Gene Profiling in Patients with Systemic Sclerosis Reveals the Presence of Oncogenic Gene Signatures

Systemic sclerosis (SSc) is a rare connective tissue disease characterized by three pathogenetic hallmarks: vasculopathy, dysregulation of the immune system, and fibrosis. A particular feature of SSc is the increased frequency of some types of malignancies, namely breast, lung, and hematological malignancies. Moreover, SSc may also be a paraneoplastic disease, again indicating a strong link between cancer and scleroderma. The reason of this association is still unknown; therefore, we aimed at investigating whether particular genetic or epigenetic factors may play a role in promoting cancer development in patients with SSc and whether some features are shared by the two conditions. We therefore performed a gene expression profiling of peripheral blood mononuclear cells (PBMCs) derived from patients with limited and diffuse SSc, showing that the various classes of genes potentially linked to the pathogenesis of SSc (such as apoptosis, endothelial cell activation, extracellular matrix remodeling, immune response, and inflammation) include genes that directly participate in the development of malignancies or that are involved in pathways known to be associated with carcinogenesis. The transcriptional analysis was then complemented by a complex network analysis of modulated genes which further confirmed the presence of signaling pathways associated with carcinogenesis. Since epigenetic mechanisms, such as microRNAs (miRNAs), are believed to play a central role in the pathogenesis of SSc, we also evaluated whether specific cancer-related miRNAs could be deregulated in the serum of SSc patients. We focused our attention on miRNAs already found upregulated in SSc such as miR-21-5p, miR-92a-3p, and on miR-155-5p, miR 126-3p and miR-16-5p known to be deregulated in malignancies associated to SSc, i.e., breast, lung, and hematological malignancies. miR-21-5p, miR-92a-3p, miR-155-5p, and miR-16-5p expression was significantly higher in SSc sera compared to healthy controls. Our findings indicate the presence of modulated genes and miRNAs that can play a predisposing role in the development of malignancies in SSc and are important for a better risk stratification of patients and for the identification of a better individualized precision medicine strategy.

Systemic sclerosis (SSc) is a rare connective tissue disease characterized by three pathogenetic hallmarks: vasculopathy, dysregulation of the immune system, and fibrosis. A particular feature of SSc is the increased frequency of some types of malig nancies, namely breast, lung, and hematological malignancies. Moreover, SSc may also be a paraneoplastic disease, again indicating a strong link between cancer and scleroderma. The reason of this association is still unknown; therefore, we aimed at investigating whether particular genetic or epigenetic factors may play a role in promo ting cancer development in patients with SSc and whether some features are shared by the two conditions. We therefore performed a gene expression profiling of peripheral blood mononuclear cells (PBMCs) derived from patients with limited and diffuse SSc, showing that the various classes of genes potentially linked to the pathogenesis of SSc (such as apoptosis, endothelial cell activation, extracellular matrix remodeling, immune response, and inflammation) include genes that directly participate in the development of malignancies or that are involved in pathways known to be associ ated with carcinogenesis. The transcriptional analysis was then complemented by a complex network analysis of modulated genes which further confirmed the presence of signaling pathways associated with carcinogenesis. Since epigenetic mechanisms, such as microRNAs (miRNAs), are believed to play a central role in the pathogenesis of SSc, we also evaluated whether specific cancerrelated miRNAs could be dereg ulated in the serum of SSc patients. We focused our attention on miRNAs already found upregulated in SSc such as miR215p, miR92a3p, and on miR1555p, miR 1263p and miR165p known to be deregulated in malignancies associated to SSc, i.e., breast, lung, and hematological malignancies. miR215p, miR92a3p, miR 1555p, and miR165p expression was significantly higher in SSc sera compared to healthy controls. Our findings indicate the presence of modulated genes and miRNAs that can play a predisposing role in the development of malignancies in SSc and are important for a better risk stratification of patients and for the identification of a better individualized precision medicine strategy. inTrODUcTiOn Systemic sclerosis (SSc) is a rare, chronic disease characterized by three pathogenetic hallmarks: vasculopathy, dysregulation of the immune system, and increased extracellular matrix deposition in the skin and internal organs, leading to extended fibrosis and to a remarkable heterogeneity in clinical features and course of the disease, resulting in high morbidity and mortality (1). Several environmental factors alongside genetic susceptibility and epigenetic mechanisms contribute to the onset of the disease (2)(3)(4)(5).
