EGOT lncRNA in head and neck squamous cell carcinomas

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer and the sixth most common cause of cancer-related mortality worldwide. Tobacco smoking, alcohol consumption and human papillomavirus (HPV) infection are the main causes of these malignancies [1, 2]. Some progress has been made towards effectively treating HNSCC, however, it remains largely unsatisfactory because of still high mortality [3, 4]. Biomarkers that have been associated with different treatment responses are needed to better combat this deadly disease.
Previous studies have correlated the regulation of microRNAs and long non-coding RNAs (lncRNAs) with tumour progression, lymph node metastases and poor prognosis in HNSCC. lncRNAs are a class of functional RNA molecules over 200-nucleotides long that modulate the activity of transcription factors and regulate changes in the chromatin structure despite not being translated into proteins. Previous studies suggest that lncRNAs have the potential to greatly improve the diagnosis, prognosis and targeted treatment of HNSCC [5, 6, 7, 8].
In this study, we focused on the expression of the eosinophil granule ontogeny transcript (EGOT) lncRNA. Human EGOT is located on the antisense strand of the intron of the ITPR1 gene. It has two known isoforms – EGO-A (unspliced) and EGO-B (spliced) – that share the same transcriptional start site and both are polyadenylated. EGOT regulates eosinophil granule protein expression during eosinophil cells developmental process and functions as a non-coding RNA [9, 10]. Numerous recent studies have been dedicated to understanding the role of EGOT in glioma [11], breast cancer [12], gastric cancer [13], haematological malignancies [14] and in viral infections [15, 16, 17] and in cardiology [18, 19]. However, the function of lncRNA EGOT remains unknown in HNSCC. Therefore, we used data made available by The Cancer Genome Atlas (TCGA) to further characterize the role of EGOT in the biology of HNSCC and determine its utility as a new biomarker in clinical practice.

Material and methods

TCGA data

The TCGA expression data for lncRNA EGOT and other selected genes was downloaded from cBioPortal (Head and Neck Squamous Cell Carcinoma, TCGA, Provisional, 530-sample dataset) [20]. Clinical data on tumour and healthy control samples were obtained from the UALCAN database (http://ualcan.path.uab.edu) [21]. All data are available online and access is unrestricted.

Data analysis

The clinical pathology parameters analysed for associations with EGOT expression levels in all localizations of the HNSCC samples include age (below vs. above 61 years), sex (women vs. men), T-stage (T1 + T2 vs. T3 + T4), N-stage (N0 + N1 vs. N2 + N3), cancer grade (G1 + G2 vs. G3 + G4), cancer stage (I + II vs. III + IV), HPV p16 marker (negative vs positive), perineural invasion (negative vs positive), angiolymphatic invasion (negative vs positive), disease surgical margin status (negative vs positive) and lymphoid neck dissection status (negative vs positive). Next, subgroups using the median of expression level of EGOT as cut-off: 1) EGOT low and 2) EGOT high were generated. Disease-free survival (DFS) and overall survival (OS) were determined in these subgroups during 180 and 210 months, respectively.

Target prediction

EGOT target prediction was performed using an online tool (http://rtools.cbrc.jp/cgi-bin/RNARNA/index.pl) [22]. Expression levels of predicted genes were compared between the EGOT low- and high-expression groups. Next, the identified target genes were classified according to biological process and cellular pathway using the Functional Annotation Tool from the DAVID 6.7 Bioinformatics Resource [23].

Statistical analysis

All statistical analyses were performed using GraphPad Prism 5 (GraphPad, San Diego, CA, USA). The Shapiro-Wilk normality test, t-test and Mann-Whitney U test were used for EGOT level depending on clinical parameters and for genes expression depending on EGOT subgroups. The expression level of EGOT depending on the cancer locations was checked using one-way ANOVA obtained using Dunn’s multiple comparisons test. For DSF and OS analyses, the Log-Rank (Mantel-Cox) test was used and median survival, Hazard Ratio (Mantel-Haenszel; HR) and 95% Confidence Interval (CI) of ratio were calculated. In all analyses, p < 0.05 was used to determine statistical significance.

