Chi-square and † Fisher's exact tests.

No associations were found between ANXA9 expression and T and N classifications or tumor recurrence (*p* = 0.91). In addition, ANXA9 expression was not associated with disease-specific survival (log rank *p* = 0.497) nor overall survival (log rank *p* = 0.406) (data not shown).

### *3.3. Expression of ANXA10 in HNSCC Specimens*

Immunohistochemical ANXA10 expression was successfully evaluated in 340 of 372 tumor samples. Positive ANXA10 expression was observed in a total of 219 (64%) cases, mainly detected in the cytoplasm of cancer cells (Figure 1E,F). Furthermore, ANXA9 and ANXA10 expression were significantly correlated (Spearman correlation coefficient 0.459, *p* < 0.001).

Similar to ANXA9, ANXA10 expression was significantly higher in oropharyngeal tumors (*p* = 0.019). Also, ANXA10 expression was significantly associated with the degree of differentiation of the tumors (decreased expression with dedifferentiation, *p* < 0.001, Figure 2B,D). No associations were observed with T and N classifications, disease stage, or tumor recurrence (Table 2). In addition, ANXA10 expression was not associated with either disease-specific (log rank *p* = 0.077) or overall survival (log rank *p* = 0.167).

**Figure 2.** ANXA9 and ANXA10 protein expression in HNSCC specimens according to the degree of differentiation. Representative examples of well-differentiated tumors showing positive expression of ANXA9 (**A**) and ANXA10 (**B**), and poorly differentiated tumors showing negative expression of ANXA9 (**C**) and ANXA10 (**D**) expression in carcinomas. Original magnification ×40.

### *3.4. In Silico Analysis of ANXA9 and ANXA10 mRNA Expression Using The Cancer Genome Atlas (TCGA) HNSCC Data*

In order to extend and confirm our results, we also performed analysis of the transcriptome data from the TCGA HNSCC cohort accessed via the original publication [22], or using the platform cBioPortal for Cancer Genomics (http://cbioportal.org/) [23] and the UALCAN web tools (http://ualcan.path.uab.edu/) [24]. Thus, ANXA9 mRNA levels were found to be significantly decreased in primary tumors compared to normal tissue samples (*p* < 0.001; Figure 3A), whilst ANXA10 mRNA levels increased in tumors versus normal tissue (*p* < 0.001; Figure 3B). These results are in good agreement with our observations at the protein level. In addition, possible correlations between ANXA9 and ANXA10 mRNA expression and the tumor grade were assessed using a homogeneous cohort of 243 HPV-negative HNSCC patients. We found that ANXA9 mRNA levels inversely and significantly correlated with the degree of histological differentiation (Spearman correlation coefficient −0.244, *p* < 0.001; Figure 3C). Consistent with our IHC protein data, ANXA9 mRNA levels were higher in well-differentiated tumors than in moderately and poorly differentiated tumors. However, ANXA10 mRNA levels did not significantly correlate with the tumor grade (*p* = 0.605; Figure 3D).

**Figure 3.** Analysis of ANXA9 and ANXA10 mRNA expression using RNAseq data from the TCGA HNSCC cohorts. Box plots comparing the mRNA expression levels of ANXA9 (**A**) and ANXA10 (**B**) in primary tumors (in red) versus normal tissue (in blue) using UALCAN online resources (http://ualcan.path.uab.edu/). The median, quartiles and range of values are shown. ANXA9 (**C**) and ANXA10 (**D**) expression (RNA seq V2 RSEM, z-score threshold ±2) was analyzed in relation to the tumor grade, categorized as well-differentiated (G1), moderately differentiated (G2) and poorly differentiated (G3) using the TCGA HPV-negative HNSCC cohort (*n* = 243). Horizontal lines (in red) represent the median values, with interquartile range. Sigma (two-tailed) *p*-values.

### **4. Discussion**

Annexins are commonly altered in cancers [9,25]. ANXA9 is a unique member of the annexin family whose intracellular activity does not appear to be regulated by calcium [10,26]. Its closest evolutionary relatives are ANXA1 and ANXA2 [1,4] and members of this clade are thought to function in the organization and regulation of membrane/cytoskeleton linkages [4,27]. As both ANXA1 and ANXA2 have been found down-regulated in head and neck squamous cell carcinoma [28–30], it was of special interest to determine whether ANXA9 showed a similar pattern of expression as this might relate to common features in the evolution, structure and function of these clade members.

We observed a weak membranous ANXA9 expression in the most differentiated cells in normal epithelium. In tumor cells, the expression is mainly membranous, similar to that observed for ANXA2 [28] and the expression of ANXA9 is mainly associated with the degree of differentiation of the tumor, with higher expression in well differentiated cases. This is consistent with elevated ANXA9 observed in differentiating keratinocytes [15]. However, ANXA9 expression was not associated with any other clinical and pathological parameter or with the prognosis in head and neck carcinomas. Analogous findings were obtained by analyzing RNAseq data from the available TCGA HNSCC cohorts. Accordingly, ANXA9 down-regulation was frequently detected in HNSCC at both mRNA

and protein levels. Moreover, ANXA9 mRNA expression in tumors was inversely correlated with the histological differentiation grade, thus confirming our IHC protein data. Hence, together these results reflect that transcriptional regulatory mechanisms contribute to the loss of ANXA9 expression in HNSCC, as we previously demonstrated for the functionally and evolutionary-related members ANXA1 and ANXA2 [29,30].

