**4. Discussion**

The pathogenesis of cSCC is complex and includes numerous intrinsic and extrinsic factors [43–45]. Carcinogenesis is a multistep process involving many cells, mediators, and signaling pathways [46,47]. In carcinogenesis, significant structural changes have been described regarding the carbohydrates in the structure of glycoproteins and glycolipids such as a high number of polylactosaminnoglycan chains, multiple branching of asparagine-linked glycans, and increased sialylation [33]. Sialic acid is the outermost monosaccharide unit in the glycan chains of glycolipids and glycoproteins [48] and is ubiquitously distributed in the human body being involved in both physiological and pathological processes. Sialic acid participates in physiological processes such as the transport of positively charged compounds or conformational changes of glycoproteins on cell membranes and mediates cell–cell interactions [21,49]. Elevated levels of sialic acid are associated with resistance to apoptosis and altered cell interactions, promoting cell survival and migration [50]. Abnormal activity of the enzymes involved in sialic acid metabolism or abnormal expression of their corresponding genes leads to aberrant sialylation of glycoproteins or glycolipids [51]. Malignant cell surface glycoproteins and glycolipids display a modified sialic acid composition [39]. Changes in the structure of these glycoproteins and glycolipids may impair processes such as cell recognition, adhesion, or antigenicity. Thus, nowadays sialylation is regarded as one of the major features of the malignant process [21,49]. Aberrant sialylation is the main cancer-associated change of glycosylation process [52,53]. Therefore, it has been suggested that the serum levels of sialic acid and enzymes of sialic acid metabolism may be useful markers in monitoring cancer patients [49]. Parmar et al. have shown higher serum levels of sialic acid in patients with head and neck SCC compared to the control group [38]. Vural et al. have detected increased serum TSA levels in patients with actinic keratoses compared to controls. When they compared the serum TSA levels between AK patients and BCC patients, no differences were obtained [39]. In the current study, we have also detected higher serum levels of TSA in the patient groups compared to controls. However, the serum levels of LSA were significantly higher only when we compared cSCC patients to the control group. In cSCC patients, we have identified higher serum levels of both TSA and LSA compared to AK patients. These results indicate that biochemical changes in glycoproteins start at an early stage of tumorigenesis. High serum levels of LSA are indicative of a premalignant change. Krishnan et al. have shown a positive correlation between TSA level and the grade of dysplasia in patients with oral leukoplakia, but no specific changes regarding LSA levels and the grade of epithelial dysplasia [54].

In various types of cancer, alteration in sialyltransferase activity has been observed [55,56]. ST6GalI seems to be the major sialyltransferase that is overexpressed in malignant tumors [57]. ST6GALI is upregulated in several cancers such as breast, ovarian, and pancreatic neoplasms, being involved in tumor aggressiveness and metastasis [27,57]. Recent studies have demonstrated the role of ST6GALI in the malignant phenotype (growth, survival, angiogenesis, apoptosis, invasion, resistance to cell stress, chemoresistance). Increased ST6GAL1 activity is determined by genetic instability and epigenetic, transcriptional, and posttranslational factors [27,30].

Although there are several studies that identified overexpression of ST6GalI in advanced disease [58,59], a study evaluating patients with oral SCC (OSCC) has revealed ST6GalI overexpression in the early stages of the disease [60]. In line with this, recently, Mehta et al. have analyzed mRNA expression of a group of sialyltransferases, including ST3GALI, ST3GALII, ST3GALIII, ST3GALIV, ST3GALVI, ST6GALI, and the plasma membrane-associated sialidase NEU3, and found down-regulation of these transcripts in OSCC samples compared to adjacent healthy tissue. In normal tissue, they observed increased mRNA levels of sialyltransferases and sialidase NEU3. These results may indicate that alterations of glycosylation are very early events during carcinogenesis. Probably in fact, in the adjacent tumor tissue, although macroscopically normal, a population of cells

with early genetic changes is present. The authors also demonstrated that elevated mRNA levels of ST3GALII and ST3GALIII may be prognostic markers in OSCC [61].