Increased frequency of a few types of cancer, namely breast, lung, and hematologic malignancies in SSc has been reported (6), and it seems to be associated with the presence of particular autoantibodies (7). Moreover, it is worthwhile mentioning that SSc may also be a paraneoplastic disease (8,9), indicating a strong link between cancer and scleroderma.
Given the high incidence of tumors in patients with SSc, we wanted to assess whether this phenomenon may be supported by a particular gene modulation that can favor cancer development in patients with SSc. Thus, we analyzed transcriptional profiles of peripheral blood mononuclear cells (PBMCs) obtained from patients with SSc to evaluate the presence of modulated genes that are involved in signaling pathways associated with malignancy. Moreover, through a complex network analysis, we verified which pathways could play a pivotal role in cancer development in SSc patients.
Besides environmental and genetic factors, epigenetic mechanisms, such as microRNAs (miRNAs), are believed to play a central role in the pathogenesis of the disease.
microRNAs are a class of small non-coding RNAs that bind 3' untranslated region of messenger RNAs (mRNAs), negatively regulating target gene expression by either the repression of translation or degradation of target mRNAs (10). miRNAs are considered an important class of epigenetics regulators in many basic cellular processes as well as in the vast majority of diseases, including cancer and autoimmunity (11,12). Little is known about the role of dysregulated expression of miRNAs in the pathogenesis of SSc (13). An altered expression of profibrotic and/or antifibrotic miRNAs has been suggested as an important factor in the development of fibrosis in SSc patients (14). Moreover, increased evidence suggests that serum miRNA levels may be promising biomarkers for the diagnosis, prognosis, and therapeutic approach in SSc (15). Serum levels of miR-21 and miR-92a have been found significantly higher in SSc samples compared to normal controls (16,17). Their function is implicated in inflammation, in regulation of immune cells (18) and in favoring fibrosis (16,17).
Since particular miRNAs have been associated with malignancies, we aimed at evaluating whether transcriptional profiles and dysregulation of particular miRNAs are shared by cancer and SSc.

Patients
We enrolled 30 patients affected by SSc, attending the Unit of Autoimmune Disease at the University Hospital of Verona, and 30 sex and age matched healthy controls. All patients fulfilled the ACR/Eular classification criteria for SSc (19). The distinction between limited (lSSc) and diffuse cutaneous SSc (dSSc) was performed according to the criteria proposed by LeRoy et al. (20). Fifteen of the 30 patients were affected by lSSc and 15 by dSSc. The clinical and demographic features of the patients enrolled in the study are summarized in Table 1.
Samples obtained from 10 patients with lSSc, 10 patients with dSSc, and 10 healthy controls were used for the gene expression analysis, whereas samples obtained from all the patients and controls were used for the microRNA study. Blood samples were collected from patients with active disease and in the absence of immunosuppressive therapies.
A written informed consent was obtained by all the participants to the study and the study protocol was approved by the Ethical Committee of the Azienda Ospedaliera Universitaria Integrata di Verona. All the investigations have been performed according to the principles contained in the Helsinki declaration.

gene expression analysis
Blood samples were collected in BD Vacutainer K2EDTA tubes using a 21-gage needle. PBMC were obtained upon stratification on Lympholyte ® cell separation density gradient (Cedarlane, Burlington, Canada). Total RNA extraction from PBMC was performed with miRNeasy mini kit following manufacturer's protocol (Qiagen GmbH, Hilden, Germany). cRNA preparation, samples hybridization, and scanning were performed following the Affymetrix (Affymetrix, Santa Clara, CA, USA) provided protocols, by Cogentech Affymetrix microarray unit (Campus IFOM IEO, Milan, Italy). All samples were hybridized on Human Clariom D (Affymetrix) gene chip and were analyzed using the Transcriptome Analysis Console (TAC) 4.0 software (Applied Biosystem, Foster City, CA USA by Thermo Fisher Scientific, Waltham, MA, USA). The Signal Space Transformation (SST)-Robust Multi-Array Average algorithm (RMA) were applied to background-adjust, normalize, and log-transform signals intensity.
Relative gene expression levels of each transcript were vali dated applying a one-way analysis of variance (ANOVA) (p ≤ 0.01) and multiple testing correction. Genes that displayed an expression level at least 1.5 fold different in the test sample versus control sample (p ≤ 0.01) were submitted both to functional classification, using the Gene Ontology (GO) annotations, and to Pathway analysis, employing the Panther expression analysis tools 1 (21). The enrichment of all pathways and functional classes associated to the differentially expressed genes compared to the distribution of genes included on the Clariom D microarray was analyzed and p values ≤0.05, calculated by the binomial statistical test, were considered as significant enrichment.

