Ó 2014 Eur J Oral Sci

Eur J Oral Sci 2014; 1–4 DOI: 10.1111/eos.12121 Printed in Singapore. All rights reserved

European Journal of Oral Sciences

Role of the MIR146A polymorphism in the origin and progression of oral squamous cell carcinoma Palmieri A, Carinci F, Martinelli M, Pezzetti F, Girardi A, Cura F, Rubini C, Scapoli L. Role of the MIR146A polymorphism in the origin and progression of oral squamous cell carcinoma. Eur J Oral Sci 2014; 00: 000–000. © 2014 Eur J Oral Sci Gene expression and cell behavior are regulated by several factors, including small non-coding RNAs. MicroRNAs affecting cell growth, differentiation, and apoptosis are thought to play an important role in tumorigenesis. The levels of miR-146 appear to be associated with cancer development and progression, including that of oral squamous cell carcinoma. The aim of this investigation was to ascertain whether the single nucleotide polymorphism, rs2910164, mapping in the MIR146A gene, has a role in oral squamous cell carcinoma progression. A genetic association study was performed with a sample set of 346 oral squamous cell carcinomas collected in Italy. Our data indicate that the rs2910164 polymorphism is not associated with tumor development. However, a slight increase in the frequency of the variant allele was observed in Stage II tumors. Further investigations are needed to verify a possible role of the variant allele or rs2910164 in oral squamous cell carcinoma progression.

Oral cavity cancers are among the most common, with an estimated worldwide annual age-standardized incidence of 3.8/100,000 and a mortality rate of 1.9/ 100,000 (1). The vast majority of these cancers are oral squamous cell carcinomas (OSCC). Recognized risk factors for OSCC, such as tobacco and alcohol consumption, are suspected to promote tumor progression by increasing the DNA damage rate (2). Oral squamous cell carcinomas generally exhibit poor prognosis. The occurrence of metastasis, which is the main cause of death, shows a significant correlation with the clinical stage, localization of the primary tumor, degree of deep infiltration, and cell differentiation. However, the prognosis of primary OSCC is difficult to predict on the basis of histopathological parameters alone. Reliable molecular markers with a predictive value could be important tools for use by clinicians during treatment planning for OSCC patients. The identification of tumor progression markers – useful for making more accurate diagnoses, treatment plans, and prognoses – was a major goal for the scientific community. However, despite extensive research efforts, until now no such markers for OSCC have been detected. Large-scale investigations have helped to identify the frequent genetic changes involved in tumor development and progression, such as chromosome aberration or variations in gene-expression profiles.

Annalisa Palmieri1, Francesco Carinci2, Marcella Martinelli1, Furio Pezzetti1, Ambra Girardi1, Francesca Cura1, Corrado Rubini3, Luca Scapoli1 1

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna; 2Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara; 3Department of Biomedical Sciences and Public Health, Section of Pathologic Anatomy and Histopathology, Polytechnic University of the Marche/United Hospitals, Torrette (AN), Italy

Dr Marcella Martinelli, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via Belmeloro, 8, 40126 Bologna, Italy E-mail: [email protected] Key words: marker; microRNA; oral squamous cell carcinoma; single nucleotide polymorphism; tumor progression Accepted for publication January 2014

Recently, a new class of molecules - microRNAs (miRNAs) - which have the ability to modulate gene expression levels, was considered appealing in terms of representing useful biomarkers of tumor progression (3). MicroRNAs, small (18–22 nucleotides) non-coding RNAs cleaved from 60- to 110-nucleotide hairpin precursors, are able to trigger the degradation of specific mRNAs (4, 5). MicroRNAs are involved in various biological processes, including cell proliferation and cell death, development, stress resistance, and metabolism (6). Because of their extraordinary tissue specificity, they have been proposed as a tool for diagnosing and classifying primary cancers and their metastases (7, 8). Our research group has recently investigated the miRNA expression profile of OSCC using oligonucleotide microarray hybridization (9). A number of aberrantly expressed miRNAs, potentially implicated in OSCC origin and progression, were identified. The subject of this investigation was miR-146a, a member of a small group of miRNAs that was found to be useful for distinguishing OSCC with lymph node metastasis. Indeed, MIR146A was found to be significantly under-expressed among patients with OSCC with lymph-node metastasis. Interestingly, a common polymorphism, with a significant functional impact, was found in the MIR146A gene. Indeed, the G>C substitution produces a mismatch in the predicted pre-miR-146a hairpin structure that results in a reduced amount of mature miR-146a. This single

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nucleotide polymorphism, named rs2910164, has been linked to papillary thyroid carcinoma (10), breast cancer (11), prostate cancer (12) and hepatocellular carcinoma (13) susceptibility. Together, all of this evidence prompted us to investigate rs2910164 in OSCC in order to test its association with tumor susceptibility and progression.