Results

Using the TCGA data available from the cBioportal and UALCAN databases, the expression level of EGOT was evaluated in HNSCC tissue (n = 520) and healthy control (n = 44) samples. We found that EGOT expression depends on the grade and localization of the HNSCC. According to the database, the expression of EGOT was slightly down-regulated in HNSCC. However, no significant difference was found in EGOT expression of the cancer and controls samples (median expression 0.097 vs. 0.221 transcripts per million respectively; p = 0.5143; Fig. 1A). An analysis of cancer stage (1-4) also failed to find any significant differences (p > 0.05; Fig. 1B). However, notable differences were observed in terms of tumour grade. Relative to healthy controls, EGOT expression was down-regulated in grade 1 (p = 0.00002), unchanged in grades 2-3 (p = 0.0813 and p = 0.2456, respectively) and significantly up-regulated in grade 4 (p = 0.0049; Fig. 1C).
The samples from patients with HNSCC (n = 522) were divided into three groups according to the National Institutes of Health (NIH) classification of tumour localization: oral cavity (n = 316), pharynx (n = 90) and larynx (n = 116). Expression analysis revealed that EGOT is down-regulated in tumours from the oral cavity (–0.4228 ±0.02226; p < 0.0001) and larynx (–0.4097 ±0.05293; p = 0.0001) compared to the pharynx (0.0566 ±0.1692). No differences were observed between the oral cavity and the larynx (p = 0.2468; Fig. 2).

EGOT levels differ depending on clinical pathology parameters

Next, EGOT expression levels were analysed according to clinical pathology parameters. Significant differences were found in terms of patients’ age (p = 0.0003), N-stage (p = 0.0173), grade (p = 0.0111), lymph node neck dissection (p = 0.0053) and were also associated with HPV p16 status (p < 0.0001); however, there were no differences in terms of sex (p = 0.0948), alcohol consumption (p = 0.0756), tobacco smoking (p = 0.7493), cancer stage (p = 0.7245), T-stage (p = 0.1699), angiolymphatic (p = 0.5111) and perineural invasions (p = 0.5186) nor disease surgical margin status (p = 0.0612). These data are summarised in Table I.

EGOT expression levels influence on DFS and OS

The HNSCC samples (n = 522) were divided into two groups according to EGOT expression using the median of EGOT expression level as a cut-off. The low expression group was defined as expression levels to –0.392 (n = 261) and the high expression group included all of the samples with expression levels above –0.392 (n = 261). We found that patients with high EGOT expression levels had longer median DFS periods than those with low levels – 71.22 vs 46.81 months respectively (p = 0.0452; HR = 0.7128, 95% CI: 0.5117-0.9931; Fig. 3A). Furthermore, patients in the high expression group had longer median OS times than the low expression group – 46.81 vs. 71.22 months (p = 0.0096; HR = 0.7008, 95% CI: 0.5352-0.9175; Fig. 3B).

Predicted EGOT targets and their function

Next, we used the available database to predict lncRNA-RNA interactions in human transcriptome for EGOT. We found 300 genes with a sumenergy of interaction between –26374.2 and –2838.8 and compared their expression levels in the low and high expression groups. Analysis using the DAVID Bioinformatics Resource revealed that, among patients with high EGOT expression levels, 107 genes were up-regulated. These genes are associated with the regulation of many cellular processes (e.g., differentiation, adhesion, development, cell communication, signal transduction, division and proliferation), protein phosphorylation and other modifications, cellular component organization, cellular homeostasis, drug response and cell motility. The 17 down-regulated genes indicated in the analysis were associated with cell cytoskeleton and filaments, localization/binding, cellular transport and protein activity (Tables II and III).

