Low let-7d and high miR-205 expression levels positively influence HNSCC patient outcome

Introduction Head and neck squamous carcinoma (HNSCC) is one of the most invasive types of cancer with high mortality. A previous study has indicated that low levels of let-7d and miR-205 in HNSCC patients are correlated with poor survival. Let-7d and miR-205 are tumor suppressors and regulators of epithelial-to-mesenchymal transition (EMT). However, it is unclear if let-7d and miR-205 together influence cancer cells. Aim To determine if let-7d and miR-205 expression levels influence HNSCC patient outcome. Methods The TCGA expression data for let-7d, miR-205 and their targets as well as clinical data were downloaded from cBioPortal and starBase v2.0 for 307 patients. The expression levels of let-7d and miR-205 were verified according to clinicopathological parameters. The let-7d and miR-205 high- and low-expression groups as well as disease-free survival (DFS), overall survival (OS) and expression levels of genes related to EMT, cancer stem cells, metastasis, cell cycle, drug response and irradiation response were investigated. Results Let-7d and miR-205 were frequently upregulated in HNSCC compared to normal samples, and ROC analysis showed high discrimination ability for let-7d and miR-205 (area 0.7369 and 0.7739, respectively; p < 0.0001). Differences between expression levels of let-7d or miR-205 and grade, angiolymphatic invasion, perineural invasion and alcohol consumption were indicated. No differences were observed in N-stage, tumor localization, gender or patient age. Patients with lower let-7d levels and higher miR-205 levels had significantly better OS (p = 0.0325) than patients with higher let-7d levels and lower miR-205 levels. In the low let-7d level and high miR-205 level group, a lower percentage of more advanced cancers was observed. The analysis of genes related to EMT, cancer stem cells, metastasis, cell cycle, drug response and irradiation response revealed a distinct phenotype of analyzed groups. Conclusions The present findings indicated that let-7d down-regulation and miR-205 overexpression create a unique cell phenotype with different behavior compared to cells with upregulated let-7d and down-regulated miR-205. Thus, let-7d and miR-205 are good candidates for new HNSCC biomarkers.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is characterized by high mortality and difficult to treat type of cancer. The main treatment of HNSCC involves surgical resection, radiotherapy and chemotherapy [1,2]. Many reports have shown a close connection between HNSCC and miRNAs [3]. miRNAs are RNAs that are 22 nucleotides long and function as posttranscriptional regulators of specific mRNA by targeting the 3' UTR of mRNA, resulting in reduced expression of the encoded proteins. These small RNAs regulate many biological processes, such as cell cycle, apoptosis, EMT, cancer-initiating cells, metastasis, drug response and irradiation response. The implication of miR-NAs in HNSCC development and progression is well documented. miRNAs may function as tumor suppressors and/or oncogenes [3,4], and they may be used as potential biomarkers in oncology [5,6]. Childs et al. proposed that low expression levels of let-7d and miR-205 are prognostic markers of HNSCC [7], but other reports have not confirmed this finding.
Expression of lethal-7d (let-7d) is dysregulated in many types of cancer, such as HNSCC, breast cancer, kidney cancer, retinoblastoma, pancreatic cancer and prostate cancer [4,8]. Let-7d plays a crucial role in cancer development, progression and metastasis, and it acts as a tumor suppressor miRNA through regulating the expression of many oncogenes [4]. However, recent studies have indicated that let-7d also acts as an oncogene [9,10]. Let-7d regulates the response to irradiation and drug exposure through changes in cell phenotype via the EMT process or by changes in multidrug resistant genes [11][12][13].
MiR-205 is altered in many cancers, including breast cancer, melanoma, renal cancer, glioblastoma, lung cancer and HNSCC [6,7,14,15]. MiR-205 may function as a tumor suppressor, and it has been described as an epithelial marker [16,17] and a modulator of the EMT process [15]. In contrast, miR-205 functions as an oncogene in breast cancer, cervical cancer and nasopharyngeal carcinoma [18,19].
The present study analyzed the expression of let-7d and miR-205 in HNSCC patients based on TCGA data. The aim of the present study was to investigate the influence of let-7d and miR-205 expression levels on HNSCC patient outcome.

TCGA data
TCGA expression data of let-7d, miR-205 and selected genes (cell cycle, apoptosis, EMT, cancer-initiating cells, metastasis, irradiation response and drug response) as well as clinical data were downloaded from cBioPortal (head and neck squamous cell carcinoma, TCGA, Provisional, 530 sample data set) and starBase v2.0 for 307 patients and presented as miRNAseq RPM (Reads Per Million) and mRNA expression z-Scores (RNA Seq V2 RSEM; z = 2, RNA-Seq by Expectation Maximization) [20,21]. All data is available online with common access.