We have detected higher serum levels of ST6GalI in cSCC patients compared to controls, whereas there were no significant differences between AK patients and controls. The soluble form of ST6GalI, possibly secreted by SCC cells, may participate in cancer progression and metastasis. This finding is supported by a previous study that showed high immunohistochemical expression of ST3GalI and ST6GalI in cutaneous epithelial lesions including keratoacanthoma, AK, BCC, and SCC. These results suggest that sialyltransferases are deregulated in skin tumors. No significant differences were found between SCC and AK samples [29]. In our study, we have found higher serum levels of ST6GalI in cSCC compared to AK. These findings show that AK and cSCC have a different pattern of sialylation that is related to tumor behavior. Only a small number of AK suffer malignant transformation [62].

Plzak et al. found that poorly differentiated SCCs are positive for 2,6-linked NeuNAc, whereas differentiated SCCs express 2,3-linked-NeuNAc [63]. The cleavage of 2,6-linked NeuNAc by sialidases allows tumor cells to be recognized by Gal-3, a useful prognostic marker in head and neck SCC. It has been shown that 2,6-NeuNAc is a more potent masking compound than 2,3-NeuNAc with regard to the binding of Gal-3 [63,64]. Melanoma is a very aggressive tumor [65] and a recent study has highlighted the role of ST3GALI in melanoma metastasis [50]. Inhibition of enzyme activity is associated with decreased invasion capacity of melanoma cells and reduced ability to hematogenously disseminate. In this process, the ST3GALI–AXL axis may have a role, where the receptor tyrosine kinase AXL represents the key effector of the ST3GALI pro-invasive function. Modulation of the ST3GAL1–AXL axis may represent a target in melanoma treatment [50].

The plasma membrane-associated sialidase NEU3 is upregulated in various types of cancer including renal, colon and prostate neoplasms [66–68]. In malignant tumors, NEU3 promotes cell survival, migration, and adhesion [69]. NEU3 induces carcinogenesis primarily by altering cell signaling at the cell surface [22]. Recent data have revealed that NEU3 modulates transmembrane signaling through the interaction with various molecules such as caveolin-1, Rac-1, integrin β4, and epidermal growth factor receptor (EGFR) [33]. In colon neoplasms, NEU3 mediates tumor cell proliferation through integrin-mediated signaling, depending on the extracellular matrix. Therefore, NEU3 produces increased adhesion to laminin, promoting cell proliferation and decreased adhesion to fibronectin [70]. On the other hand, in renal cancer, increased levels of NEU3 mRNA have been identified in association with high levels of IL-6, a cytokine that acts as an activator of NEU3, and in turn, NEU3 drives IL-6-mediated signaling via the PI3K/Akt pathway leading to a malignant phenotype characterized by reduced apoptosis and increased cell mobility [67].

Hata et al. suggested that the serum levels of NEU3 are correlated with serum gangliosides, which have been detected in patients with neoplasms [22]. It seems that NEU3 is released from the cell surface in response to the accumulation of gangliosides in serum. It should be noted that NEU3 is an important enzyme for ganglioside degradation [22]. NEU3 acts on gangliosides that are located on the same cell membrane (cis-activity) or on the membrane of neighboring cells (trans-activity), regulating the interactions between cells [71]. The overexpression of *NEU3* and *GD3* synthase genes has been reported in cutaneous melanoma. NEU3 and GM1 and GM2 synthases, involved in ganglioside metabolism, were associated with melanoma cell proliferation and invasion [72]. NEU3 regulates transmembrane signaling through the modulation of ganglioside catabolism. NEU3 hydrolyzes polysialic acid-containing gangliosides to GM1 ganglioside and is involved in the increase in GM1 levels [73]. In addition, in some instances, gangliosides are not efficiently hydrolyzed by plasma membrane sialidases and this explains why gangliosides may accumulate [72].

