SC144 - a promising small-molecule gp130-inhibitor of IL-6 and Oncostatin-M signaling for treatment of pancreatic cancer

doi:10.21203/rs.3.rs-1602630/v1

Background: Interleukin-6 (IL-6), Oncostatin-M (OSM) and the major downstream effector STAT3 are pro-tumorigenic agents in many cancers, including pancreatic ductal adenocarcinoma (PDAC). Glycoprotein 130 (gp130) is a compound of the IL-6 and OSM receptor complex that triggers STAT3 signalling. Recently, a novel small molecule gp130 inhibitor named SC144 was discovered with a broad-spectrum anticancer activity. In this study, we examine the in vitro effects of SC144 in PDAC cell lines and the expression of gp130 in human PDAC specimens.

Methods: Growth inhibition by SC144 in human PDAC cell lines AsPC-1 and L3.6pl was assessed using BrdU and MTT assays. Western blotting was performed to identify the inhibitory effect of SC144 on IL-6- or OSM-stimulated STAT3 signaling. Tissue microarrays were constructed from 175 human tumor specimens after pancreatic resection for PDAC. The expression of gp130 in tumor epithelium and tumor stroma was determined by immunohistochemistry. The gp130 expression was then correlated with clinicopathological parameters and survival analysis was performed.

Results: SC144 inhibited cell proliferation and viability in human PDAC cells. Treatment with SC144 suppressed IL-6- and OSM-stimulated STAT3^Y705 phosphorylation in PDAC cells. gp130 was expressed in epithelium of 78.8% of the tumor samples and in stroma of 9.4% of the tumors. The median overall survival for patients with no epithelial gp130 expression was 15.9 months, whereas for patients with gp130 expression was 16.7 months (p = 0.830). Patients with no stromal gp130 expression showed a poorer survival than patients with stromal gp130 expression in PDAC (median 16.2 and 22.9 months, respectively), but this difference did not reach significance (p = 0.144).

Conclusions: SC144 potently inhibits PDAC progression in vitro through binding and inhibition of gp130 and, thereby, causes abrogation of IL-6 or OSM/gp130/STAT3 signaling. Although gp130 is expressed in the epithel of most human PDAC, stromal expression is rare. These results suggest gp130 as a novel drug target and blocking gp130/STAT3 signaling by SC144 as a potential new therapeutical approach for pancreatic cancer.

SC144

pancreatic cancer

PDAC

interleukin 6

IL-6

oncostatin M

OSM

gp130

STAT3

inhibition

Malignancies of the pancreas account for about three percent of all cancers but remain nowadays the fourth most common cause of cancer-related death in both sexes in the western world (Torre et al., 2015). By 2030, total deaths from pancreatic cancer are supposed to increase dramatically and become the second leading cause of cancer-related deaths after lung cancer (Rahib et al., 2014). Despite the continuous progress in the field of chemotherapy, the medical treatment’s impact on the natural history of the disease remains pure (Denley et al., 2013). Thus, novel therapeutic agents are urgently required to improve the prognosis of pancreatic cancer.

In pancreatic ductal adenocarcinoma (PDAC), key signaling pathways are dysregulated, contributing to pancreatic tumorigenesis. Recent studies have shown that interleukin-6 (IL-6) and the major downstream effector signal transducer and activator of transcription-3 (STAT3) are pro-tumorigenic agents in a variety of human cancers, including PDAC (Taher et al., 2018). High serum IL-6 levels have been proposed as a negative prognostic marker in patients with PDAC (Lesina et al., 2014).

IL-6 is a pleiotropic cytokine with biological effects on a wide variety of cells regulating many cellular functions, including cell proliferation, cell differentiation, immune defense mechanisms, and hematopoiesis (Lesina et al., 2014). IL-6 acts either by affecting the tumor cells directly or modulating the tumor microenvironment. A study in KRAS-mutated mice demonstrated the crucial role of IL-6 in PDAC, showing that IL-6 presence and STAT3 activation are necessary for the early PanIN lesions to be developed to PDAC (Lesina et al., 2011). STAT3 is a transcription factor and a member of the STAT protein family. It is encoded by the STAT3 gene, an oncogene expressed in several human cancers, including pancreatic, having a well-established role in tumorigenesis (Corcoran et al., 2011; Yu et al., 2009). Elevated serum levels of IL-6 are found in patients with PDAC and correlated with poor survival, weight loss and cachexia, which are also negative prognostic factors for these patients (Bellone et al., 2005; Falconer et al., 1994; Okada et al., 1998). IL-6 is elevated not only in serum but also in tumor tissues isolated from patients with pancreatic cancer.