UICC stage group (I, II, III, or IV), histological grading (G1, G2, or G3), and histological grading in the deep invasive front, patient gender, and age. Logistic regression analysis was performed using SPSS v. 14 (IBM, New York, NY, USA).

Results Material and methods The sample included 346 OSCC patients diagnosed at the Section of Pathologic Anatomy and Histopathology, Polytechnic University of the Marche/United Hospitals, Ancona, Italy. Most were part of a cohort previously described (14). Tumor sites were limited to the oral cavity, as defined by the Union Internationale Contre le Cancer (UICC) code ICD-O C02–C06, which includes tongue cancer and excludes tonsil and laryngeal cancers (15). The informed consent of all patients who participated in the experimental investigation was obtained according to the Declaration of Helsinki. The Ethical Committee of the United Hospitals of Ancona, Italy, approved this study. The patients ranged in age from 18 to 95 yr, with a mean  SD age of 65  13 yr; as expected, men (n = 252) were more prevalent than women (n = 94). The TNM staging of tumors is shown in Table 1. Genomic DNA was purified from paraffin-embedded specimens, as described previously (14). Genotypes of rs2910164 were obtained with a specific Applied Biosystems assay using the TaqMan chemistry and the ABI PRISM 7500 Real-Time PCR System. Control Italianpopulation genotypes were obtained from a public database [i.e. the ‘Toscani in Italia’ (TSI) population from the HapMap project] and from published investigation data (16–18). Hardy–Weinberg equilibrium was tested by comparing the observed and expected genotype frequencies using the Pearson’s chi-square test. Allelic association was investigated by 2 9 2 contingency tables and the Mid-P exact test. Data were further investigated using 2 9 2 contingency tables by combining the heterozygous genotype with either one of the homozygous genotypes in a dominant or recessive mode of inheritance. The ORs were obtained by conditional maximum-likelihood estimation. To account for multiple comparisons, the Bonferroni correction for significance level was adopted. A logistic regression analysis was conducted to predict rs2910164 C-allele carriers using, as predictors, tumor localization (tongue, cheek, gingiva, mouth floor, palate, lip, base of tongue, and other parts of the mouth), tumor size (T1, T2, T3, or T4), node metastasis (N0, N1, or N2),

Table 1 Tumor staging of the sample study

T1 T2 T3 T4 Total

N0

N1

N2

Total

143 75 8 29 255

12 26 3 3 44

12 26 4 5 47

167 127 15 37 346

N, lymph node involvement; T, tumor size.

A case–control association study was performed to verify whether the inherited polymorphism, rs2910164, in the MIR146A gene could influence the risk of development of OSCC or be related to metastasis production or cancer stages. The rs2910164 genotyping assay did not produce a valid result for nine samples. Failure to produce the rs2910164 genotype was not related to a particular clinical aspect or to the locus itself, but rather to poor DNA quality. Genotypes obtained from 337 OSCC specimens were distributed according to Hardy–Weinberg equilibrium. The allelic association test indicated that there was no significant difference in the allele frequency at each locus between the patients and four independent control groups of the Italian population (Table 2). No deviation from the null hypothesis was observed with the genotypic association tests using dominant or recessive models. ANOVA indicated that age at diagnosis did not vary among groups of different genotype, F(2, 336) = 0.21, P = 0.81. In order to test association between rs2910164 and tumor progression, genotypes were stratified based on tumor dimension (T), node metastasis (N), and UICC

Table 2 Distribution of genotypes at rs2910164, and testing for allelic association Variable

n

Study groups OSCC 337 Controls Tuscany 88 206 MARINO et al. (18) 543 MANNA et al. (17) FENOGLIO et al. (16) 339 Tumor size T1 162 T2 124 T3 14 T4a-T4b 37 Lymph node metastasis N0 248 N+ 89 N1 42 N2 47 Stage group Stage I 139 Stage II 73 Stage III 46 Stage IV 79

GG

GC

CC

MAF

P

197

121

19

0.24

Reference

50 105 297 195

31 84 206 115

7 17 40 29

0.26 0.29 0.26 0.26

0.58 0.06 0.20 0.41

100 67 7 23

55 46 6 14

7 11 1 -

0.21 0.27 0.29 0.19

Reference 0.09 0.37 0.65

144 53 22 31

89 32 18 14

15 4 2 2

0.24 0.22 0.26 0.19

Reference 0.68 0.66 0.31

85 36 25 51

48 29 18 26

6 8 3 2

0.22 0.32 0.25 0.19

Reference 0.04 0.37 0.52

MAF, minor allele frequency; OSCC, oral squamous cell carcinoma.