Protein-Protein interaction (PPi) network construction and network clustering
The Search Tool for the Retrieval of Interacting Genes (STRING version 10.5 2 ) is a web-based database which comprises experimental as well as predicted interaction and covers >1,100 completely sequenced organisms (22). DEGs were mapped to the STRING database to detect protein-protein interactions (PPI) pairs that were validated by experimental studies (23) and the corresponding PPI network was constructed. A score of ≥0.7 for each PPI pair was selected to design the PPI network.
To detect high-flow areas (highly connected regions) of the network, we used the CFinder software tool, based on the Clique Percolation Method (CPM) (24). Finally, Cytoscape software (25) was used to define the topology of the built networks.

cell-Free microrna expression in ssc sera
To isolate cell-free circulating miRNA (cf-miRNA) in serum, we used miRNeasy serum/plasma kit (Qiagen, GmbH, Hilden, Germany). For all the samples, RNA was extracted from 200 µL of serum in accordance with manufacturer's instructions. In order to minimize the technical variation between samples in downstream PCR analysis we added, for all isolations, 0.90 fmol of spike-in non-human synthetic miRNA cel-39-3p into the sample after the addition of the lysis/denaturant buffer to the serum. After extraction, RNA was eluted in 14 µL of nuclease-free water.

resUlTs gene array analysis
In order to gain new insights into the pathogenesis of SSc, we compared the gene expression profiles of PBMC samples obtained from 10 lSSc and 10 dSSc patients with 10 PBMC samples obtained from age and sex matched healthy donors. Raw data have been deposited in the ArrayExpress database at EMBL-EBI 3 under accession number E-MTAB-6531.
When we analyzed lSSc samples, we found that 829 differently expressed genes (DEGs) satisfied the Bonferroni-corrected p value criterion (p ≤ 0.01) and the fold change criterion (FC ≥ |1.5|), displaying robust and statistically significant variation between SSc and healthy controls samples. In particular, 736 and 93 transcripts resulted to be over-and underexpressed, respectively. The complete list of modulated genes can be found in Table S1 in Supplementary Material.
When the same Bonferroni-corrected p value and fold change criteria were used, 456 transcripts were differentially modulated in dSSc patients compared to healthy controls, 327 and 129 transcripts resulted to be up-and downregulated, respectively (Table  S2 in Supplementary Material).
The Gene Ontology analysis of modulated genes in lSSc patients, showed that a large number of the modulated transcripts can be ascribed to biological processes that may play a role in SSc, including: apoptotic process, cell proliferation, growth factor and growth factor binding, inflammatory response, immune response, angiogenesis, endothelial cell activation, cell adhesion, and extracellular matrix organization process.
Moreover, a large number of modulated genes were ascribed to well-known signaling pathways encompassing Type I interferon, epidermal growth factor (EGF) receptor, transforming growth factor (TGF)-beta, interleukin, Wnt, glycolysis, platelet derived growth factor (PDGF), FAS, and Phosphoinositide 3 (PI3)-kinase signaling pathway. Table 2 shows a selection of DEGs within the above-mentioned categories and also includes public gene accession numbers and fold changes.
Interestingly, all these molecules have been previously associated to different type of cancer (27)(28)(29)(30). Moreover, we found downregulation for cullin 3 (CUL3) a tumor suppressor molecule (31). All these observations may suggest that a dysregulation of proliferative genes in patients with SSc may predispose to the development of malignancies.
We also found upregulation of growth factors encoding molecules such as platelet-derived growth factor alpha polypeptide (PDGFA), bone morphogenetic factor 6 (BM6), and myeloid differentiation primary response 88 (MYD88) a fibrogenic molecule that controls pericyte migration and trans-differentiation of endothelial cells to myofibroblasts (32).
The initial stages of SSc are accompanied by an angiogenic response to tissue ischemia and vascular damage, that is later replaced by a deficient wound healing and by fibrosis (36). Indeed, several genes involved in angiogenesis were upregulated in lSSc samples including calcium and integrin binding 1 (CIB1); integrin beta 3 (ITGB3), platelet-derived growth factor alpha polypeptide (PDGFA), and protein kinase C, alpha (PRKCA). Interestingly, PDGF stimulates tumor cells, promotes angiogenesis, and the development of cancer associated fibroblasts (37) leading to tumor progression. In addition, aberrant expression of PKCA is associated with a range of malignancies and has recently become a target for anti-cancer therapies (38).
