Increased SPON1 promotes pancreatic ductal adenocarcinoma progression by enhancing IL‐6 trans‐signalling

Abstract Objectives This study investigated the specific molecular mechanism and the roles of extracellular matrix protein Spondin 1 (SPON1) in the development of pancreatic ductal adenocarcinoma (PDAC). Materials and Methods The expression pattern and clinical relevance of SPON1 was determined in GEO, Ren Ji and TCGA datasets, further validated by immunohistochemical staining and Kaplan‐Meier analysis. Loss and gain of function experiments were employed to investigate the cellular function of SPON1 in vitro. Gene set enrichment analysis, luciferase assay, immunofluorescence and Western blot and immunoprecipitation were applied to reveal the underlying molecular mechanisms. Subcutaneous xenograft model was used to test the role of SPON1 in tumour growth and maintenance in vivo. Results SPON1 is significantly upregulated in PDAC tumour tissues and correlated with progression of PDAC. Loss and gain of function experiments showed that SPON1 promotes the growth and colony formation ability of pancreatic cancer cells. Combining bioinformatics assays and experimental signalling evidences, we found that SPON1 can enhance the IL‐6/JAK/STAT3 signalling. Mechanistically, SPON1 exerts its oncogenic roles in pancreatic cancer by maintaining IL‐6R trans‐signalling through stabilizing the interaction of soluble IL‐6R (sIL‐6R) and glycoprotein‐130 (gp130) in PDAC cells. Furthermore, SPON1 depletion greatly reduced the tumour burden, exerted positive effect with gemcitabine, prolonging PDAC mice overall survival. Conclusions Our data indicate that SPON1 expression is dramatically increased in PDAC and that SPON1 promotes tumorigenicity by activating the sIL‐6R/gp130/STAT3 axis. Collectively, our current work suggests SPON1 may be a potential therapy target for PDAC patient.

therapy, the prognosis of pancreatic cancer patients is still not optimistic, and the 5-year survival rate is only 8%. 2,3 Moreover, pancreatic cancer was the fourth leading cause of cancer-related deaths in Europe in 2020 4,5 and is projected to become the second leading cause of cancer-related deaths by 2030. 6 The tumour microenvironment (TME), the complex environment in which tumour cells survive, is mainly composed of a variety of different extracellular matrix (ECM) components and stromal cells. 7 Structural and non-structural proteins are the main components of the ECM. 8 Among them, non-structural ECM proteins have many biological activities and play important roles in chronic inflammation, tumour growth, invasion and metastasis and immune microenvironment regulation. 9,10 Therefore, exploring the roles of ECM proteins in the occurrence and development of pancreatic cancer is expected to provide new insights into the diagnosis and treatment of pancreatic cancer.
The ECM protein SPON1, also known as F-spondin or vascular smooth muscle cell growth-promoting factor (VSGP), is a member of the thrombospondin family encoded by a highly conserved gene. 11 SPON1 is a secreted ECM protein derived from the floor plate of vertebrate embryos. It was reported that SPON1 promotes the growth of neural axon cells while inhibiting the migration of neural crest cells. 12,13 In terms of disease research, SPON1 was regarded as a novel candidate hypertension gene, and its expression increased over time in spontaneously hypertensive rats. 14 In addition, SPON1 could bind the amyloid precursor protein and inhibit its cleavage by β-secretase, resulting in cognitive decline in Alzheimer's disease. 15 SPON1 has been also reported to be abnormally expressed in solid tumours. Previous studies showed that SPON1 triggers focal adhesion kinase 1 (Fak) and tyrosine-protein kinase Src signalling and promotes distant metastasis in osteosarcoma. 16 Another study reported that SPON1 promotes tumour invasion and metastasis in liver cancer. 17 However, these studies have not clearly elucidated the specific molecular mechanism, and the roles of SPON1 in the development of pancreatic cancer remain unknown.
In the present study, we discovered that SPON1 is markedly upregulated and correlated with malignant progression in PDAC. Functional experiments revealed that SPON1 silencing significantly inhibited the proliferation and colonization of PDAC cells. Mechanistically, SPON1 exerts its pro-growth function in maintaining IL-6R trans-signalling by stabilizing the interaction of sIL-6R/gp130 in PDAC cells. Using an in vivo PDAC model, we demonstrated that targeting SPON1 profoundly hindered the tumorigenesis of PDAC. More interestingly, the combination of gemcitabine treatment and SPON1 depletion more profoundly delayed tumour growth and significantly extended the survival of tumour-bearing mice. Taken together, our results pave the way for developing novel therapeutic strategies for pancreatic cancer based on targeting SPON1.