Data analysis
The expression levels of let-7d and miR-205 were analyzed in all HNSCC sample localizations depending on the following clinicopathological parameters: age (< 60 vs. > 60), gender (women vs. men), T-stage (T1 + T2 vs. T3 + T4), N-stage (N0 vs. N1 + 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). The group of 307 patients was divided into high and low expression subgroups for let-7d and/or miR-205. The following groups were created based on cut-off values for miRNA expression of healthy samples: i) high let-7d + low miR-205 (N = 24 cases); ii) high let-7d + high miR-205 (N = 217 cases); iii) low let-7d + low miR-205 (N = 12 cases) and iv) low let-7d + high miR-205 (N = 54 cases). Disease free-survival (DFS) and overall survival (OS) were analyzed in the four subgroups. The selected clinicopathological parameters (cancer grade, cancer stage, T-stage, perineural invasion, angiolymphatic invasion and disease surgical margin status) were compared between the low let-7d level and high miR-205 level group and the high let-7d level and low miR-205 level group. The expression levels of selected genes related to cell cycle, apoptosis, EMT, cancer-initiating cells, metastasis, drug response and irradiation response were analyzed. Let-7d and miR-205 predicted targets were verified using the miRDB database [22] and target genes were compared between patients with the low let-7d level and high miR-205 level group and the high let-7d level and low miR-205 level group.

Statistical analysis
All statistical analyses were performed by Graphpad Prism 5 (GraphPad Software San Diego, CA, USA) using the Shapiro-Wilk normality test, T-test, Mann-Whitney U test or one-way ANOVA with Dunn's multiple comparison test. For DSF and OS analyses, the log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used. In all analyses, a p-value < 0.05 was considered as significant. The heat map and clustering were generated using MORPHEUS, an online versatile matrix visualization and analysis software tool (https://software.broadinstitute.org/morpheus/).
The correlations between let-7d and miR-205 were also investigated. For normal samples, Spearman correlation analysis indicated no association between the let-7d and miR-205 miRNAs (R = − 0.1762, p = 0.2769).

Expression levels of let-7d and miR-205 influence patient survival
The disease-free survival (DFS) and overall survival (OS) of HNSCC patients defined after 36 months of observation were analyzed in groups of different expression levels of let-7d and miR-205. There were no statistically significant differences between groups of patients with specified low and/or high expression levels of let-7d and miR-205. However, patients with low let-7d levels and high miR-205 levels had significantly better OS than patients with opposite phenotype -high let-7d levels and low miR-205 levels (p = 0.0325). These results are shown in Fig. 2a and b. Due to differences in survival between these groups, it was checked whether they differ in terms of other clinical features.
Patients with low let-7d and high miR-205 expression levels have better prognosis The clinicopathological parameters were analyzed in the low let-7d and high miR-205 expression level group and the high let-7d and low miR-205 expression level group. The low let-7d and high miR-205 expression level group had lower percentages of more advanced cancers compared to the high let-7d and  Fig. 3.

Patients with low let-7d and high miR-205 expression level have a distinct tumor phenotype
For the high let-7d and low miR-205 expression level group (let-7d H + miR-205 L) and the low let-7d and  Groups were described in the material and methods section; p < 0.05 was considered significant, nsno significant, * p < 0.05 high miR-205 expression level group (let-7d L + miR-205 H), the expression levels of genes related to metastasis, EMT, cancer-initiating cells, cell cycle, apoptosis, irradiation response and drug response were examined and compared between these two groups. A distinct expression pattern of these genes was observed for each group. The let-7d L + miR-205 H group showed the following statistically significant (p < 0.05) changes compared to the let-7d H + miR-205 L group: 29 down-regulated and 6 upregulated genes related to metastasis; 31 down-regulated and upregulated 8 genes related to EMT; 28 down-regulated and 5 upregulated genes related to cancer-initiating cells; 17 down-regulated and 4 upregulated genes related to cell cycle; 24 down-regulated and 10 upregulated genes related to apoptosis; 32 down-regulated and 5 upregulated genes related to irradiation response; and 44 down-regulated and 9 upregulated genes related to drug response (Figs. 4 and 5). Next, target genes for let-7d and miR-205 were selected from the studied processes using the miRDB database. Twelve targets, including 11 upregulated targets, were identified for let-7d (patients with high let-7d vs. low let-7d) in the set of genes related to metastasis, EMT, cancer-initiating cells, cell cycle, apoptosis, irradiation response and drug response. The following targets were upregulated: DAPK1 0.3994 ± 0.1293; p < 0.0001), which is related to cell cycle arrest (Fig. 6a). The following three molecular targets were identified for miR-205 (low miR-205 vs. high miR-205 expression): LTA (0.1778 ± 0.1251 vs. -0.2701 ± 0.07117; p = 0.0014), which is a positive regulator of apoptosis; ZEB1 (1.294 ± 0.2672 vs. -0.05386 ± 0.1134; p < 0.0001), which is a regulator of cell migration, metastasis, growth and proliferation; and CDH11 (1.092 ± 0.2816 vs. -0.1691 ± 0.09423; p < 0.0001), which is related to cell adhesion (Fig. 6b).

