covering number of structures
One of the frequently diagnosed malignancies, globally, is head and neck cancer. Frequent relapses and distant metastasis have been observed in patients with advanced disease stages, resulted in an endurance rate of 5 years in almost 60% patients despite of substantial advances in multimodality rehabilitation . Mucosal malignancy of head and neck is a potent melanoma with poor prognosis. The nasal pit, paranasal sinuses, and oral cavity are the most widely recognized areas. Survival rates of 1, 3, and 5 years were 63, 30, and 20%, respectively, around 2000–2007. All interpretations suggested that cigarette smoking is hazardous. Clinical marks and clues are typically nonspecific for it. Surgery is regarded as the backbone of treatment for most mucosal melanomas of the head and neck supplemented with radiotherapy . The relationship amongst alcohol consumption and head and neck disease is quite clear and the outcomes reliably demonstrated an expanded head and neck tumor chance related with alcohol drinking. Bagnardi et al. reported a positive relationship in between alcohol drinking and head and neck cancer risk . In Western nations, around 39% of head and neck malignancy can be ascribed to alcohol utilization (4% for only alcohol drinking and 35% for the joint impact of alcohol and tobacco) . Head and neck squamous cell carcinomas (HNSCCs) cause more than 300,000 deaths globally every year. Locoregional and removed recurrences are more important prognostic indicators and acknowledged surrogate markers of patients’ survival. No legitimate biomarker and rescue treatment exist to recognize and treat patients at high‐danger of recurrence . Despite of reduction in smoking and alcohol utilization, the rate of oropharyngeal squamous cell carcinoma (OPSCC) is rising. It refers to human papilloma virus (HPV) infection contamination . HPV is an entrenched prognostic marker for OPSCC . Oropharyngeal tumors are firmly connected with HPV‐positivity . Oral tumor constitutes the dominant part of head and neck diseases, which is the fifth most basic malignancy around the world, representing 984,430 cases in 2012. During 2000 and 2010, there were 1916 instances of OSCC in New Zealand with a male to female proportion of 1.85:1, and an age‐institutionalized rate of 42 for every 1,000,000 people . Liquor utilization, trailed by tobacco, is considered the most common hazard in New Zealand. Given the high pervasiveness of these two hazardous elements and their synergistic impact, it is vital for specialists to boost smoking cessation and limited liquor consumption. More research should be conducted to confirm use of tobacco and water‐pipe smoking in New Zealand, particularly because of changing demography and increments in transient populaces. UV radiation is an additionally imperative hazard element . Laryngeal squamous cell carcinoma (LSCC), being a potent threat, is amongst the most regularly analyzed malignant sorts of head and neck SCC around the world. Rates of LSCC have been estimated to escalate recently . Salivary gland pleomorphic adenoma (SGPA) is also one of the most widely recognized types of salivary organ tumor. In China, particularly in the South, nasopharyngeal cancer (NPC) is another widely recognized threatening tumors and hence remained unregistered even by National cancer registries, since little is known about its epidemiology . The occurrence represented around 40% of the world’s new cases as indicated by the World Health Organization’s GLOBOCAN revealed information of 2012 . Individuals with a family history of NPC have a generously higher danger of NPC . NPC’s mortality indicates marked distinction between endemic (highly vulnerable territories), where nonkeratinizing carcinoma (NKC) is pervasive, and nonendemic (safe districts), where the keratinizing squamous cell carcinoma (KSCC) is more frequent. Fluctuations in smoking and alcohol consumption amongst genders and geographic regions may clarify the diverse rates and patterns fully observed for KSCC and partially for NKC. Dietary patterns and improvement in disease management can also be accountable for observed trends .