| Knockdown and overexpression assay
The lentivirus against SPON1 was purchased from Gene Pharma (Shanghai, China), and the sequences targeting SPON1 were: sh-1, 5 0 - Real-time PCR system (Applied Biosystems) at the recommended thermal cycling settings: one initial cycle at 95 C for 10 min followed by 40 cycles of 15 s at 95 C and 60 s at 60 C. Relative mRNA expression was calculated by the 2 ÀΔΔCt method and normalized to 18S mRNA levels. Primer sequences are listed as follows: sIL-6R 5 0 -CATGTGCGTCGCCAGTAGT-3 0 ,

| Western blotting
The lysates of protein samples were collected by using RIPA lysis and extraction buffer (ThermoFisher, 89900), separated by SDS-PAGE in polyacrylamide gels, and transferred to nitrocellulose membranes.
Subsequently, membranes were washed with TBST (50 mM TRIS + 150 mM sodium chloride + 0.1% Tween 20, pH 7.4) and blocked using 5% nonfat milk solution in TBST at least 1 h at room temperature. Membranes were then incubated with primary antibodies:

| Colony-formation assays
In the colony-formation assays, indicated cells (3000 cells/ml) were seeded in six-well plates. The colonies were collected after incubation for 2 weeks and then fixed with 4% paraformaldehyde fix solution and stained with 0.5% (w/v) crystal violet, followed by calculation with Image J. This experiment was repeated twice.

| EdU (5-ethynyl-2 0 -deoxyuridine)
The indicated cells were seeded into chambered coverslips (80826, ibidi) and incubated at 37 C with 5% CO 2 condition for 48 h, and then added EdU working solution (10 μM, Yeasen) into culture medium at 37 C with 5% CO 2 condition for overnight. After that, the chambers were fixed with 4% paraformaldehyde fix solution for 10 min at room temperature.
After following the manufacturer's instruction, DAPI (G1012, Servicebio) were used to counterstain nuclei for 5 min. Confocal microscopes (Leica, Germany) were used to capture digital images.

| Immunohistochemistry (IHC) assay
All the patients were supplied with written informed consent before enrollment, and the study was approved by the Research Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Scoring was calculated based on the percentage of positivestaining cells: 0%-5% scored 0, 6%-35% scored 1, 36%-70% scored 2, and more than 70% scored 3; and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2, and strongly staining scored 3. The final score was showed using the percentage score Â staining intensity score as follows: "À" for a score of 0-1, "+" for a score of 2-3, "++" for a score of 4-6 and "+++" for a score of > 6. The median SPON1 expression was selected as the threshold to define low and high expression of SPON1. Low expression was defined as a total score < 4 and high expression with a total score ≧ 4. These

| Immunofluorescence
The indicated cells were seeded into chambered coverslips (80826, ibidi) and incubated at 37 C with 5% CO 2 condition for 48 h. After that, the chambers were fixed with 4% paraformaldehyde fix solution for 10 min at room temperature (necessarily, treated with 0.1% Triton X-100 for 5 min at RT). After blocking with 5% BSA for 1 h, the chambered cover- were used to counterstain nuclei for 5 min. Confocal microscopes (Leica, Germany) were used to capture digital images.

| Measurement of sIL-6R
For measurement of sIL-6R level in the culture medium, a total of 5 Â 10 6 indicated PDAC cells were seeded into 10 cm-plates and allowed to attach overnight. Then cells were incubated for another 12 h in a humidifier at 37 C, and the culture medium was collected.
For preparation of tissue homogenates, 14 PDAC tissues and paired non-tumour tissues were washed with PBS to remove blood components. Following homogenization in a standard ELISA buffer, the tubes were centrifuged at 12,000 g for 30 min at 4 C. The supernatant was carefully removed and transferred for further measurement of sIL-6R.
For preparation of platelet-poor-plasma samples, 10 ml blood was collected into an EDTA plasma tube and centrifuged at 1000 g for 30 min at 4 C. Then plasma was aliquoted, labelled and stored at Values from each sample in tissues were normalized to total protein content as detected by BCA assay (Thermo Pierce).