Our previous studies showed a high expression of IL-6/STAT3 pathway proteins in human PDAC specimens (Pozios et al., 2018) and promising results for tumor growth inhibition by blocking this signaling (Pozios et al., 2020). IL-6 mediates part of its functions through the IL-6-receptor complex (IL-6R). Glycoprotein 130 (gp130) as a compound of the IL-6R complex plays a crucial role in this signaling cascade and is expressed in almost all organs playing a fundamental role in development, hematopoiesis, cell survival, and growth (Xu and Neamati, 2013). Interestingly, gp130 is a common receptor part of the IL-6 family cytokines, as IL-6 is only one of minimum eleven known cytokines of IL-6 family, which also activate the same signaling cascade with similar downstream effects. Oncostatin M (OSM) belongs to IL-6 family cytokines, is associated with poor prognosis in PDAC and contributes to epithelial–mesenchymal transition of PDAC cells (van Duijneveldt et al., 2020).

The above data indicate IL-6 or OSM/gp130/STAT3 signaling as a possible target in chemotherapy for pancreatic cancer. Several antibodies against IL-6R, soluble IL-6R (sIL-6R), gp130, JAK, and STAT3 are in clinical development as anti-inflammatory and anticancer therapeutics reporting promising results in both disease groups (van Duijneveldt et al., 2020; Waetzig and Rose-John, 2012; Yao et al., 2014). A novel small molecule gp130 inhibitor named SC144 was recently discovered with a broad-spectrum anticancer activity without significant toxicity to normal tissues (Oshima et al., 2009; Xu et al., 2013). In the present study, we investigated the efficacy of this gp130 inhibitor in PDAC cells in vitro as a novel therapeutic approach for PDAC.

Cell lines and reagents

AsPC-1 cells were purchased from the American Type Cell Culture Collection (Virginia, USA). L3.6pl is a secondary, highly metastatic human PDAC cell line of an orthotopic mouse xenograft model (Bruns et al., 1999). Cell lines were maintained in Dulbecco’s Minimal Essential Medium (Invitrogen GmbH, Karlsruhe, Germany), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were incubated in a humidified atmosphere of 5% CO₂ at 37°C.

SC144 (a quinoxalinhydrazide derivative) was purchased from Sigma-Aldrich (Schnelldorf, Germany) and was solved in 0.1% dimethyl sulfoxide (DMSO). IL-6 was purchased from Invitrogen GmbH (Karlsruhe, Germany) and was dissolved in acetic acid. OSM was obtained from Cell Signaling Technology, Frankfurt am Main and dissolved in phosphate buffered saline. Anti-phosphorylated-STAT3 (Y705), anti- STAT3, and anti-gp130 antibodies were purchased from Cell Signaling Technology (Frankfurt am Main, Germany). Anti-β-actin was purchased from Sigma-Aldrich (Taufkirchen, Germany).

Measurement of cell proliferation and viability

AsPC-1 or L3.6pl cells were seeded in 96-well plates (5,000 cells/well) and were allowed to grow overnight. Then cells were incubated with increasing concentrations of SC144 in serum-free medium at 37°C for 48 hours. The effect of SC144 on cell proliferation was measured using the 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay, according to the manufacturer’s instructions (Roche Applied Science, Penzberg, Germany).

Cytotoxicity was assessed by a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma-Aldrich, Missouri, USA) as previously described (Carmichael et al., 1987). MTT reagent (at a final concentration of 0.5 mg/ml) was added to each well, and cells were incubated for four hours at 37°C. After removing the medium, 10% sodium dodecyl sulfate (SDS) was added, and the absorbance was read at 550 nm. All assays were conducted in sextuplicate.

Target identification using DARTS assay

Drug Affinity Responsive Target Stability (DARTS) assay was performed to identify and assess the protein-ligand interaction between SC144 and gp130. DARTS assay identifies potential protein targets for small molecules via the protease protection from pronase as described previously (Lomenick et al., 2009, 2011). Briefly, L3.6pl cells were lysed using M-PER (Thermo Fisher) supplemented with protease and phosphatase inhibitors. The cell lysate, containing 3 µg/µL total proteins, was treated with SC144 or with solvent alone at room temperature for one hour, followed by proteolysis for 5 minutes at room temperature with pronase (1:5,000 ratio; Roche Applied Science, Penzberg, Germany) as described previously (Lomenick et al., 2011). Proteolysis was stopped by adding SDS loading buffer and heating to 95°C for five minutes. Afterward, the samples were analyzed by Western blotting as described below.