2. MicroRNAs discovery
MicroRNAs (miRNAs) are single‐stranded, small RNA molecules whose presence was reported for the first time in 1993 in nematode
3. MicroRNAs biology
miRNAs are small ncRNAs of about 19–25 nucleotides and have been discovered recently in all metazoans tested so far [41, 42]. They comprised of a small fraction of expressed genome; however, genes encoding them are present in either introns or exons of coding as well as noncoding genes throughout the genome. miRNAs are involved in regulating number of processes including differentiation, maintenance of homeostasis, migration, programmed cell death, and morphogenesis [42–45]. miRNAs are transcribed with the aid of RNA polymerase II enzyme from the miRNAs genes, which in turn transcribed into primary microRNA transcripts following transformation into pre‐miRNAs which are then ultimately results in miRNAs [32, 46–50]. In recent years, a novel class of molecules naming miRNAs has showed a boom. With advancements in genomic technologies and methodologies, miRNAs gene family is rising as new members discovered day by day. In 2005, it was speculated that there are about 1000 miRNAs genes in human genome . However, 2042 mature miRNAs have been reported in miRBase up to date (version 19). miRNAs are characterized by having 21–23 nucleotides in length and are exclusively found in eukaryotes. They show partial complementarity to specific regions in targeted mRNA and hence are involved in gene regulation of 50% of human genome at posttranscriptional level . Each of these noncoding RNAs can have sequence complementary to a large number of transcripts and regulate expression of an enormous number of genes. This can be achieved by multiple mechanisms including an increased mRNA degradation, site‐specific cleavage, and translational inhibition . miRNAs dysregulation has been associated with a number of different types of cancer, their initiation, and proliferation, since the first ever study claiming the association between chronic lymphoid leukemia and miR‐15a and miR‐16‐1 in the year 2002 [54–56]. Therefore, they may prove as potent therapeutic targets for cancers including HNSCC as well as biomarkers for timely diagnosis, prognosis, and recurrence of cancers as well. Two different pathways have been reported for the biogenesis of miRNAs. Usually, a canonical pathway is used for their biogenesis as most of the miRNA genes are intergenic (Figure 1). These miRNAs when transcribed possess a local hairpin structure and are named as primary transcripts (pri‐miRNA) and have characteristics to that of mRNA, as there is a 5′cap and a 3′poly‐A tail. Drosha (a protein complex having nuclease activity) and Pasha (an RNA‐binding protein) process these pri‐miRNAs to pre‐miRNAs having 70‐nucleotide stem‐loop structure. pre‐miRNAs are converted into a double‐stranded miRNA/miRNA* duplex by an RNase III endonuclease (Dicer) followed by transportation in cytoplasm by Exportin‐5. Further, a helicase destabilizes the duplex miRNA/miRNA* to a mature miRNA and miRNA*. Finally, the mature miRNA then integrates into RNA‐induced silencing complex (RISC) also called miRNA ribonucleoprotein complex (miRNP).
The resultant miRNP is involved in RNA interference which is initiated by both siRNA and miRNA. In case of intronic stem‐loops‐derived miRNA, no Drosha activity is involved for the maturation of miRNAs [32, 57] (Figure 2).
4. MicroRNAs deregulation in head and neck cancer
The first ever study involving miRNAs in HNC was published in 2005 . miRNAs showed greater cancer‐related potential than expected. miRNAs have been associated with all processes of physiological and pathological nature. Expression profiles of miRNAs have reported to be more specific to cancer tissue origin and have greater potential for early diagnosis and provided more information than mRNAs [59, 60]. Association between dysregulation of miRNAs and tumorigenesis in head and neck has been documented previously [61–63]. Many biological and molecular mechanisms cause resistance to the tumors radiotherapy. The principle mechanisms include changes in intracellular pathways that are required in damaging DNA as well as its repair, apoptosis, proliferation, and angiogenesis. The regulation of these perplexing procedures is regularly controlled by microRNAs. miRNAs are short endogenous RNA molecules that posttranscriptionally modulate gene expression. Their impaired expression has been seen in numerous tumors including head and neck cancer. Particular expression patterns of miRNAs have additionally been appeared to anticipate prognosis and therapeutic response in head and neck cancer . Development and progression of different sorts of malignancies in human is attributed to the deregulation of miRNAs. Oncogenic part of miR‐214 is proposed in NPC. Bax inhibition initiated by siRNA weakens the