| Luciferase assay
As the characteristics of chemiluminescence reaction using the combination of luciferase and substrate, the indicated cells were transfected with vectors, STAT2, STAT3 and hedgehog using jetPRIME (Polyplus transfection). Next, the cells were assayed for firefly luciferase activities via a luciferase system (Promega, Madison, WI, USA) according to the manufacturer's instructions.   All error bars in this study represent the mean ± SD, except for bioluminescent emission, whose error bars represent the mean ± SEM (n.s, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). PDAC patient cohort. We found that SPON1 expression was associated with the sensitivity of PDAC to chemotherapy. SPON1 expression in the progressive (PG) group was significantly higher than that in the complete remission (CR) and partial remission (PR) groups ( Figure 1C). In addition, we found that SPON1 expression increased with increasing T stage, and SPON1 expression increased sequentially in T1, T2 and T3 PDAC specimens ( Figure 1D). To further validate our results at the protein level, we performed immunohistochemical (IHC) staining. Expression was scored based on staining intensity and area. As shown in Figure 1E, a higher staining score was exhibited by the tumour tissues. According to clinical data obtained from Ren Ji cohort, our results suggested that samples which are larger than 3 cm in size show higher staining score than the  Figure 1F). As expected, Kaplan-Meier analysis revealed that patients with higher SPON1 expression had a poorer prognosis ( Figure 1G). These data suggested that SPON1 plays an important role as an oncogene in PDAC progression.

| SPON1 promoted the growth of PDAC cell in vitro
To gain further insight into the roles of SPON1 in PDAC, we examined SPON1 expression in six pancreatic cancer cell lines at the protein level ( Figure 2A). We selected PANC-1 and SW1990 cells, which exhibit relatively high SPON1 expression, for genetic inhibition of SPON1 by short hairpin RNA (shRNA) transfection. After verifying SPON1 expression at the protein level ( Figure 2B), cell proliferation was assessed. It was shown that knockdown of SPON1 expression impaired cell growth in vitro ( Figure 2C-F). Meanwhile, we selected two cell lines that express SPON1 at relatively lower levels for SPON1 overexpression, and SPON1 expression was examined at the mRNA and protein levels ( Figure 2G). As expected, cell proliferation and colony formation were enhanced by SPON1 overexpression (Figure 2H-I). The above results indicated that the level of SPON1 expression has crucial effects on PDAC cell growth.