Western blot

L3.6pl cells were treated with different concentrations of SC144 or solvent in a serum-free medium. Phosphorylation was stimulated by IL-6 (100 ng/ml) or OSM (50 ng/ml) three hours after the addition of SC144 (1, 2, 3 and 5 µM). After an incubation period of six or 24 hours, cells were washed with ice-cold phosphate buffer saline and lysed in radioimmunoprecipitation assay lysis buffer containing phosphatase and protease inhibitors. Protein concentration was determined by Quantipro BCA (bicinchoninic acid) protein assay kit (Sigma-Aldrich, Taufkirchen, Germany) according to the manufacturer’s instructions. The proteins were fractionated by SDS polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Perkin Elmer, Boston, USA). Membranes were blocked (Tris-buffered Saline with Tween-80, 5% Bovine Serum Albumin, and 0.02% sodium azide) and incubated with specific primary antibodies overnight at 4°C. Then, they were subsequently probed with horseradish peroxidase-conjugated secondary antibody. Signals were visualized by luminescence imaging (Peqlab Biotechnologie GmbH, Erlangen, Germany) using an enhanced chemiluminescent substrate system (SuperSignal West Pico PLUS, Thermo Fisher, Ulm, Germany).

Patients

In total, 211 patients who underwent surgical therapy of PDAC at the Department of Surgery at the Hospital of the Ludwig-Maximilians-University of Munich between 2003 and 2010 were considered for this study. Exclusion criteria were perioperative mortality (patients dying within 30 days after curative resection), the presence of macroscopic residual disease after resection and periampullary tumors other than PDAC, e.g., ampullary, distal cholangiocarcinomas, and duodenal adenocarcinomas. Finally, 36 patients were excluded and 175 patients were included in the analysis. Data on clinicopathological parameters and follow-up information were extracted from the local tumor registry and clinical records. The study was approved by the Ethics Committee of the Hospital of the Ludwig-Maximilians-University of Munich.

Tissue microarrays

Archival tumor specimens (paraffin tissues) from the Institute of Pathology of the Ludwig-Maximilians-University of Munich were analyzed and tissue micro-arrays (TMAs) were constructed according to standard procedures as previously described (Knösel et al. 2005, 2006). Two TMAs containing 422 samples from 211 patients were constructed. The PDAC TMA was assembled using 0.6-mm punch biopsies from all 211 samples. In total, 422 specimens of pancreatic tissue including normal mucosa were evaluated.

Immunohistochemistry

Immunohistological staining of TMAs was performed according to standard procedures. Briefly, the TMA slides were pretreated and then incubated with the antibodies, followed by antibody detection via biotinylated anti-mouse secondary antibody and a biotin–streptavidin amplified detection system (Biogenex, San Ramon, CA, USA). Staining was visualized using a Fastred chromogen system (DAKO, Hamburg, Germany). The TMA slides were evaluated by a person blinded for the clinical data. The staining was scored semiquantitatively by a four-tier scale (0, negative; 1, weak; 2, moderate; 3, strongly positive) according to standard procedures (Knösel et al. 2006). This was reduced also to a two-tier system (0, negative; 1–3, positive) for the statistical analysis of protein epithelial and stromal expression and its correlation with clinicopathological parameters including survival.

Statistical analysis

Data were analyzed with SPSS software, version 20.0 (IBM Corp., Armonk, NY, USA). Statistical analysis and p-value determinations were carried out by t-test with a confidence interval of 95% for determination of significant differences between treatment groups. Chi-square or Fisher’s exact tests were used for the categorical variables to compare independent groups. Fisher’s exact test was used to analyze categorical data when the sample size was small. p values lower than 0.05 were considered statistically significant. Kaplan–Meier curves were performed for each investigated parameter. Survival curves were compared and assessed using the log-rank test.

SC144 inhibits pancreatic cancer cell proliferation and viability in a dose-dependent manner.

Tumor growth depends on proliferation and viability of cancer cells. Therefore, the SC144 effect on proliferation and viability of AsPC-1 and L3.6pl pancreatic cancer cell lines was assessed in BrdU and MTT assays, respectively. In both AsPC-1 and L3.6pl pancreatic cancer cell lines, SC144 inhibits significantly cell proliferation and viability in a dose-dependent manner. A low dose of 0.5 µM SC144 was already effective to reduce proliferation and viability. In both cell lines, the maximum inhibitory effect was achieved with 2 µM SC144 and could not be enhanced by higher doses of 5 or 10 µM (Fig. 1a-d).