The upregulation of genes such as chemokine (C-X-C motif) ligand 10 (CXCL10), forkhead box P1(FOXP1), protein arginine methyltransferase 5(PRMT5), and selectin P (SELP) is consistent with the endothelial cells activation that is typical of SSc and is accompanied by the overexpression of cell adhesion molecules involved in endothelial cells and leukocytes interactions as well as in blood cells extravasation. We indeed found upregulation for intercellular adhesion molecule 2 (ICAM2), protocadherin 9 (PCDH9), integrin beta 3 (ITGB3), integrin beta 7 (ITGB7), and CD151 molecule that, importantly, is an emerging possible poor prognostic factor for solid tumors (39).
Systemic sclerosis is characterized by connective tissue fibrosis of skin and internal organs that is sustained by extracellular matrix (ECM) remodeling (40). Accordingly, we found an increased expression of transcripts that play a role in ECM organization including laminin gamma 1 (LAMC1), collagen, type XIX (COL19A1), bone morphogenetic protein 6 (BMP6), cystatin C (CST3), and secreted protein, acidic, cysteine-rich (SPARC), a secreted protein that is overexpressed in the fibroblasts of skin biopsy from patients with SSc (41). Noteworthy, remodeling of the stromal ECM by cancer-associated fibroblasts is crucial for tumor cell migration and invasion (42) and SPARC has been associated to these events (43). Interestingly, an elevated preoperative CST3 level was demonstrated to be related with worse survival in patients with renal cell carcinoma (44).
The pathway enrichment analysis that we performed to find signaling network that were overrepresented by modulated genes in lSSc samples, showed an enrichment in apoptosis, glycolysis, PDGF, 5HT4 type receptor, Fas cell surface death receptor (FAS), histamine H2 receptor, serine glycine biosynthesis, beta 3 adrenergic receptor, angiotensin (through G proteins and betaarrestin), and interleukin signaling pathways (Table 3).
Interestingly, several of the abovementioned enriched pathways are involved in cancer development.
Other modulated transcripts belong to the epidermal growth factor (EGF), transforming growth factor (TGF) beta, Wnt, and PI3 kinase signalings, all involved not only in the development of the SSc associated fibrosis (45,46) but also in cancer development (47)(48)(49)(50). In particular, we observed overexpression of the AKT2 member of the EGF signaling pathway and of the FOXO1 component of the PI3K pathway. The deregulation of these genes has been associated with tumor progression and metastatic spread (29,51). The functional classification of DEGs in dSSc samples shows that they reflects the gene modulation observed in lSSC samples (see Table 4).
Endothelial activation was well represented in dSSc samples by the overexpression of gene encoding for endothelin 1 (EDN1) that is associated with diseases characterized by endothelial dysfunction and fibrosis (55).
Finally, we found upregulation of three transcripts that play a role in the ECM organization including ADAM metallopeptidase domain 2 (ADAM2), CST3 (increased also in lSSc samples), and basigin (BSG). BSG, also named ECM metalloproteinase inducer (EMMPRIN), is expressed on the surface of tumor cells and induces fibroblasts to synthesize matrix metalloproteinases (56).
As found in lSSc samples, among other DEGs involved in signal transduction, several transcripts were involved in Wnt, PDGF, and type I interferon signaling pathway and, their role in cancer development has been already stressed in our dissection of genes modulated in lSSc. Not surprisingly, type I interferon pathway accounted for a large amount of DEGs (14) and the evidence of the modulation of this molecular signaling both in lSSc and in dSSc further underlines its role in the pathogenesis of the disease. The gene expression profiling of SSc patients was complemented with a network analysis. With this purpose, by a bioinformatic analysis, we selected all the functional and experimentally validated interactions between the protein products of modula ted genes and we constructed the two protein-protein interaction (PPI) networks that were representative of lSSc and dSSc dataset. The lSSc-PPI network comprised 440 genes (nodes) and 1351 pairs of interactions (edges) (Figure 1A), whereas the dSSc included 225 genes and 870 pairs of interactions ( Figure 1B).
The PPI networks were then submitted to a modular analysis to find set of highly interconnected nodes (modules) that participate in multiple activities in a coordinated manner and that are expected to play a prominent role in the development of biological phenomena.