| SPON1 activates the IL-6/JAK/STAT3 pathway in PDAC cells
To further understand the underlying mechanism by which SPON1 promotes PDAC cell proliferation, we first performed gene set enrichment analysis (GSEA) of samples in TCGA divided into two groups based on SPON1 expression. As shown in Figure 3A, the HALLMARK_ IL2_STAT5_SIGNALLING, HALLMARK_IL6_JAK_STAT3_SIGNALLING and HALLMARK_HEDGEHOG_SIGNALLING datasets were sifted. Further, luciferase assay was performed to figure out which pathway may mediate the pro-growth effect of SPON1. The results showed that JAK-STAT3 pathway activation was much higher than others pathway upon SPON1 overexpression in PDAC cell ( Figure 3B). To verify the effects of SPON1 on this pathway, we then examined the protein level of phospho- 3.4 | SPON1 activated IL-6 trans-signalling to triggering IL-6/JAK/STAT3 pathway IL-6 activates downstream signalling pathways by forming complexes with its receptors, including two subunits: IL-6R (also called IL-6α or CD126) and signal-transducing glycoprotein-130 (also called gp130, IL-6β, CD130). In addition, IL-6 signalling pathway models may vary in different contexts: in the classic IL-6 signalling pathway, extracellular IL-6 binds membrane-bound IL-6R (mIL-6R) to yield a complex to which gp130 binds, forming a complex consisting of two IL-6, two IL-6R and two gp130 molecules 18 ; alternativly, IL-6 trans-signalling that follows the classical pathway except that IL-6 binds soluble IL-6R (sIL-6R) rather than mIL-6R can also occur. 19 The third model, termed IL-6 trans-presentation, has recently been identified and is specific to dendritic cells. 20 IL-6 level was measured by ELISA assays after administration of F-spondin, and were not significantly different ( Figure 4A).
As shown in Figure 4B, we examined the IL-6 related receptors after treatment with F-spondin. It was shown that the level of sIL-6R was markedly increased, while the mIL-6R level was not. In addition, SPON1 KD cells showed reduced sIL-6R protein levels; however, mIL-6R levels were not reduced ( Figure 4C).
To further explore the potential mechanism of SPON1-induced STAT3 phosphorylation, we then performed cell proliferation assays to determine which model is responsible for this effect. Soluble gp130 (sgp130) blocks IL-6 trans-signalling by binding the IL-6/sIL-6R. 21 Further, the PDAC cell lines were treated with F-spondin, sgp130 or Fspondin plus sgp130. The results revealed that sgp130 impaired cell proliferation. Also, sgp130 could diminish the F-spondin-induced proliferation advantage ( Figure 4D,E). In line with this, the immunofluorescence results showed that F-spondin could promote the nuclear translocation of p-STAT3, which was impaired by sgp130 treatment ( Figure 4F). We speculated that SPON1 activates the IL-6 trans-signalling pathway by competitive binding with sgp130. That is, sIL-6R may be affected by SPON1 in some ways, resulting in changes to cell proliferation.
3.5 | SPON1 enhances the IL-6/JAK/STAT3 pathway by stabilizing the sIL-6R/gp130 complex We next investigated why sIL-6R was increased with increasing F-spondin doses. In general, sIL-6R proteins were translated from alternatively spliced IL-6R mRNA. Moreover, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and metalloproteinase domaincontaining protein 17 (ADAM17) can cleave mIL-6R to generate sIL-6R. 22 Therefore, we examined the mRNA level of sIL-6R after the addition of Fspondin, but the difference was not statistically significant ( Figure 5A).
Then, ADAM10 and ADAM17 were assessed at the protein level and showed no significant changes as well ( Figure 5B). Intriguingly, F-spondin decreased the sIL-6R level in the culture medium ( Figure 5C). The results of immunoblot analysis showed decreased P-STAT3 levels after treatment with IL-6 for increasing durations ( Figure 5D,E). Given the above results, we speculated that SPON1 contributes to the formation of the sIL-6R/gp130 complex, thus sustaining IL-6 trans-signalling and STAT3 activation. To test our hypothesis, we performed endogenous immunoprecipitation assays to confirm the interaction between SPON1 and sIL-6R/gp130 ( Figure 5F). Consistently, immunofluorescence co-localization analysis and immunofluorescence staining were used to further verify the interaction between SPON1 and sIL-6R/gp130 ( Figures 5G and S1). The results showed that both SPON1 and sIL-6R/gp130 were co-localized in human PDAC tumour tissues ( Figure 5H). Besides, we detected the changes of sIL-6R located in cell membrane. The cell membrane of SPON1 KD cell and control cells were isolated by subcellular protein fractionation kit, following ELISA detection. As expected, the membrane location of sIL-6R dramatically decreased upon SPON1 knockdown. The CO-IP results showed that much less of sIL-6R interacted with gp130 in SPON1 KD cells when compared with the control (Figure 5I-J). Conversely, extopic SPON1 expression could enhance the membrane location of sIL-6R/gp130 and its interaction ( Figure 5I,J). Taken together, these data showed that SPON1 stabilizes the sIL-6R/gp130 complex to sustaining the IL-6 trans-signalling pathway.  reported that IL-6-STAT3 signalling could crosstalk with IL2-STAT5 pathway. 30,31 In line with this, our data showed that IL2-STAT5 axis had some extent activation, not as stronger as IL-6-STAT3 signalling. In summary, our data reveal the roles of SPON1 in signalling and as a mediator of pancreatic cancer.

| CONCLUSION
We have shown that SPON1 expression is dramatically increased in PDAC and that SPON1 promotes tumorigenicity by activating the sIL-6R/gp130/STAT3 axis. We have also demonstrated that targeting SPON1 may represent an attractive therapeutic strategy. Collectively, our findings provide not only new insights into the role of SPON1 in cancer progression but also a novel and effective therapeutic approach for the eradication of PDAC, which remains a life-threatening disease without an effective, targeted therapeutic strategy.