SC144 binds specifically to gp130

DARTS assay was performed to identify and assess the protein-ligand interaction between SC144 and gp130. Western blotting (Fig. 2a) and densitomtric quatification revealed that the gp130 protein signal was 65±23% more pronounced in SC144-treated samples compared to solvent-treated (DMSO) controls (set 100%) (Fig. 2b). This proves that SC144 binds specifically to gp130 in L3.6pl cell lysates and by this reduces the protease susceptibility of gp130.

SC144 suppresses IL-6 and OSM-induced STAT3 signaling.

To test whether IL-6 or OSM could stimulate STAT3 phosphorylation, L3.6pl cells were firstly treated with IL-6 or OSM respectively. We found that 100 ng/ ml IL-6 (Fig. 3a) and 50 ng/ml OSM (Fig. 3b) stimulated STAT3^Y705 phosphorylation in L3.6pl cells after short (6 hours) and long time (24 hours) incubation. Then, we evaluated different SC144 concentrations (1–5 µM) to inhibit the IL-6- or OSM-induced STAT3 phosphorylation. 5 µM SC144 suppressed the phosphorylation of STAT3^Y705 signficantly in both IL-6 and OSM challenged cells. IL-6 stimulated STAT3^Y705 phosphorylation was significantly decreased after 24 hours treatment but did not reach significance after six-hour treatment (Fig. 3a and b). Inhibitory SC144 effects on OSM stimulated STAT3^Y705 phosphorylation were significant after six hours, but no more after 24 hours (Fig. 3b and 3d).

Clinicopathological parameters

The study population consisted of 94 males and 81 females ranging from 32 to 88 years (median 68.4 years). Most patients were older than 60 years (76%) and underwent either a Whipple procedure (34.9%) or a pylorus-preserving partial pancreatoduodenectomy (44.6%) for tumors in the head of the pancreas. As shown in Table 1, most of tumor samples showed advanced tumor infiltration (pT3 or pT4 = 84.6%) and lymph node involvement (pN1 = 64%), whereas 8.6% of the patients had already developed distant metastases. The median number of lymph nodes analyzed was 13 (range 0–41). The histopathological examination showed high-grade tumors (G3) in the majority (66.9%) of tissue samples and microscopic residual disease after resection in 42.3% of the tumors. Most patients underwent either perioperative chemotherapy (33.2%) or chemoradiotherapy (45.1%), whereas 21.7% of the patients had no adjuvant therapy. The characteristics of the study subjects are summarized in Table 1.

Table 1

Clinicopathological parameters of 175 patients with resected pancreatic ductal adenocarcinoma associated with epithelial and stromal gp130 expression.
Characteristics		n	gp130 expression* (%)
		175	epithelial	p	stromal	p
Age	≤ 60 years	42	80.5	0.765	9.8	1.000
	> 60 years	133	78.3		9.3
Sex	Male	94	79.3	0.856	9.8	0.857
	Female	81	78.2		9.0
Tumor infiltration	T1-2	17	87.5	0.527	6.3	1.000
	T3-4	158	77.9		9.7
Lymph node status	N0	63	78.7	0.974	8.2	0.685
	N1	112	78.9		10.1
Metastasis	M0	160	78.7	1.000	9.7	1.000
	M1	15	80.0		6.7
Staging	0-IIa	54	76.9	0.687	9.6	0.952
	IIb-IV	121	79.7		9.3
Grading	G1-2	58	83.6	0.288	7.3	0.587
	G3	117	76.5		10.4
Residual Tumor	R0	97	71.6	0.015	9.5	0.820
	R1	74	87.3		8.5
Chemotherapy	No	38	83.8	0.404	8.1	1.000
	CTx	137	77.4		9.8
Radiochemotherapy	No	96	77.7	0.680	9.6	0.936
	RCTx	79	80.3		9.2
^*gp130 expression includes following immunohistological staining scores: 1 (weak), 2 (moderate) or 3 (strongly positive); Staging according to UICC 2010; CTx, Chemotherapy; RCTx, Radiochemotherapy.

Immunohistochemical analysis

Epithelial gp130 expression was found in 78.8% of the tumor samples and stromal gp130 expression in 9.4% of the tumors. Representative examples of immunohistochemical staining of PDAC tissue microarrays for gp130 proteins are shown in Fig. 4a. Apart from residual tumor status (R status), no significant correlation of clinicopathological parameters with the epithelial or stromal gp130 expression was found (Table 1).