Few studies have analyzed the expression of ANXA9 in cancers. One study showed that *ANXA9* gene expression is associated with bone metastasis in breast cancer [31]. In colorectal cancer, patients with high *ANXA9* gene expression also had lower overall survival [32]. ANXA9 protein expression in colorectal cancer was higher than in normal mucosa, and associated with invasion and lymphatic metastasis and, consequently, a worse prognosis [13]. These studies suggest a role for ANXA9 in invasion and metastasis, but this role could not be confirmed in head and neck cancers.

Several studies have identified ANXA10 as a tumor suppressor, diagnostic marker, potential therapeutic target, or prognostic factor in various malignancies, including bladder cancer, hepatocellular carcinoma, acute myeloid leukemia, gastric carcinoma, oral squamous cell carcinoma, pancreatobiliary adenocarcinoma, and urothelial carcinoma [33–37]. Studies have shown that ANXA10 was down-regulated in hepatocellular carcinoma and was associated with a poor prognosis [34,35]. ANXA10 has recently been identified as a marker with high specificity for the serrated histology of colorectal cancer [33,38]. The physiological importance of abundant ANXA10 expression specific to the stomach mucosa and intestinal M-cells is currently unknown.

Only one previous study has analyzed ANXA10 in head and neck cancer; Shimizu et al. [17] showed that ANXA10 is overexpressed frequently in oral squamous cell carcinomas and that this overexpression is associated with tumor size. They suggested that ANXA10 expression may be associated with tumor progression by promoting cell-cycle progression in the G1 phase through activation of the ERK/MAPK signaling pathway, leading to decreased expression of cyclin-dependent kinase inhibitors (CDKIs). While further studies are needed to study the interaction of ANXA10 and the ERK/MAPK signaling pathway, these data suggested that ANXA10 plays an important role in cellular proliferation.

We also observed that ANXA10 was not visibly expressed in normal epithelium, while it was variably expressed in the cytoplasm of cancer cells. Consistent with this, analysis of the transcriptome data from the TCGA HNSCC also demonstrated the up-regulation of ANXA10 mRNA expression in tumors compared to the corresponding normal tissue. In addition, we found that ANXA10 expression, as ANXA9, was lower in poorly differentiated tumors, but it was not related to other clinicopathologic parameters or prognosis. However, we were unable to confirm the correlation of ANXA10 protein expression with the histological grade using RNAseq data. Nevertheless, these apparently contradictory results may reflect the contribution of additional regulatory mechanisms (e.g., translational or post-translational) leading to the frequent up-regulation of ANXA10 protein in over 60% of tumor samples.

### **5. Conclusions**

These original results indicate that the expression of annexins A9 and A10 is frequently altered in HNSCC at both mRNA and protein level, suggesting that they could be implicated in the pathogenesis or compensatory mechanisms of these cancers. Additional studies are ongoing to establish the pathogenic roles of these proteins in the progression of squamous cell carcinomas of the head and neck and especially, to determine whether their altered expression is a cause or consequence of the cancerous state. The association of ANXA9 with pathogenic prognosis in colorectal cancer [13] contrasts with a proposed tumor suppressor role for ANXA10 in gastric cancer [36]. The unique, calcium-independent actions of these two annexins may also contribute to a better understanding of their underlying mechanisms. Since these particular annexins are poorly expressed in general but exhibit highly tissue-specific expression, it will undoubtedly be important to explore the role of epigenetic regulatory changes responsible for their selective expression in normal versus cancer tissues. *J. Clin. Med.* **2019**, *8*, 229

**Author Contributions:** Conceptualization, J.P.R. and J.M.G.-P.; formal analysis, C.L. and J.P.R.; funding acquisition, J.P.R. and J.M.G.-P.; Investigation, C.S., S.Á.-T., E.A., L.d.V. and M.S.F.-G.; methodology, M.P.F., R.O.M., E.A. and A.V.; project administration, J.M.G.-P.; resources, M.P.F., R.O.M. and A.V.; supervision, J.M.G.-P.; visualization, J.P.R.; writing—original draft, C.S.; writing—review and editing, M.P.F., R.O.M., C.L., J.P.R. and J.M.G.-P.

**Funding:** This study was supported by grants from the Plan Nacional de I+D+I 2013-2016 ISCIII (PI13/00259), RD12/0036/0015 of Red Temática de Investigación Cooperativa en Cáncer (RTICC), PI16/00280 and CIBERONC (CB16/12/00390 and CB16/12/00228), the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), PCTI-Asturias (GRUPIN14-003), Fundación Bancaria Caja de Ahorros de Asturias-IUOPA and the FEDER Funding Program from the European Union.

**Acknowledgments:** We thank the samples and technical assistance kindly provided by the Principado de Asturias BioBank (PT13/0010/0046), financed jointly by Servicio de Salud del Principado de Asturias, Instituto de Salud Carlos III and Fundación Bancaria Cajastur and integrated in the Spanish National Biobanks Network.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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### *Article*