In the lSSc-PPI network, we identified nine modules that are graphically represented in Figure 2. Moreover, a functional enrichment analysis was applied to find the associations between each module and relevant enriched "GO terms" and pathways. All the significantly enriched (p < 0.05) biological processes (BPs) and pathways in each module are showed in Table S3 in Supplementary Material and Table 6 shows a selection of the most relevant terms. We observed that the most enriched BPs in module M0, were the signaling of G-protein coupled receptors (GPCRs) and mediated by chemokine. Interestingly, the GPCRs pathway is involved in cancer initiation and progression and GPCRs are emerging as anti-cancer drug targets (64). In the same module M0, we could highlight, among other, an enrichment in inflammation mediated by chemokine and cytokine, PI3 kinase and endothelin signaling pathway. Module M1 showed an enrichment in the positive regulation of type I interferon production BP whereas, among different enriched pathways, we found the glycolysis pathway and again, the endothelin signaling pathway. Module M2 and M4 were the most representative of the type I interferon response. In M2, the GO terms associated to the type I interferon signaling were the most enriched biological processes, followed by terms associated to the innate immune response (i.e., innate immune response, defense response to virus, interferon gamma mediate signaling) and to the adaptive immune response (i.e., antigen processing and presentation of exogenous peptides). Interestingly, the enriched pathways in M2 were related to the Jak/Stat and Interleukin signaling, both associated to tumor development as previously remarked.
In M4, the most enriched BPs were interferon-gammamediated signaling pathway and cellular response to interferongamma. Besides the BPs associated to type I interferon response, we found an enrichment in terms referred to the lymphocyteassociated immune response (i.e., regulation of lymphocyte activation, regulation of T cell activation, regulation of T cell proliferation and antigen processing, and presentation of peptide antigen via MHC class I and II). Most enriched pathways in M4 were referred to interferons (alpha, beta and gamma) pathways. Interestingly, in M4, we observed an enrichment in the PD-1 signaling, an immune-inhibitory-checkpoint that acts as crucial mediator for the escape phase of cancer immune editing (65). Module M3 and M5 were enriched in terms related to translation and other metabolic processes of proteins and, in module M6 the most enriched BPs and pathways were referred to the glycolytic pathway. In module M7, we observed an enrichment of many BPs associated to positive and negative regulation of apoptosis and concordantly, an enrichment in the apoptotic FAS and p53 pathways signaling. Moreover, in M7, several cancer-associated signaling pathways were included, including PI3 kinase, Ras, Interleukin, FGF, and EGF receptors signaling pathways. Finally, in module M8, the protein ubiquitination BP and the ubiquitin proteasome pathway were the most over-represented. Interestingly, tumor cells have a high dependency on the proteasome for survive and proteasome deregulation is frequently induced by many types of tumors (66).
From the dSSC-PPI network, we could extract seven modules (Figure 3) that were studied by a functional enrichment analysis.
The results of this analysis are fully presented in Table S4 in Supplementary Material and the most relevant terms are showed in Table 7.
The BPs that we found enriched in the module M0 were mainly referred to the immune response. In particular, we observed an enrichment in the regulation of tumor necrosis factor superfamily cytokine production process. Moreover, the over-represented pathways in M0 reassumed nearly all signalings that were enriched in the entire dSSc dataset, including inflammation mediated by chemokine and cytokine, JAK/ STAT, p53, oxidative stress response, Ras, interleukin, EGF receptor, and angiogenesis signaling pathway. Module M1 was largely enriched in ATP and purine ribo/nucleoside metabolic processes, whereas M2 was mostly represented by BPs referred  to the type I interferon signaling followed by BPs involved in both innate and adaptive immune response. In addition, DEGs in M2 mainly play a role in ubiquitin proteasome, Jak/Stat, interleukin, EGF receptor, and PDGF signaling pathways. In M3, the most enriched BP was cellular response to TNF and, not surprising, the most over-represented pathway was the apoptosis signaling. Other pathways, statistically representative of module M3 were toll-like receptor (TLR), ubiquitin proteasome, and p53 pathway. In particular, the enrichment of TLR signaling is interesting since it is well known that inducing pro-inflammatory cytokines and co-stimulatory molecules, it contributes to the development of an excessive inflammatory response, leading to both autoimmune disorders and tumor growth (67). Module M4 was mostly enriched in G-protein coupled receptor signaling, and we also observed enriched BPs related to leukocyte/lymphocyte chemotaxis, whereas the most over-represented pathway was inflammation mediated by chemokine and cytokine signaling pathways. The Wnt signaling pathway was preeminent in module M5. In addition, DEGs in this module were also primarily involved in the p53 pathway. Response to virus was the most representative BP of module M6: is worthwhile mentioning that numerous viruses have been proposed as possible triggering factors in SSc (68), and indeed, it has been estimated that up to a quarter of human tumors are connected to infection or infection-associated chronic inflammation (69). A large number of BPs involved in immune cells activation, proliferation, and differentiation and in type I interferon signaling, were prevalent in M6. The over-represented pathways in this module were again signalings also involved in cancer and already

Micrornas in ssc sera
Systemic sclerosis is associated with an increased risk of malignancies, and in the present study, we found a modulation of genes encoding for molecules that have been previously associated to different types of cancers. An important role of miRNAs in human cancers is well established (12). The higher incidence of cancer in SSc patients prompted us to investigate whether specific cancer-related miRNAs could be deregulated in the serum of SSc patients as compared to healthy controls.