Discussion
Head and neck squamous cell carcinoma (HNSCC) is a group of poor outcome cancers due to late diagnosis, aggressiveness and high capability of metastasis to lymph nodes and distant sites [1,2,23]. HNSCC treatment is based on surgery, chemotherapy and radiotherapy, but these treatment strategies are still not personalized and not matched to molecular features of every patient's cancer [2,5]. Moreover, HNSCCs contain a small The epithelialto-mesenchymal (EMT) process is one of the factors responsible for maintenance of this cell phenotype as well as metastasis or poor response to irradiation, chemotherapeutic drugs or other agents [24]. This cell phenotype and behavior are, in part, controlled by miR-NAs [3].
Childs et al. proposed that low level miR-205 is associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. Moreover, these researchers postulated that combination of low levels of let-7d and miR-205 expression is associated with poor survival and that these two miRNAs can be used as prognostic markers of HNSCC [7]. However, these results have not been validated using different/larger cohort of patients and the molecular basis of this observation has not been elucidated. The present study used TCGA data and analyzed 307 patients with HNSCC to investigate the diagnostic utility of let-7d and miR-205. The present results contradicted those reported by Childs and colleagues. In the present study, patients with low let-7d and high miR-205 levels had significantly better survival than patients with opposite phenotype -high let-7d and low miR-205 levels. Due to differences in survival between these groups, it was checked whether they differ in terms of other clinical features. We observed, that in the low let-7d and high miR-205 expression level group, there was a lower percentage of more advanced cancers.
It should be noted, that unexpectedly, the small group of patients with low let-7d and miR-205 expression levels was characterized by the best survival. However, these patients comprised 4% of all analyzed cases and due to the small number of cases, this group was not further analyzed by us.
Our analysis confirmed that both let-7d and miR-205 were frequently upregulated or remained unchanged in HNSCC. Other studies have also indicated that let-7d and miR-205 are upregulated in HNSCC [6] and in other cancers [4,18,19].
We observed, that patients with higher levels of let-7d and lower levels of miR-205 had worse outcome and more advanced cancers than the group with low let-7d and high miR-205 expression levels.  5 Heat map and clustering of significantly changed (p < 0.05) expression levels of genes related to cell cycle, apoptosis, irradiation response and drug response (mRNA expression z-Scores, RNA Seq V2 RSEM; z = 2). Patients belong to the let-7d high and miR-205 low expression group (let-7d H + miR-205 L) marked as green and patients belong to the let-7d low and miR-205 high expression group (let-7d L + miR-205 H) marked as red More advanced tumors (higher N-stage and grade) that generally metastasize to blood and lymph vessels had higher levels of let-7d. Similarly, Hilly et al. found that higher expression of let-7d (and other members of the let-7 family) is associated with more aggressive oral tongue carcinoma in young patients [25]. Based on an in vitro model, Di Fiore et al. demonstrated that let-7d also acts as an oncogene [10]. Our previous study using a FaDu cell line (squamous cell carcinoma of the hypopharynx) with upregulated let-7d expression showed that higher levels of let-7d causes more irradiation-resistant cells but not chemotherapeutic drug-resistant cells [9].
In contrast, down-regulation of let-7d promotes EMT and higher self-renewal of cancer-initiating cells as well as influences the chemoresistant property of oral cancer cells [13]. However, the present results based on TCGA data indicated that let-7d can act as oncogene.
MiR-205, the second analyzed microRNA may function as a tumor suppressor or an oncogene [18,19]. miR-205 is an epithelial marker [16,17], and it is a well known modulator of the EMT process [15]. Jamali et al. performed a meta-analysis and showed that down-regulation of miR-205 is associated with poor prognosis in HNSCC [26]. Our results demonstrated that high expression of Fig. 6 Expression of predicted targets for let-7d (A) and miR-205 (B) in HNSCC patient groups with high let-7d and low miR-205 expression levels (let-7d H + miR-205 L) and low let-7d and high miR-205 expression levels (let-7d L + miR-205 H); unpaired T-test; mean of mRNA expression z-Scores (RNA Seq V2 RSEM; z = 2) with SEM; * p < 0.05; ** p < 0.01; *** p < 0.001 miR-205 was related to less aggressive tumors, less metastasis to blood, less metastasis to lymph vessels and lower ability of perineural invasion. The present findings also indicated that patients with high miR-205 expression had no dissection of neck lymph nodes.