Survival analysis

The median overall survival of patients with PDAC was 16.3 months [interquartile range (IQR) 8.7–37.7] and the mean overall survival 33.3 months. The median progress-free survival (PFS) was 15.5 months (IQR 8.1–30.1) and the mean progress-free survival 34.6 months.

Correlation of epithelial gp130 expression in human PDAC tissue with patient survival

The median overall survival for PDAC patients with no epithelial gp130 expression was 15.9 (IQR 10.7–30.0) months, whereas for patients expressing epithelial gp130 in the pancreatic tumors was 16.7 (IQR 8.5–37.7) months (p = 0.830). Patients expressing epithelial gp130 showed a trend for poorer progress-free survival (median 15.0 months, IQR 7.1–29.7) than patients with no epithelial gp130 expression (median 19.1 months, IQR 10.5–31.8), but without significant prognostic relevance (p = 0.247). The corresponding survival curves are shown in Fig. 4.

Correlation of stromal gp130 expression in human PDAC tissue with patient survival

The median overall survival for PDAC patients with no stromal gp130 expression was 16.2 (IQR 8.7–34.2) months, whereas for patients expressing stromal gp130 in the pancreatic tumors was 22.9 (IQR 12.2–117.6) months (p = 0.144). While patients with no stromal gp130 expression showed a poorer survival than patients with PDAC stromal gp130 expression, this difference did not reach significance. The median progress-free survival for patients without and with stromal gp130 expression was 15.0 (IQR 7.7–29.8) and 18.4 (IQR 9.6–33.7) months, respectively (p = 0.469). Stromal gp130 expression showed no significant prognostic relevance). The corresponding survival curves are shown in Fig. 4.

In the present study, we identified gp130 as a novel potential drug target for PDAC therapy. We showed that the small molecule gp130 inhibitor SC144 suppresses PDAC cell viability and proliferation and inhibits IL-6 or OSM-induced gp130/STAT3 signaling in vitro. Furthermore, we proved that gp130 is expressed in the epithelium of the majority of human PDAC samples, making it a valid candidate for therapeutically targeting by SC144.

IL-6/gp130/STAT3 signaling has an established role in the aggressive and metastatic phenotype of PDAC and constitutes one of the essential signaling cascades in pancreatic cancer initiation and progression (Lesina et al., 2014). IL-6 mediates part of its functions through the IL-6-receptor complex (IL-6R). The IL-6-receptor is a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL-6R-subunit (chain α) and the signal transducer gp130 (chain β). gp130 (also known as CD130), as a compound of the IL-6R complex, plays a key role in this signaling cascade and, in contrast to IL-6R-subunit, is ubiquitously expressed. Although initially identified as the β subunit of the IL-6R complex, gp130 also transmits signals of other members of the IL-6 family cytokine receptors. gp130 is the signaling transducer subunit of IL-11, IL-27, IL-31, OSM, cardiotrophin-1, cardiotrophin-like cytokine, ciliary neurotrophic factor and leukemia inhibitory factor (Heinrich et al., 2003). Through binding to the cytokine receptor, gp130 transmits the signal intracellular and activates the tyrosine kinases janus kinases JAK1, JAK2, and tyrosine kinase TYK2, which leads to the phosphorylation of STAT1 and STAT3. Phosphorylated STAT1 and STAT3 translocate to the cell nucleus (Hirano, 1992; Kishimoto et al., 1992; Rincon, 2012; Syed et al., 2002; Yamamoto et al., 2000) and regulate the transcription of target genes involved in proliferation, apoptosis, survival, cell cycle progression, angiogenesis and immunosuppression, playing a pivotal role in many cellular processes (Scheller et al., 2006).

SC144 was recently discovered as a novel small molecule gp130 inhibitor with a broad-spectrum anticancer activity (Oshima et al., 2009). Because gp130 is the ubiquitous signaling transducer receptor subunit of all IL-6 family cytokines, SC144 could potentially inhibit STAT3 phosphorylation globally through every gp130-mediated cytokine, achieving a better result on blocking STAT3 effects.