Since SSc patients are mainly affected by breast, lung, or hematological malignancies (70), we selected miRNAs with a solid evidence in literature for deregulation in these kind of cancers. We focused on miR-155-5p, miR-126-3p, and miR-16-5p. Furthermore, we decided to analyze miR-21-5p and miR-92a, since they play key roles in many cancers, and to confirm their upregulation in our cohort of SSc patients ( Table 8). Cell-free miRNAs (cf-miRNA) in limited and diffuse SSc and in healthy sera was evaluated by real time PCR, as represented in Figure 4. A significant higher expression of 4/5 of the miRNAs tested in SSc sera was found as compared to healthy controls. miR-126-3p expression also showed a trend of upregulation in SSc samples although it did not reach statistically significant differences in the samples tested compared to controls. Moreover, no significant differences between limited and diffuse SSc were found in our analysis. miR-21-5p and miR-92a-3p were upregulated,    . Expression values of mature miRNAs were calculated using the comparative ΔCt method and normalized to spikein cel393p. Histograms represent mean ± SD. pvalues (ns = not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001) were determined using the Mann-Whitney rank sum test.
as reported in literature, further supporting the hypothesis of their involvement in SSc pathogenesis. To our knowledge, upregulation of miR-155-5p and miR-16-5p in SSc sera has not been reported yet, and it suggests a role of these miRNAs in the disease.

DiscUssiOn
Systemic sclerosis represents a major medical challenge and many unmet needs in the treatment of the disease still remain. However, advances have been made thanks to the increased understanding of the pathogenesis of SSc and the overall patients' survival is improved.
The increased risk of malignancies in SSc is a major source of concern and the identification of risk factors for both disorders may have implications for the prognosis and for the treatment.
We therefore wanted to carry out a gene expression profile and an epigenetic analysis in scleroderma patients to verify the presence of common bases for the development of malignancies and SSc.
We have noticed that the functional classes, to which the genes modulated in limited and diffuse forms of SSc belong, were virtually overlapping and, in both cases, we observed the modulation of genes and pathways previously associated with malignancy. Although it has been reported a higher incidence of some tumors in patients with dSSc than in those with lSSc, our data seem to suggest that in both forms of the disease there is a genetic modulation that may be linked to the onset of a neoplastic transformation. Indeed, from our analysis has emerged that the various classes comprising genes potentially linked to the pathogenesis of SSc (such as apoptosis, endothelial cell activation, extracellular matrix remodeling, immune response, and inflammation), include genes that directly participate in the development of malignancies or that are involved in pathways known to be associated with carcinogenesis.
In this regard, several pathways enriched in lSSc and/or dSSc (apoptosis, glycolysis, PDGF, Fas cell surface death receptor, FAS, angiogenesis, interleukin, Ras, Jak/Stat, and EGFR signaling pathways) are involved in cancer development.
Indeed, altered apoptosis has a crucial role in the induction of a malignant phenotype, and it is well known that some oncogenic mutations block apoptosis, leading to cancer progression (79). Moreover, some cancer cells express FAS ligand (FasL) and the activation of FAS pathway may favor immune privilege to tumors by inducing apoptosis of anti-tumor lymphocytes (80).
The modulation of genes involved in the glycolytic pathway is not surprising, given that upregulation of glycolysis is a welldocumented property of cancers, and this mechanism confers to tumor cells a significant growth advantage (81).