advancing impact of miR‐214 deregulation on NPC cell apoptosis, recommending that Bax is a downstream effector in miR‐214 that is intervened NPC cell proliferation and death. Bax expression level is deregulated in NPC tissues . miR‐145 expression in LSCC is downregulated, and its overexpression creates hindrance in multiplication and relocation of Hep‐2 cells through cell cycle arrest and apoptotic induction. SOX‐2 is overexpressed in tumor samples and exhibit restricted expression in miR‐145 overexpressed Hep‐2 cells . miRNAs deregulation plays a key role in HNSCC progression . miR‐93‐5p (HN2092) and miR‐425‐5p (HN1957) are top candidates of downregulated miRNAs in primary HNSCC cell cultures and blood plasma of patients . miRNAs deregulation plays a noteworthy part in head and neck/oral cancer . miR‐10b and miR‐196a, not formerly linked with HNSCC, may show a role in oncogenesis through the deregulation of cell proliferation . Deregulation of miRNA genes (such as miR‐138) plays a critical part in HNSCC. While downregulation of miR‐138 has been observed in HNSCC and other cancer types often; however, the exact role of miR‐138 in tumorigenesis is unknown. Current bioinformatics analyses and
|Downregulated miRNAs||Sample used||Assay||Type of cancer||Reference|
|miR‐145||Tissue||qRT‐PCR and Western blot analysis||Laryngeal SCC|||
|Blood plasma||qRT‐PCR||HNSCC patients|||
|Blood plasma||qRT‐PCR||HNSCC patients|||
|miR‐99 family||Meta analyses||RT‐PCR, microarray||HNSCC|||
|UNG, FUCA2, DERA, GMFB, TF, and SNX2||Cell lines||RT‐PCR||Head and neck squamous cell carcinoma|||
|miR‐21 and miR‐31||Saliva, Plasma||q‐PCR||Tongue epithelium|||
|miR‐29c||Cell lines, clinical specimens||Microarray, qRT‐PCR||NPC|||
|miR‐9||Cell lines, tissues||qRT‐PCR||NPC|||
|miR‐378||Cell lines, tissues||qRT‐PCR||NPC|||
|miR‐200 family||Cell lines||Microarray||NPC|||
|MiR‐99a||Tissue, cell lines||Luciferase assay||Oral cancer|||
|miR‐24||Tissue, cell lines||RT‐PCR||Oral cancer|||
|miR‐133a, miR‐133b, miR‐100, miR‐138||Tissues||qRT‐PCR||TSCC|||
|miR‐133b, miR‐138, miR‐137, miR‐184||Cell lines||qRT‐PCR||OSCC|||
|miR‐138, miR‐222||Cell lines||Microarray||TSCC||[85, 86]|
|miR‐342, miR‐21||Cell line||Microarray||TSCC|||
|miR‐342, miR‐346, miR‐373||Cell lines||Microarray||HNSCC|||
5. miRNAs as oncogenes in oral squamous cell carcinoma (OSCC)
A number of miRNAs have been reported to act as oncogenes and were found to be upregulated in OSCC. miR‐21 was found to be highly expressed and to regulate several biological activities in OSCC [68, 92–94]. Presence of an upregulated level of miR‐21 in oral premalignant lesions was an indication that variations in miR‐21 level may be prior event in OSCC development . miR‐21 plays an oncogenic role in the progression of OSCC by promoting cell proliferation , antiapoptotic activity , invasion [93, 97], and chemoresistance  in both
Oral squamous cell carcinoma (OSCC) is a typical cause of cancer‐related death. A number of efforts have been made in investigating new medications and impressive progress in multimodality treatment; however, remedial tolls have not yet been achieved. The trouble of timely detection and more pervasiveness of metastasis associated with OSCC results in poor prognosis. In recent couple of decades, growing information from tumor biology and clinical trials prompted development of ncRNAs prognostic biomarkers that are believed to be promising biomarkers in this regard. miRNAs are one of the most studied ncRNAs in terms of their biogenesis, function, and significance in carcinogenesis . An association between severity of pathogenesis and increment of miR‐31 and miR‐21 has been reported through staining in 4NQO‐induced injury in tongue epithelium. A dynamic rise in the level of
6. MicroRNAs in human nasopharyngeal carcinoma (NPC)
Paul Ahlquist’s group working at National Cancer Institute (NCI) published the first ever study of global profiling of miRNAs involved in NPC in 2008 . They discovered a number of deregulated miRNAs, using a micro‐array‐based approach, in laser‐capture micro dissected (LCM) 31 NPCs and 10 epithelial samples as control . miRNA downregulation and change in pathways have been involved in NPC which is profoundly invasive and metastatic widespread in Southern China. miR‐9 is commonly downregulated in NPC with significant functional consequences. Diminished expression of miR‐9 is contrary to clinical stages and denotes the movement from locoregional to metastatic tumors. CpG island hypermethylation adds to inhibition of miR‐9 in NPC cell lines and tissues. Ectopic miR‐9 expression significantly hinders proliferative, transient, and obtrusive limits of NPC cells, both
7. miRNAs as prognostic indicators