Within the present study, we demonstrated that SC144 binds specifically to gp130, inhibited proliferation and reduced viability in human PDAC cells. We observed that SC144 suppresses both IL-6 and OSM signaling in a dose-dependent manner in human L3.6pl pancreatic cancer cells. Inhibition of IL-6 and OSM stimulated STAT3^Y705 phosphorylation was observed by 5 µM SC144. The potential inhibitory effect of SC144 on STAT3 signaling, induced by two different cytokines is promising. However, the role of the other IL-6 family cytokines in pancreatic cancer is mostly unknown and remains to be further analyzed. Previous studies have investigated SC144 in other human carcinoma cell lines and showed its cytotoxity in e.g., ovarian cancer cells, lung adenocarcinoma cell lines, human breast cancer lines or colorectal cancer cells (Plasencia et al. 2009). Tumor growth inhibition by SC144 was reported from xenograft mouse models of breast, colorectal (Plasencia et al. 2009) and ovarian cancer (Xu et al. 2013) so far.

Our previous study on tissue microarrays from a cohort of 175 patients with PDAC showed that STAT3, phosphorylated STAT3 and IL-6 were expressed in more than half of the examined pancreatic tumors, supporting the importance of this pathway in pancreatic cancer (Pozios et al., 2018). However, the expression of gp130 was not examined in pancreatic tumors so far. In the present study, we proved in a cohort of 175 patients that gp130 was expressed in the epithelium of most of the examined pancreatic tumors which validates the role of gp130 as a promising chemotherapeutic target in patients with PDAC. Our survival analysis demonstrated a poorer prognosis for patients with gp130 expression in tumor epithelium, but a trend for better prognosis for tumors expressing gp130 in tumor stroma. However, these differences did not reach significance in our cohort. A tumor epithelial gp130 expression may indicate an activation of IL-6/gp130/STAT3 pathway, which acts as a tumorigenic factor in PDAC, while gp130 stroma expression may contribute to an inflammatory reaction of tumormicroenvironment to inhibit tumor spreading. Epithelial gp130 expression was significantly more frequent in patients with residual tumor (R1 status) indicating an aggressive tumor biology, while no other significant correlation of clinicopathological parameters with the gp130 expression was found (Table 1).

The above data indicate IL-6/gp130/STAT3 pathway as a promising chemotherapeutic target in patients with PDAC. In a previous study of our group, we identified raloxifene to exert an inhibitory effect on IL-6/gp130/STAT3 signaling in PDAC cells and on tumor growth in a xenograft mouse model (Pozios et al., 2020). Recently, different IL-6-directed humanized, murine or chimeric antibodies are under investigation in phase I, II, or III clinical trials as anti-inflammatory or anticancer therapeutic agents (Olokizumab, Sirukumab, Siltuximab, Clazakizumab, PF-423691, MED15117, and Elsilimomab) (Yao et al., 2014). For example, the humanized anti-IL-6R-subunit antibody tocilizumab (Actemra, RoActemra in the EU) that has been approved in more than 100 countries for rheumatoid arthritis and other autoinflammatory diseases, is currently examined in combination with gemcitabine/nab-paclitaxel as first-line treatment in patients with locally advanced or metastatic pancreatic cancer (MD, 2022). Regarding the fact that gp-130/STAT3 signaling can be activated by numerous different cytokines and growth factor, it can be hypothesized that therapeutically targeting the gp130 subunit might be more effective than blocking IL-6 solely.

In the present study, we found that SC144 potently inhibits PDAC progression in vitro through binding and inhibition of gp130 and, causes abrogation of IL-6 or OSM/gp130/STAT3 signaling. Moreover, we proved gp130 to be expressed on the majority of human pancreatic ductal adenocarcinoma. Taken together, these results suggest gp130 as a novel drug target and SC144 as a potential new therapy for pancreatic cancer. The interactions with the other IL-6 related cytokines are to be further investigated.

IL-6: Interleukin-6

OSM: Oncostatin-M

PDAC: pancreatic ductal adenocarcinoma

gp130: Glycoprotein 130

STAT3: signal transducer and activator of transcription-3

IL-6R: IL-6-receptor complex

sIL-6R: soluble IL-6 receptor

DMSO: dimethyl sulfoxide

BrdU: 5-Bromo-2′-deoxyuridine (BrdU)

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

SDS: sodium dodecyl sulfate

DARTS: Drug Affinity Responsive Target Stability (DARTS)

IQR: interquartile range

Ethics approval and consent to participate

The TMA study was approved by the Ethics Committee of the Hospital of the Ludwig-Maximilians-University of Munich.

Consent for publication

All authors have agreed to be published.

Availability of data and materials

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

All authors declare that they have no conflict of interest.

