*3.4. Correlations between Calumenin, S100A6 and Cytohesin 2*

Calumenin levels were positively correlated with S100A6 levels (r = 0.400, *p* < 0.001), while a positive correlation was observed between S100A6 levels and cytohesin 2 levels (r = 0.262, *p* = 0.02).

#### **4. Discussion**

The results of the current study confirmed circulatory calumenin, S100A6 and cytohesin 2 as biomarkers for SSc patients. To the best of our knowledge, this is the first study that has evaluated circulatory levels of calumenin, S100A6, and cytohesin 2 on an independent cohort of Ssc patients.

Calumenin is a protein that binds calcium being localized in the endoplasmic reticulum. It belongs to a CREC chaperone alongside with Reticulocalbin 1, Cab45, and ERC-55, being involved in protein folding and the secretory pathway. Calumenin was described

to be secreted in the surrounding medium [9]. It has been widely evaluated in various cancers by influencing tumorigenesis and therapeutic response [10]. Increased expression of calumenin was associated with poorer outcome in gliomas [11]. As such, this molecule could play a prognostic role for survival and for the therapeutic response in cancer. Secreted calumenin proved to have autocrine and paracrine effects, being linked to thrombosis and wound healing by modifying the fibroblast phenotype. [12]. Previous data suggest that calumenin is also secreted by activated thrombocytes and is localized in atherosclerotic lesion [13]. Having such an influence upon wound healing via fibroblast activity modulation calumenin could be an interesting and promising biomarker for Ssc patients as it has been previously isolated from biological samples from Ssc patients using mass spectrometry [14]. In this study, calumenin was confirmed as a circulatory biomarker and was associated with diffuse cutaneous fibrosis, inflammation, and low complement level. These data suggests that patients with higher inflammatory activity (that is continuously sustained by complement consumption) associate higher circulatory calumenin levels. However, no association was found between Ssc disease activity score and serum levels of calumenin and between patients with diffuse and localized Ssc. There was also a tendency for higher calumenin levels in patients with pulmonary fibrosis. Although the results did not reach statistical significance, this was most probably due to the relatively small sample size. The regulatory function of calumenin in tissues that are affected by the processes of cytoskeleton rearrangement points out calumenin as a candidate prognostic biomarker in Ssc patients, like has been previously suggested for another member of the CREC family (reticulocalbin 1) [15]. Future prospective studies including early Ssc patients should evaluate the prognostic capacity of circulatory calumenin in Ssc patients.

S100A6 (calcyclin) was extensively evaluated as a biomarker for digestive cancers. Serum S100A6 has a relatively good diagnostic capacity for cholangiocarcinoma [16]. Significant expression of S100A6 was observed also in placenta of women diagnosed with preeclampsia, and its expression is increased in oxidative stress response. [17]. S100A6 was much higher in Ssc patients compared to healthy controls. There were no associations with clinical Ssc features except for higher S100A6 levels in patients with active digital ulcers. Also, serum S100A6 positively correlated with the number of active digital ulcers. These data suggest that S100A6 could be a biomarker associated with more aggressive Ssc pattern. Another striking observation is the fact that S100A6 expression was a few folds higher in Ssc patients compared to controls. S100A6 is linked to the RAGE (Receptor for Advanced Glycation Endproducts) family by interacting with these molecules and acting as cytokines. It seems that S100A6 binding activates the NF-kB pathway and leads to apoptosis [18]. RAGE are known to be associated with various inflammatory cutaneous diseases (both autoimmune and infectious) [19]. RAGE pathway was reported to be involved in Ssc ulcerations development [20]. As a promotor for RAGE pathway, S100A6 could initiate and sustain the inflammatory process that is associated with digital ulceration. Also, very high levels of S100A6 were found in Ssc patients, which points out the fact that this specific molecule could be a potential future therapeutic target at least for prevention of digital ulcer development/aggravation. This hypothesis needs further testing in specific clinical settings on independent Ssc cohorts.