Despite significant improvement in multimodal treatment, the prognosis of advanced HNC is quite poor. Recent studies are promising regarding the potential role of miRNAs as prognostic indicators. Downregulation of Let‐7 (a family of tumor suppressing miRNAs) has been reported in HNSCCs by many researchers [74, 92, 95, 103, 130–138]. Similarly, underexpression of miR‐146a, miR‐155, and Let‐7 has been correlated with the progression of cancer . Moreover, a decreased level of Let‐7 miRNA in nasopharyngeal carcinoma cells was also suggestive of regulating the proliferation of carcinoma cells via c‐MYC downregulation . Furthermore, role of Let‐7 in the Kirsten rat sarcoma (KRAS) regulation has been demonstrated recently by some studies on nonsmall cell lung cancer . A variant allele in the V‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) 3′untranslated region (KRAS‐LCS6) has been demonstrated to be involved in high expression of KRAS and decreased levels of Let‐7. Presence of KRAS‐LCS6 variant has been reported in Squamous cell head and neck cancer (SCHNC) and was correlated with poor prognosis . TP53, a tumor suppressor gene, encodes a protein product that is involved in cell cycle regulation. It is a major change involved in cancer induction with a remarkable frequency of 53% as reported by Poeta et al. in a hefty cohort of SCHNC. The ratio of TP53 mutations were 75 and 56.7% in patients suffering from hypopharynx and larynx tumor, respectively. Furthermore, a strong association was described between TP53 mutations in SCHNC and high risk of recurrence and poor survival . Ganci et al. has previously reported a strong link among 49 miRNAs and TP53 status. Among them, a more specific correlation of a subset of 12 miRNAs was correlated with a brief recurrence free‐survival while four of these were correlated with comparatively lower cancer‐specific survival . Expression of some particular miRNAs, such as miR‐375 and miR‐210, has been correlated with the outcome of SCHNC patients. A low expression of miR‐375 results in poor survival and distant metastases while high expression of miR‐210 results in locoregional recurrence [143, 144].
8. miRNAs as biomarkers
Diagnosis of HNSCC more frequently occurs in advanced stage when it metastases to regional lymph nodes. Moreover, there is a high risk of recurrence even in patients with a combination of different therapeutic approaches. Therefore, for an early diagnosis, the most important goal would be the detection of biomarkers. Recently, some miRNAs have been discovered to fulfill the goal of diagnosis. In fact, miRNAs are found circulated stably in different body fluids, i.e., urine, blood, saliva, as well as in breath. Hence, these miRNAs can be assessed easily with noninvasive methods.
miRNAs can be utilized as biomarkers or novel therapeutic targets. Further investigation is needed to test its utilization . Circulating miRNAs (miR‐425‐5p, miR‐93‐5p) are easily approachable and have turned out to be valuable prognostic markers in cancer patients. The prognostic worth of this exciting perception requires affirmation using an independent patient cohort that includes clinical follow‐up data. Changes of miRNAs succeeding radio‐chemotherapy in the blood plasma are related with the tumor response to therapy, and they might signify novel biomarkers for therapy monitoring . The investigations of microRNA modifications in HNSCC are a fundamental stride to the mechanistic comprehension of tumorigenesis and could prompt the disclosure of clinically pertinent biomarkers . For NPC diagnosis, plasma miRNAs expression proves to be a helpful biomarker . Pathway improvement analysis of these four miRNAs (miR‐34c, miR‐140, miR‐154, and miR‐449b) sign posted a role in cell cycle regulation, highlighting a possibly important role for markers of cell cycle enactment as prognostic indicators in NPC . miR‐200b expression is mostly connected with distant metastasis, while miR‐155 associated with local recurrence. miR‐155 and miR‐146a were recognized as surrogate markers for tumor‐invading lymphocytes in HNSCC . miR‐21 expression could be an imperative tool for treatment planning and prognostic predictor for HNSCC patients . Genetic variants of miR‐146a and miR‐1269b are biomarkers for improvement of oral premalignant lesions (OPLs) and oral squamous cell carcinoma (OPSCC) . hsa‐miR‐375‐3p appears to be a comparatively promising diagnostic marker in HNSCC but is not appropriate for prognosis of patients . Overexpression of TP53 mutation-associated miR-182 may contribute to proliferation and migration of tumour cell in HNSCC, hence suggest a possible biomarker for prognosis of tumour recurrence . miR‐30a is a possible biomarker for metastasis in NPC patients . miR‐148a and miR‐375 are significantly upregulated during LSCC and are conceivable biomarkers for early diagnosis of LSCC . A list of miRNAs is given below which has been identified as potential biomarker by a number of researchers in HNC (Table 2).