Funding

This study was supported by Grant of the Oskar-Helene-Heim Foundation (approval No. 48, Pozios, PI).

Authors’ contributions

All authors participated in the drafting of the article or its critical revision for important intellectual content and approved the publication of the final version of the manuscript. IP, NH and HS conceived and designed the study. IP, NH, EG and MA performed the cell biology and molecular biology experiments. IP, SE, TK and CB cooperate for the TMA construction and data collection. IP, NH and EG analyzed the data. IP and NH organized figures and wrote the manuscript. GM, CK, MK, KB and HS revised the manuscript. All authors have read and approved the manuscript.

Acknowledgments

Part of this was conducted in the context of a PhD study at Charité Universitätsmedizin Berlin, Germany and will be published as a part of a full thesis in the library of the Freie Universität Berlin.

Bellone, G., Smirne, C., Mauri, F.A., Tonel, E., Carbone, A., Buffolino, A., Dughera, L., Robecchi, A., Pirisi, M., and Emanuelli, G. (2005). Cytokine expression profile in human pancreatic carcinoma cells and in surgical specimens: implications for survival. Cancer Immunol Immunother 55, 684–698.
Bruns, C.J., Harbison, M.T., Kuniyasu, H., Eue, I., and Fidler, I.J. (1999). In Vivo Selection and Characterization of Metastatic Variants from Human Pancreatic Adenocarcinoma by Using Orthotopic Implantation in Nude Mice. Neoplasia 1, 50–62.
Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., and Mitchell, J.B. (1987). Evaluation of a Tetrazolium-based Semiautomated Colorimetric Assay: Assessment of Chemosensitivity Testing. Cancer Res 47, 936–942.
Corcoran, R.B., Contino, G., Deshpande, V., Tzatsos, A., Conrad, C., Benes, C.H., Levy, D.E., Settleman, J., Engelman, J.A., and Bardeesy, N. (2011). STAT3 Plays a Critical Role in KRAS-Induced Pancreatic Tumorigenesis. Cancer Res 71, 5020–5029.
Denley, S.M., Jamieson, N.B., McCall, P., Oien, K.A., Morton, J.P., Carter, C.R., Edwards, J., and McKay, C.J. (2013). Activation of the IL-6R/Jak/Stat Pathway is Associated with a Poor Outcome in Resected Pancreatic Ductal Adenocarcinoma. J Gastrointest Surg 17, 887–898.
van Duijneveldt, G., Griffin, M.D.W., and Putoczki, T.L. (2020). Emerging roles for the IL-6 family of cytokines in pancreatic cancer. Clin Sci (Lond) 134, 2091–2115.
Falconer, J.S.F.R.C.S., Fearon, K.C.H.M.D., Plester, C.E.B.S., Ross, J.A., and Carter, D.C.M.D. (1994). Cytokines, the Acute-Phase Response, and Resting Energy Expenditure in Cachectic Patients with Pancreatic Cancer. Annals of Surgery 219, 325–331.
Heinrich, P.C., Behrmann, I., Haan, S., Hermanns, H.M., Müller-Newen, G., and Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374, 1–20.
Hirano, T. (1992). The biology of interleukin-6. Chem. Immunol. 51, 153–180.
Kishimoto, T., Akira, S., and Taga, T. (1992). Interleukin-6 and its receptor: a paradigm for cytokines. Science 258, 593–597.
Lesina, M., Kurkowski, M.U., Ludes, K., Rose-John, S., Treiber, M., Klöppel, G., Yoshimura, A., Reindl, W., Sipos, B., Akira, S., et al. (2011). Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer. Cancer Cell 19, 456–469.
Lesina, M., Wörmann, S.M., Neuhöfer, P., Song, L., and Algül, H. (2014). Interleukin-6 in inflammatory and malignant diseases of the pancreas. Seminars in Immunology 26, 80–87.
Lomenick, B., Hao, R., Jonai, N., Chin, R.M., Aghajan, M., Warburton, S., Wang, J., Wu, R.P., Gomez, F., Loo, J.A., et al. (2009). Target identification using drug affinity responsive target stability (DARTS). PNAS 106, 21984–21989.
Lomenick, B., Jung, G., Wohlschlegel, J.A., and Huang, J. (2011). Target identification using drug affinity responsive target stability (DARTS). Curr Protoc Chem Biol 3, 163–180.