Platelet derived growth factors and PDGF receptors have substantial role in regulating cell growth during embryonal development. An enhancement of PDGF receptor signaling, may also sustain tumor cell growth. Moreover, fibroblasts, and myofibroblasts of solid tumors stroma express PDGF receptors, and PDGF stimulates these cells promoting tumorigenesis (82). Noteworthy, PDGF enhances c-myc expression and stimulates the c-myc promoter (83). This gene, found overexpressed in our lSSc dataset, coordinates cell growth and cell proliferation and its deregulation is a near-universal property of primary and metastatic cancers (84).
Ras proteins are key elements in malignant transformation; moreover, the Jak/stat signaling pathway sustains epithelial mesenchymal transition and generates a pro-tumorigenic microenvironment. The EGF receptor signaling pathway modulates migration and survival of cancer cells.
It has been established that inflammation caused by either chronic disease or infection is an important risk factor in cancer development and that a variety of interleukins are involved in both inflammation and carcinogenesis (85). In particular, among genes involved in the interleukin signaling pathways that was enriched in lSSc modulated genes, we found overexpression of the interleukin 2 receptor alpha (IL2RA) gene. The soluble form of this molecule is released from neoplastic cells and is expressed on the surface of both lymphoid and non-lymphoid cancer cells (86).
Pathway analysis also highlighted the modulation of a large number of genes (11) involved in the Type 1 interferon signaling pathway. This pathway is a hallmark of many systemic autoimmune diseases (87) including SSc (88) and has also been considered as a "double-edged sword" in cancer. Indeed, in cancer, it promotes both T cell responses and a negative feedback leading to immunosuppression. Tumor cells can take advantage from this counter-regulatory effects induced by IFN type I to avoid immune cell killing (89).
This suggests that there are multiple points of contact between the signaling pathways leading to scleroderma and those that predispose to the development of a malignancy. However, these pathways may lead to different outcomes in different tissues in which they are expressed, and indeed in patients with scleroderma, some malignancies are more frequent such as those affecting breast, lung, and lymphoid tissue.
Endothelial cell apoptosis is among the first manifestations of vasculopathy associated with the development of SSc, and fibroblasts activation and proliferation lead to the fibrotic characteristics of the disease. Gene expression profiles obtained from the PBMC of patients with SSc indicate an altered modulation of genes involved in apoptosis and in cell proliferation that could, at tissue level, exert a mitogenic effect on some cellular populations or lead to down-modulation of oncosuppressor genes, as suggested by the downregulation of CUL3 (31) in both lSSc and dSSc.
Our data also suggest the presence of a genetic modulation that can favor angiogenesis, a common feature associated to the development and progression of any type of cancer.
Modulation of many genes involved in the immune response, including genes inducible by type I interferon, reflects the strong immune system dysregulation associated with excessive antigenic stimulation typical of autoimmune diseases. Such dysregulation can either induce proliferation of cells of the immune system, leading to malignancy in susceptible individuals or modify the immune system's regulatory mechanisms that inhibit the development of naturally occurring cancers.
Several modulated genes participate in the remodeling process of the extracellular tissue matrix. Excessive deposition of ECM promotes the development of fibrosis, hallmark of SSc, and the fibrotic and inflammatory processes of the lung have been considered the basis for the eventual onset of lung cancer in SSc. On the other hand, within these functional classes, we have also observed over-expression of metalloproteases (i.e., ADAM2) or ECM metalloproteases inducers (i.e., BSG) typically induced by several tumor cells (56,90). Moreover, genes associated with the migration of tumor cells such as SPARC (43), a glycoprotein associated with ECM, are involved in the development of non-small cell lung cancer. Therefore, while ECM deposition and related profibrotic events may favor carcinogenesis through different mechanisms such as blocking lymphatic channels and creating niches in which carcinogens may eventually accumulate, on the other hand, there is a genetic modulation that favors cancer cells spreading.
A crucial issue in basic and clinical research is to understand gene modulation in terms of biological networks since proteins usually function in protein-protein interacting networks. We therefore wanted to dissect meaningful relationships among modulated genes in SSc, analyzing the PPI network in which their protein products can be involved. Moreover, since it is known that the deregulation of protein expression may provoke more drastic biological effects when genes/proteins with more interacting partners are involved, we focused our attention on the most connected genes/proteins that "as a matter of fact" (by definition) are included in the network areas called modules. This is particularly important when studying gene regulation in certain diseases in order to identify the molecular pathways that are most relevant in disease pathogenesis. The pathway enrichment analysis of modulated genes included in the modules confirmed the enrichment of signaling pathways (i.e., the aforementioned Jak/stat, glycolysis, Ras, PDGF, EGF receptor, Wnt, and type I interferon signaling pathways) associated with carcinogenesis in both datasets.