|miRNA biomarker||Type of cancer||References|
|miR‐34c, miR‐140, miR‐154, and miR‐449b||NPC|||
|miR‐155 and miR‐146a||HNSCC|||
|miR‐146a and miR‐1269b||Oral premalignant lesions (OPLs) and oral squamous cell carcinoma (OPSCC)|||
|miR‐148a and miR‐375||LSCC|||
9. Novel therapeutic targets
miRNAs are key therapeutic agents (depends upon the type of mRNA affected by them) in addition to their prognostic and diagnostic value . miRNAs are efficient molecules to be targeted as they regulate a number of biological activities by interacting with numerous other molecules . Use of synthetically designed “miRNAs sponges” , miR mimics (agomiRs) , miR antagonists (“antagomiRs”) [155, 156], and miR inhibitors (antimiRNAs oligonucleotides)  is an innovative strategy to modulate oncogenic and tumor‐suppressive pathways. MicroRNAs regulate each step of cell cycle and unraveling their altered expression may prove fruitful in designing new drugs and can open treatment regimes. One piece of evidence is a previous study abduction of miR‐122, which has been observed in a novel therapy for HCV patients by Miravirsen (first microRNA targeted drug) in clinical phase2a. This therapy resulted in diminution of HCV RNA levels in a dose‐dependent manner . This research has raised a lot of hope for employing miRNAs therapies in number of malignancies despite the disparity of the diseases from cancer. Some other miRNAs therapies are in preclinical and clinical phase 1. These approaches are anxiously awaited to be reported beyond the mere documentations. Few miRNAs, though not verified clinically, are very good applicants for being named for these therapies. Apoptotic pathway is one of the most significant candidates for novel anticancer therapies, as the neoplastic cells usually lose the ability to undergo programmed cell death. Therefore, any powerful proapoptotic agent may directly or indirectly reduce cancer progression by enhancing the apoptosis.
Nevertheless, for an efficient and target specific miRNAs‐based drug delivery system, there lies enormous challenges [22, 53]. To access the targeted sites, therapeutic RNA must travel across the plasma membrane to enter in cytoplasm by leaving the circulatory system and avoiding endosomal vesicles to entering in the cell . Moreover, nonconjugated therapeutic RNA molecules are either cleared by the kidney (<50kDa) or by the immune cells (7‐20kDa) . Levels of mature miRNAs may be modulated or reduced by delivery of synthetic double‐stranded hairpin exogenously by complexing with lipids or proteins. Delivery of miR‐34a may prove effective in HNSCC cells as suppression of cellular proliferation and apoptosis was observed in two cancer cell lines (colon) and experimental lung metastasis of murine melanoma upon miR‐34a delivery [164, 165]. Use of unmodified dsRNAs in environments, where local administration is possible, is of limited value
10. Future challenges
For last 3 decades, dysregulation of tumor suppressor genes and protein‐coding oncogenes were thought to be involved in cancer. However, discovery of noncoding genes, i.e., miRNAs raised a question on the notion that this mechanism is solely responsible for cancer. To unveil the involvement of miRNAs in cancer, massive efforts have been made but still enormous challenges lie ahead. Detection of the exact pathways and genes regulated by these miRNAs will be of prime importance. A better understanding of the wide‐ranging effects of these novel molecules will equip the researchers in the selection of specific pathways for treatment of cancers. Our understanding of miRNAs involvement in HNC is in its infancy. However, studies have been confirmed the dysregulation of a number of miRNAs in both benign and malignant HNC. There are real prospects that miRNAs in near future may be used as prognostic and diagnostics of HNC to improve its treatment strategies. Growing evidences make these miRNAs viable targets for the development of new and better anticancer therapies. However, before translation to be carried out in clinical settings, a more clear insight of efficacy and “off‐target” effects of miRNAs is necessarily suggested. It is quite fascinating that recently a number of miRNAs including miR‐21, miR‐155, and let‐7b have been nominated as key players of human carcinogenesis including head and neck tumors. The discovery of miRNAs heralds a complete new paradigm in the understanding of exact molecular pathways involved in HNC development. More detailed studies are required for better understanding and therapeutic targets to treat HNC.
However, a number of efforts have been made but truly comprehensive profiling of miRNAs is still needed in HNSCC. Even, technology utilized for profiling also needs to be improved. Use of Polymerase chain reaction (PCR) and microarrays is a common practice for profiling but for a better picture and more deep insight next‐generation sequencing (NGS), cross‐platform analysis and new approaches like NanoString nCounter system must be applied. Functional studies are growing, highlighting the potentially targetable miRNAs and some related pathways. Tumor cells survival and their development have been reported to be affected by the forced expression or even the inhibition of a number of miRNAs