Clinical and pathological parameters indicate specific, different phenotypes between these groups. To check this, we decided to analyze some important processes such as: cell cycle, apoptosis, EMT, cancer-initiating cells, metastasis, irradiation response and drug response between these groups. Next, we checked the role of let-7d and miR-205 in these processes. We observed differences in the expression of genes associated with the analyzed processes between patients with higher levels of let-7d and lower levels of miR-205 than the group with low let-7d and high miR-205 expression levels.
In the group of patients with low let-7d and high miR-205, the analysis of predicted targets for let-7d showed that the predicted targets, with the exception of CDKN1A (p21), were down-regulated and that these genes were related to apoptosis, EMT, cell cycle, cell proliferation and DNA damage and repair. Llanos et al. indicated that HNSCC patients with p21-and phosphoS6-positive tumors present a better disease-specific survival [27]. Moreover, patients with p21-positive tumors have better overall survival than those with p21-negative tumors [28]. However, immunohistochemical staining of p21 and Ki-67 has shown that coexpression of p21/Ki-67 is a strong negative prognostic factor in HNSCC and may be important in patients treated by primary radiotherapy [29]. p21 can function as a suppressor or oncogene, and it is an example of an antagonistic duality molecule, in which function is determined by particular cellular context [30]. In contrast, the present results suggested that let-7d influences a specific phenotype of HNSCC through direct regulation of CDKN1A and results in better patient survival.
In the analyzed processes we observed three miR-205 targets -LTA, ZEB1 and CDH11, which were down-regulated in the group of patients with low let-7d and high miR-205 compared to the group of patients with high let-7d and low miR-205 expression levels. The LTA gene-encoded tumor necrosis factor-beta (TNF-β), which is a member of the large family of cytokines, is down-regulated in HNSCC compared to normal samples, but TGF receptors RI and RII are upregulated in HNSCC [31]. Buhrmann et al. reported that TNF-β increases the capacity of cell survival and invasion as well as positively influences the EMT process and maintenance of the cancer-initiating cell-like phenotype in colon cancer. Moreover, TNF-β promotes the chemoresistance of colon cancer cells to 5-FU [32].
Based on the HNSCC samples, the high expression of ZEB1 and ZEB2 was associated with advanced and metastatic cancer, and high levels of ZEB1 and ZEB2 predicted poor outcome. ZEB1 and ZEB2 are key modulators of cancer-initiating cell properties in HNSCC, EMT process, metastasis and cisplatin resistance [33]. Kurihara et al. showed that BMI1 and ZEB1 are important factors for promotion of EMT and invasion of tongue cancer. Expression of ZEB1 positively correlates with vimentin, but no significant correlation between ZEB1 and E-cadherin has been identified [34]. The present analysis indicated that the group of patients with better survival had lower levels of cadherin-11 (CDH11). Ma et al. showed that the expression of CDH6, CDH11 and CD44 is upregulated in cancer tissue compared to normal mucosa and is highly increased in OSCC patients with lymph node metastasis. Moreover, OSCC patients with high coexpression of CDH6, CDH11 and CD44 exhibit lower disease-specific survival time, and these molecules may be used as biomarkers of metastases and prognosis [35]. In addition, downregulation of CDH11 promotes proliferation and enhances invasion, and CDH11 has tumor suppressor function. However, overexpression of CDH11 is cancer-specific [36].

Conclusion
In conclusion, the present study clearly shows that patients with low let-7d and high miR-205 expression levels had less advanced cancers and better survival compared to patients with high let-7d and low miR-205 expression levels. These results contrasted the model presented by Childs and colleagues [7], but the present observations were based on a larger group of patients and were consistent with other studies that describe let-7d as an oncogene [9,10]. Down-regulation of let-7d and overexpression of miR-205 create a unique cell phenotype with different behavior that influences patient survival compared to cells with upregulated let-7d and down-regulated miR-205. Thus, let-7d and miR-205 are responsible for the observed phenomenon by direct regulation of CDKN1A (let-7d) and by regulation of LTA, ZEB1 and CDH11 (miR-205). However, other mechanisms may better explain the observed results. In conclusion, let-7d and miR-205 are good candidates for new biomarkers in the personalization of HNSCC treatment.