MIR146A in OSCC

stage group (Table 2). Among these evaluations, significant association was observed only for the comparison of Stage I with Stage II; in fact, the rare C allele was more frequent in Stage II (P = 0.04). Carriers of the variant allele showed a higher probability of progressing at Stage II, with an OR of 1.62 (95% CI: 0.91–2.87); in contrast, the OR for homozygotes was 3.15 (95% CI: 1.02–9.73). However, none of the tests found a significant deviation from the null hypothesis when the threshold level was adjusted to account for multiple comparisons. A logistic regression analysis was performed to test if carriers of rs2910164 variant alleles (GG = 0; GC and CC = 1) can be predicted based on clinical data. As shown in Table 3, the model including eight clinical factors was unable to predict rs2910164 variant-allele carriers (P = 0.28), and none of the factors produced a significant contribution.

Discussion An increasing body of evidence relating miR-146a expression levels to cancer has recently been published. Overexpression of miR-146a was observed in OSCC and cervical cancer, a finding that was associated with a worse survival rate for squamous cell lung cancer (19–21). On the other hand, a low level of expression of miR-146a was found in cervical cancer cell lines and hormone-refractory prostate cancer (20, 22). Ectopic expression of miR-146a was able to inhibit migration, invasion, and the occurrence of metastasis in different experimental models (23, 24). Our previous data suggest that the miRNA precursor, mir-146, could play a critical role in OSCC development and progression, being overexpressed in OSCC without lymph node metastasis and down-regulated in lymph node-positive tumors (9). The evidence that a polymorphism in the MIR146A gene can influence expression of the active form of miR-146a (10) prompted us to investigate Table 3 Logistic regression analysis for prediction of the rs2910164 C-allele carrier Independent variable N T UICC stage group G G advanced front Localization Gender Age Constant Model

B

e.s.

0.12 0.28 0.01 0.01 0.06 0.77 v2 = 23.29 d.f.

Wald

d.f.

3.17 3.90 4.21 1.20 2.25 6.28 0.19 0.24 0.01 = 20 P =

2 3 3 2 2 6 1 1 1 0.28

Sign.

Exp(B)

0.21 0.27 0.24 0.55 0.33 0.39 0.66 0.62 0.93

0.88 1.01 0.94

The dependent variable is allele C coded so that 0 = GG and 1 = GC and CC. d.f., degrees of freedom; Exp(B), exponentation of the B coefficient, also called odd ratio; e.s., error standard; Sign., P-value; UICC, Union Internationale Contre le Cancer.

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whether it could also influence the development and progression of OSCC. The results obtained from 337 OSCCs with valid genotype data indicate that rs2910164 in the MIR146A gene is not associated with cancer development; in fact, the allele and genotype frequencies of patients were similar to those of the Italian population controls. Publicly available data indicate that the rs2910164 C-allele frequency varies among populations of different ethnicity. The mean C-allele frequency from the ‘1000 Genomes Project’ is 0.38, with a higher value observed in Asians (0.60) and a lower value observed in Europeans (0.22). The C-allele frequency in 337 OSCCs from Italian patients was close to the value reported in the 1,000 Genomes Project for Europeans. Although a decrease in miR-146a expression was found to be related to the occurrence of lymph node metastasis, this important step in tumor progression was not found to be associated with any rs2910164 allele or genotype. However, a slight increase in the frequency of the variant allele was observed among Stage II tumors. This difference was significant at a nominal level, but not at the more stringent level corrected for multiple comparisons. Additional investigation could verify if this trend was caused by chance or whether it has a biological basis. One could speculate that the rarer C allele of rs2910164 may favor progression of the tumor from Stage I to Stage II; however, this effect becomes negligible when compared with other, more severe, mutations capable of promoting faster progression to Stages III and IV. Interestingly, in a previous investigation, the C allele was found to be more prevalent among OSCC patients with N2 nodal involvement (25). We could not confirm this data; in fact, in this investigation, the C allele was slightly less frequent among N2 patients. A second statistical approach was performed in an attempt to correlate rs2910164 to tumor progression. The multivariate analysis failed to predict rs2910164 C-allele carriers using a model with eight clinical factors, and did not find any OSCC factor related to the C allele. Thus, this approach weakens the hypothesis that rs2910164 might influence OSCC progression. The effect of rs2910164 appears to be more relevant in other tumors. Indeed, it seems to predispose to papillary thyroid carcinoma (10), prostate cancer (26), breast cancer (12), and cervical squamous cell carcinoma (27); however, conflicting data have been reported (28). Recently published meta-analyses indicated that the association between pre-MIR146A polymorphism and cancer susceptibility was either not detectable or was barely evident and influenced by ethnicity (29). For this reason, studies involving a large sample size, but limited to a specific cancer and ethnicity, are advisable. Data obtained in this investigation indicate that the common polymorphism, rs2910164, did not significantly alter the risk for developing oral cancer, although it might be involved in tumor progression.

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Conflicts of interest – The authors declare that they have no conflicts of interest.

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