MD, I.C. (2022). A Multinational, Randomized, Phase II Study of the Combination of Nab-Paclitaxel and Gemcitabine With or Without Tocilizumab, an IL-6R Inhibitor, as First-line Treatment in Patients With Locally Advanced or Metastatic Pancreatic Cancer. (clinicaltrials.gov).
Okada, S., Okusaka, T., Ishii, H., Kyogoku, A., Yoshimori, M., Kajimura, N., Yamaguchi, K., and Kakizoe, T. (1998). Elevated Serum Interleukin-6 Levels in Patients with Pancreatic Cancer. Jpn. J. Clin. Oncol. 28, 12–15.
Oshima, T., Cao, X., Grande, F., Yamada, R., Garofalo, A., Louie, S., and Neamati, N. (2009). Combination effects of SC144 and cytotoxic anticancer agents. Anticancer Drugs 20, 312–320.
Pozios, I., Knösel, T., Zhao, Y., Assmann, G., Pozios, I., Müller, M.H., Bruns, C.J., Kreis, M.E., and Seeliger, H. (2018). Expression of phosphorylated estrogen receptor beta is an independent negative prognostic factor for pancreatic ductal adenocarcinoma. J Cancer Res Clin Oncol 144, 1887–1897.
Pozios, I., Seel, N.N., Hering, N.A., Hartmann, L., Liu, V., Camaj, P., Müller, M.H., Lee, L.D., Bruns, C.J., Kreis, M.E., et al. (2020). Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERβ and IL-6/gp130/STAT3 signaling. Cell Oncol (Dordr).
Rahib, L., Smith, B.D., Aizenberg, R., Rosenzweig, A.B., Fleshman, J.M., and Matrisian, L.M. (2014). Projecting Cancer Incidence and Deaths to 2030: The Unexpected Burden of Thyroid, Liver, and Pancreas Cancers in the United States. Cancer Res 74, 2913–2921.
Rincon, M. (2012). Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends in Immunology 33, 571–577.
Salama, C., Han, J., Yau, L., Reiss, W.G., Kramer, B., Neidhart, J.D., Criner, G.J., Kaplan-Lewis, E., Baden, R., Pandit, L., et al. (2021). Tocilizumab in Patients Hospitalized with Covid-19 Pneumonia. New England Journal of Medicine 384, 20–30.
Scheller, J., Ohnesorge, N., and Rose-John, S. (2006). Interleukin-6 trans-signalling in chronic inflammation and cancer. Scand. J. Immunol. 63, 321–329.
Syed, V., Ulinski, G., Mok, S.C., and Ho, S.-M. (2002). Reproductive Hormone-Induced, STAT3-Mediated Interleukin 6 Action in Normal and Malignant Human Ovarian Surface Epithelial Cells. JNCI J Natl Cancer Inst 94, 617–629.
Taher, M.Y., Davies, D.M., and Maher, J. (2018). The role of the interleukin (IL)-6/IL-6 receptor axis in cancer. Biochem Soc Trans 46, 1449–1462.
Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., and Jemal, A. (2015). Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians 65, 87–108.
Waetzig, G.H., and Rose-John, S. (2012). Hitting a complex target: an update on interleukin-6 trans-signalling. Expert Opinion on Therapeutic Targets 16, 225–236.
Xu, S., and Neamati, N. (2013). gp130: a promising drug target for cancer therapy. Expert Opinion on Therapeutic Targets 17, 1303–1328.
Xu, S., Grande, F., Garofalo, A., and Neamati, N. (2013). Discovery of a Novel Orally Active Small-Molecule gp130 Inhibitor for the Treatment of Ovarian Cancer. Mol Cancer Ther 12, 937–949.
Yamamoto, T., Matsuda, T., Junicho, A., Kishi, H., Saatcioglu, F., and Muraguchi, A. (2000). Cross-talk between signal transducer and activator of transcription 3 and estrogen receptor signaling. FEBS Letters 486, 143–148.
Yao, X., Huang, J., Zhong, H., Shen, N., Faggioni, R., Fung, M., and Yao, Y. (2014). Targeting interleukin-6 in inflammatory autoimmune diseases and cancers. Pharmacology & Therapeutics 141, 125–139.
Yu, H., Pardoll, D., and Jove, R. (2009). STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 9, 798–809.
Cancer statistics, 2022 - Siegel - 2022 - CA: A Cancer Journal for Clinicians - Wiley Online Library.

originalblotsIL6andOSM.pptx

SC144 - a promising small-molecule gp130-inhibitor of IL-6 and Oncostatin-M signaling for treatment of pancreatic cancer

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