Cytohesin 2 is a guanine-nucleotide exchange factor (GEF) for ARF6 (ADP-ribosylation factor 6). It was previously showed that cytohesin 2 is localized on the plasma membrane and endosome [21]. It is involved in a multitude of signaling and trafficking pathways as it plays a major role in granule secretion in thrombocytes, chromaffin, and pancreatic β-cells [22]. Cytohesin 2 is an activator of both EGF and IGF-1 signaling pathways by direct interaction with the cytoplasmic domains of the activated EGF-R. In vitro studies on colorectal cells pointed out that cytohesin 2 blockade associated with a decreased cell proliferation, migration, and invasion of tumor cells, indicating cytohesin 2 as a potential therapeutic target in colorectal cancer [23]. A recent report also showed that inhibition of cytohesin 2 prevented the morphological changes in vascular smooth muscle cells due to resistin. Cytohesin 2 blockade modified the secretome of the vascular smooth muscle by decreasing matrix metalloproteinase expression [24].

IGF-1 and vascular smooth muscle cells are important pathogenic factors in Ssc, as they also seem to be involved in the progression from preclinical stages of Ssc to early stages [25]. Serum cytohesin 2 levels were increased in Ssc patients, but the prognostic value of these observed changes is not clear. Associations with some cutaneous features of Ssc were pointed out in this study (telangiectasia) alongside with positive correlations with inflammatory markers (especially CRP). Higher cytohesin 2 levels were associated with higher PAP but otherwise no associations with pulmonary Ssc involvement were found, although previous proteomic studies identified cytohesin 2 as a proteomic biomarker from BALF and suggested it as a candidate biomarker for pulmonary involvement in Ssc [14]. This is probably due to the fact the significant changes of cytohesin 2 levels are local rather than systemic. Confirmation studies for cytohesin 2, as a biomarker for IPF from BALF, are needed.

As previous findings pointed out that cytohesin 2 blockade is associated with a more quiescent cellular phenotype, it suggests that cytohesin 2 overexpression could also have a pathogenic effect in Ssc patients. Cytohesin 2 is localized at the crossroad of multiple pathogenic pathways in Ssc patients, and future in vitro studies on fibroblasts derived from Ssc samples are needed in order to evaluate cytohesin 2 blockade effects in Ssc derived cells and define cytohesin 2 as a potential Ssc therapeutic target.

The present study has important limitations. Firstly, the sample size is relatively small, especially for associations between serum levels of the biomarkers and all clinical characteristics of Ssc patients. This was due to the fact that the study was unicentric and Ssc is a rare disease. Secondly, this was only a confirmatory study of the proposed candidate biomarkers, without follow-up and dynamic evaluation of patients in order to validate them as biomarkers in Ssc patients. Thirdly, nailfold capillaroscopy data were not collected for these patients. As such, associations between nailfold capillaroscopy features and biomarkers were not evaluated. Future prospective validation studies on larger Ssc cohorts that also include nailfold capillaroscopy analysis are needed. Prospective studies evaluating the prognostic value of these biomarkers that include Ssc patients with early disease are of great interest.

#### **5. Conclusions**

In conclusion, this study confirmed circulatory cytohesin 2, calumenin and S100A6 as biomarkers in Ssc patients and found associations between the serum levels of these biomarkers and clinical characteristics, especially with severe cutaneous involvement (necrosis and diffuse sclerosis). Among all these biomarkers, calumenin had the best predictive capacity for cutaneous diffuse manifestation, while S100A6 was associated with digital ulcers. Independent confirmation of candidate biomarkers is the first essential step, and once the biomarkers are validated, it will eventually lead to a personalized approach of Ssc patients.

**Author Contributions:** Conceptualization, P.B., C.B. and A.B.; methodology, P.B.; formal analysis, P.B.; investigation, P.B. and E.B. writing—original draft preparation, P.B., A.B. and E.B.; writing review and editing, P.B. and C.B.; supervision, C.B.; project administration, P.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant of theMinistry of Research, Innovation and Digitization, CNCS/CCCDI–UEFISCDI, project number PN-III-P1-1.1-PD-2019-0118, within PNCDI III.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Colentina Clinical Hospital (decision No. 10/11 September 2020).

**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 upon reasonable request from the corresponding author.

**Acknowledgments:** This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI–UEFISCDI, project number PN-III-P1-1.1-PD-2019-0118, within PNCDI III.

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