The genes participating to these pathways are indeed comprised in network areas (modules), whereas gene interactions are concentrated and generally underline significant biological processes.
These molecular pathways also emerged from our first global analysis of the two datasets, but the network analysis further underlined their involvement in SSc. Indeed, members of these signaling networks are concentrated in modules were the molecules that are supposed to play a prominent role in shaping the typical features of the disease, are positioned.
Another aspect we have investigated is the expression of microRNAs in SSc. Altered expression of miRNAs in SSc, as well as their involvement in inflammation and fibrosis, has been described (14,91). Similarly, the implication of miRNAs in human cancers is well established (12). A fascinating hypothesis is that a dysregulated epigenetic control mediated by miRNAs in SSc could promote tumorigenesis. Deregulated expression of miRNAs has been found in blood of SSc patients and it could exert oncogenic effects at distant sites from SSc lesions. Indeed, the upregulation of miR-21 and miR-92a in SSc (16,92) may support this hypothesis, since these miRNAs play a role in many tumors, repressing important oncosuppressor genes (74,93). Thus, the higher incidence of breast, lung and hematological malignancies in SSc patients (70) prompted us to investigate whether specific cancer-related miRNAs could be deregulated in the serum of SSc patients. We found that miR-21-5p, miR-92a-3p, miR-155-5p, and miR-16-5p expression was significantly higher in SSc sera compared to healthy controls. miR-21 can play a role in SSc since it is also upregulated in SSc fybroblasts and it is implicated in TGF-β-regulated fibrosis (17). Interestingly, miR-21 upregulation promotes proliferation, migration, and invasion in lung cancer (71). Elevated miR-92a levels in serum and in dermal fibroblasts of SSc patients have also been reported (16), and this miRNA is implicated in angiogenesis and proliferation in lung cancer and it can promote leukemogenesis (74,75). MiR-155-5p expression was also increased in SSc sera. Notably, miR-155 was found upregulated in SSc fybroblasts, and it was associated to the progression of lung fibrosis in dSSc patients (94,95). miR-155 upregulation also sustains survival and proliferation in hematological disorders and breast cancer (72,76). We found increased miR-16-5p levels in SSc sera despite this miRNA is frequently associated to oncosuppressor functions and it is frequently absent in human leukemias (78). Since this miRNA inhibits cell proliferation and promotes apoptosis (96), it could participate to the apoptotic process of endothelial cells, considered the first pathogenic event in SSc. A release of miR-16 in the bloodstream from apoptotic endothelial cells could explain the increased levels of the miRNA in SSc sera. We also decided to evaluate miR-126 in SSc sera since it was found downregulated in breast and lung cancer (76,77). Moreover, miR-126 plays an important role in angiogenic signaling and in vascular integrity (97), and we found several genes involved in angiogenesis upregulated in SSc by microarray analysis. We did not find statistically significant differences in the miR-126 expression levels between SSc patients and healthy controls, although we observed a trend toward upregulation in SSc samples. In conclusion, we describe here interesting findings on deregulated cancer-related miRNAs in SSc patients. However, further studies are needed to elucidate the potential role of these miRNA in SSc. In particular, since the levels of circulating micro-RNAs can be affected by different cell types, interesting points will be to elucidate which cells mainly contribute to these circulating oncogenic miRNAs and whether they can play an active role in the increased tumorigenesis associated to the disease.
Taken together, our data suggest the presence of modulated genes and miRNAs that can play a predisposing role in the development of malignancies in SSc. The findings of genetic and epigenetic features that are shared by SSc and cancer shed new light on the pathogenesis of the disease and strengthen the idea that autoimmunity plays a central role in the initiation and progression of SSc, since the presence or development of malignancies is associated with particular autoantibodies. These aspects are central to a better risk stratification of patients and to develop an individualized precision medicine strategy.

eThics sTaTeMenT
This study was carried out in accordance with the recommendations of "local ethical committee Azienda integrata Università di Verona e Ospedale Borgo Roma Verona, Italy" with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the "name of committee. "

aUThOr cOnTribUTiOns
APu, CL, MD, APe, and APu conceived and designed the experiments. MD, APe, and PF performed the experiments. MD, APe and APu analyzed the data. GP and ET contributed reagents. ET, GP, and CL contributed materials. MD and APe wrote the paper.