In our study, we have found higher serum levels of NEU3 in cSCC patients when compared to the control group and as well as when compared to AK patients. The results were similar between AK patients and the control group. The study by Shiga et al. has

revealed that mRNA levels of NEU3 are upregulated in head and neck SCC with lymph nodes metastases compared to normal cells. In addition, the study has shown that NEU3 stimulates cell mobility and invasion. NEU3 increases the phosphorylation of EGFR, which may lead to ERK activation and subsequent release of metalloproteinases (MMP-2 and MMP-9) that promote cell invasion [40]. It has also been shown that NEU3 modulates the phosphorylation of EGFR and its dimerization in HeLa cells, activating the Ras cascade, which will promote cell survival [69].

The results of the present study show that there is an altered degradation rate of sialoglycoconjugates in cSCC patients compared to AK and control groups. Hypersialylation and increased glycoconjugate catabolism are features of patients with cSCC compared to AK patients and controls. Our study has revealed that the ST6GalI/NEU3 ratio is significantly altered in patients with cSCC and could represent a potential molecular target in these patients.

This is necessary to determine markers for SCC diagnosis and management [74]. In our study, the tumor diameter, depth of invasion, and Ki67 were associated with higher levels of TSA and LSA. Inal et al. also found increased levels of LSA that correlated with tumor size in patients with head and neck SCC [75]. There was no correlation between NEU3 and tumor aggressiveness. As mentioned above, NEU3 plays an important role in tumor invasion and metastasis, but metastasis in cSCC is very rare. In line with this, in head and neck SCC, NEU3 has been associated especially with lymph node metastasis [40]

These results support the role of these compounds in tumor progression and invasiveness. A potential role of sialidases as therapeutical targets in the management of cancer has been highlighted. For example, oseltamivir phosphate, an extensively used anti-influenza drug and a viral sialidase inhibitor, targets human NEU1 showing encouraging results in preventing tumor cell metastasis [76,77] The inhibition of NEU1 expression by oseltamivir phosphate leads to overexpression of E-cadherin and downregulation of N-cadherin expression, which may limit the ability of cells to metastasize and increase their sensitivity of antineoplastic drugs [78].

Monitoring serum levels of total sialic acid, lipid-bound sialic acid, sialyltransferases, and sialidases in correlation with tumor characteristics may have useful clinical applications for the diagnosis of cSCC. Future research is required to find reliable markers for the management of cSCC.

## **5. Conclusions**

The current study reveals aberrant sialylation in cSCC patients, but not in AK patients. We have found a significant increase in serum levels of TSA, LSA, ST6GalI, and NEU3 in the cSCC group compared to the control group, whereas in patients with AK only the serum level of TSA was significantly higher compared to the control group. When the cSCC and AK groups were compared, significant differences between the serum levels of TSA, LSA, ST6GalI, and NEU3 were found. In conclusion, the serum ST6GalI/NEU3 level may represent a potential molecular factor to distinguish cSCC patients from noncancer patients, pending further validation. Further studies are needed to understand sialylation-related changes in cSCC, supporting further improvements in the diagnostic and treatment approach.

**Author Contributions:** All authors have equally contributed to the writing and editing of the manuscript. Conceptualization, C.D.E. and I.N.; methodology, I.N.; software, C.D.E.; validation, C.D.E., I.N., S.R.G. and C.E.; formal analysis, I.N. and C.E.; investigation, C.D.E.; resources, M.T., S.R.G. and C.M.; data curation, C.D.E. and S.R.G.; writing—original draft preparation, C.D.E., C.I.M. and M.I.M.; writing—review and editing, C.D.E., C.M. and M.T.; visualization, S.R.G., C.I.M., M.I.M. and C.M.; supervision, S.R.G., I.N. and M.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** The article processing charges were funded by Carol Davila University of Medicine and Pharmacy.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the "Victor Babe¸s" Clinical Hospital for Infectious Diseases, Bucharest (3/10.03.2018).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

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