Discussion

Poor prognosis and high resistance to radio- and chemotherapy are characteristic of HNSCC. Various treatment strategies have been employed in the past couple of decades but none have been great breakthroughs. Improved clinical outcomes will occur once molecular diagnostics and personalized treatments are determined for the different sub-types of this cancer [3, 4]. To date, great effort has been dedicated to the discovery of clinical biomarkers, particularly in oncology, and lncRNAs have been identified as one of the new promising classes of molecules [7, 8]. A previous study investigated the role of lncRNAs, including HOTAIR, UCA1, LET, MEG3, MALAT1, H19 and NAG7, in the biology of HNSCC in addition to their suitability as biomarkers [7]. This study as the first revealed the biological role of lncRNA EGOT in HNSCC. We found that EGOT expression levels depend on tumour grade and location. EGOT is slightly down-regulated in lower grades (1-3) relative to healthy tissue but up-regulated in the case of grade 4. The expression of EGOT is lower in the oral cavity and larynx but higher in the pharynx. Similarly, Xu et al. found that EGOT expression was lower in breast cancer cells compared to non-cancerous samples and varies according to the molecular subtypes of breast cancer. Furthermore, they showed that low EGOT expression levels significantly correlate positively with tumour size, lymph node metastasis and Ki-67 expression. This evidence shows that the down-regulation of EGOT is involved in the progression of more invasive types of cancer [12]. In glioma, the expression of EGOT is significantly lower in the cancer than in the adjacent non-cancerous tissues [11]. An in vitro study determined that the over-expression of EGOT inhibits cell proliferation and migration and promotes cell apoptosis by increasing protein expression levels of caspase-3, caspase-9 and cytochrome c in U251 and U87 glioma cell lines [11].
Similarly, in renal cell carcinoma, the expression of EGOT is down-regulated in tumour samples compared to paired, healthy tissues. In vitro study found that the up-regulation of EGOT expression suppresses proliferation, migration and invasion and induces apoptosis in 786-O and ACHN renal cell lines [24].
Taken together, these results suggest that EGOT serves as a suppressor gene.
Surprisingly, our analysis of clinical pathology parameters in patients with HNSCC indicated that a low expression level of EGOT is observed in the group of patients with lower N-stage, lower grade, with a lymph node neck dissection and with higher age of patients. However, some evidence has shown that EGOT might also have oncogenic properties. For example, a study of samples from patients with gastric carcinoma and an MKN-45 gastric cancer cell line revealed that EGOT is up-regulated in this cancer and high expression levels are associated with lymphatic metastasis and higher TNM stage. Furthermore, down-regulated EGOT expression in vitro results in the inhibition of the hedgehog signalling pathway, cell proliferation and cycle progression arrest in the case of gastric cancer cell line [13].
We observed that patients with high EGOT expression levels have better prognoses (longer DFS and OS) than those to those with low expression. The same association was identified in breast cancer patients: lower EGOT expression levels are indicators of lower OS times [12]. However, the opposite pattern was observed for gastric cancer in which higher EGOT expression was associated with shorter survival times [13].
Analysis of the predicted EGOT targets indicated an association between this lncRNA and mRNAs the of genes connected with many important cellular processes. We analysed the changes in these genes between two groups of patients divided according to expression level of EGOT. Our results showed that the genes that were up-regulated in the high expression group are involved in the regulation of cellular processes (differentiation, adhesion, developmental process, cell communication, signal transduction, division and proliferation), protein phosphorylation and other modifications, cellular component organization, cellular homeostasis, drug response and cell motility. The genes that were down-regulated in this group related to cell cytoskeleton and filaments, localization/binding, cellular transport and protein activity. The changes in these processes have either an indirect or direct influence on the treatment response and survival of patients with HNSCC.
It must also be noted that the location of HNSCC is a crucial clinical factor in terms of treatment strategy and survival prediction. The observed up-regulation of EGOT expression in the pharyngeal cancers is probably due to HPV infection, which is characteristic of the oropharynx (tonsils and base of tongue) but is sometimes also associated with other locations [25]. Indeed, our observations confirmed that EGOT expression is mostly up-regulated in HPV p16 positive HNSCC. It has been shown that the expression of some other lncRNAs is associated with viral infections [15, 26]. It has also been shown that EGOT expression is up-regulated in HPV positive HNSCC [17] but no role and exact mechanism for its involvement in HPV infection was proposed [17]. Carnero et al. found that the high levels of EGOT expression are required for hepatitis C virus replication in samples from patients with hepatocarcinoma (HCC) and in HCC cell lines; moreover, cells with lower EGOT expression produced fewer viral genomes [15]. Cytoplasmatic viral replication is probably required to induce EGOT expression and EGOT level dramatically decreases after viral inhibition in the cells e.g., by exposure to sofosbuvir, daclatasvir or ribavirin [15]. The expression of EGOT is up-regulated in response to pathogen-associated molecular patterns (dsRNA or synthetic analogues and viral RNA) and upon TNFα (tumour necrosis factor α) treatment. TNFα induces NF-kB (nuclear factor-κB), which likely stimulates EGOT expression by binding to its promoter in the case of liver cells [15].
Our analysis suggests that EGOT is involved in the progression of HNSCC. Furthermore, it seems likely that the role of EGOT is connected to HPV infection, given the association between high EGOT expression levels and pharyngeal tumours, younger patients, better DFS and OS and p16 expression, as these are all characteristic of HPV positive HNSCC cases. We supposed that EGOT could be potentially a new biomarker of HPV infection and probably has important role in viral response and biology of HPV positive HNSCC cancers.

Availability of data and materials section

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Raw data are available on the cBioPortal and UALCAN databases.

This work was supported by Greater Poland Cancer Centre – grant no.: 21/2015 (113) and grant no.: 13/2016 (128).
The authors declare no conflict of interest.

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