E3 Ubiquitin Ligase NEDD4L Negatively Regulates Skin Tumorigenesis by Inhibiting IL-6/GP130 Signaling Pathway.

IL-6 signaling plays a crucial role in the survival and metastasis of skin cancer. NEDD4L acts as a suppressor of IL-6 signaling by targeting GP130 degradation. However, the effects of the NEDD4L-regulated IL-6/GP130 signaling pathway on skin cancer remain unclear. In this study, protein expression levels of NEDD4L and GP130 were measured in tumor tissues from patients with cutaneous squamous cell carcinoma. Skin tumors were induced in wild-type and Nedd4l-knockout mice, and activation of the IL-6/GP130/signal transducer and activator of transcription 3 signaling pathway was detected. The results indicated a negative correlation between the protein expression levels of NEDD4L and GP130 in cutaneous squamous cell carcinoma tissues from patients. Nedd4l deficiency significantly promoted 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin tumorigenesis and benign-to-malignant conversion by activating the IL-6/GP130/signal transducer and activator of transcription 3 signaling pathway, which was abrogated by supplementation with the GP130 inhibitor SC144. Furthermore, our findings suggested that NEDD4L can interact with GP130 and promote its ubiquitination in skin tumors. In conclusion, our results indicate that NEDD4L could act as a tumor suppressor in skin cancer, and inhibition of GP130 could be a potential therapeutic method for treating this disease.

Targeting Interleukin-6/Glycoprotein-130 Signaling by Raloxifene or SC144 Enhances Paclitaxel Efficacy in Pancreatic Cancer.

Interleukine-6 plays a key role in the progression and poor survival in pancreatic ductal adenocarcinoma (PDAC). The present study aimed to clarify if targeting the interleukin-6/glycoprotein-130 signaling cascade using the small-molecule gp130 inhibitor SC144 or raloxifene, a non-steroidal selective estrogen receptor modulator, enhances paclitaxel efficacy. MTT/BrdU assays or TUNEL staining were performed to investigate cell viability, proliferation and apoptosis induction in L3.6pl and AsPC-1 human pancreatic cell lines. In vivo, effects were studied in an orthotopic PDAC mouse model. Tumor specimens were analyzed by qPCR, immunohistochemistry and ELISA. Combination of paclitaxel/raloxifene, but not paclitaxel/SC144, enhanced proliferation and viability inhibition and increased apoptosis compared to single treatment in vitro. Synergy score calculations confirmed an additive influence of raloxifene on paclitaxel. In the PDAC mouse model, both combinations of raloxifene/paclitaxel and SC144/paclitaxel reduced tumor weight and volume compared to single-agent therapy or control. Raloxifene/paclitaxel treatment decreased survivin mRNA expression and showed tendencies of increased caspase-3 staining in primary tumors. SC144/paclitaxel reduced interleukin-6 levels in mice's tumors and plasma. In conclusion, raloxifene or SC144 can enhance the anti-tumorigenic effects of paclitaxel, suggesting that paclitaxel doses might also be reduced in combined chemotherapy to lessen paclitaxel side effects.

Gp130 is expressed in pancreatic cancer and can be targeted by the small inhibitor molecule SC144.

PURPOSE: Interleukin 6 (IL-6), Oncostatin M (OSM), and downstream effector STAT3 are pro-tumorigenic agents in pancreatic ductal adenocarcinoma (PDAC). Glycoprotein 130 (gp130) is a compound of the IL-6 and OSM receptor complex that triggers STAT3 signaling. SC144 is a small molecule gp130 inhibitor with anticancer activity. This study examines the gp130 expression in human PDAC specimens and the in vitro effects of SC144 in PDAC cell lines. METHODS: Tissue micro-arrays were constructed from 175 resected human PDAC. The gp130 expression in tumor epithelium and stroma was determined by immunohistochemistry, and survival analysis was performed. Growth inhibition by SC144 was assessed in vitro using BrdU and MTT assays. Western blotting was performed to evaluate the SC144 effect on IL-6 and OSM signaling. RESULTS: Gp130 was expressed in the epithelium of 78.8% and the stroma of 9.4% of the tumor samples. The median overall survival for patients with or without epithelial gp130 expression was 16.7 months and 15.9 months, respectively (p = 0.830). Patients with no stromal gp130 expression showed poorer survival than patients with stromal gp130 expression (median 16.2 and 22.9 months, respectively), but this difference did not reach significance (p = 0.144). SC144 inhibited cell proliferation and viability and suppressed IL-6- and OSM-stimulated STAT3Y705 phosphorylation in PDAC cells. CONCLUSION: Gp130 is expressed in the epithelium of most human PDAC, but stromal expression is rare. The small molecule gp130 inhibitor SC144 potently inhibits PDAC progression in vitro and may abrogate IL-6 or OSM/gp130/STAT3 signaling. These results suggest gp130 as a novel drug target for pancreatic cancer therapy.

Macrophage-NLRP3 Activation Promotes Right Ventricle Failure in Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) often results in death from right ventricular failure (RVF). NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3)-macrophage activation may promote RVF in PAH. Objectives: Evaluating the contribution of the NLRP3 inflammasome in RV macrophages to PAH RVF. Methods: Rats with decompensated RV hypertrophy (monocrotaline [MCT] and Sugen-5416 hypoxia [SuHx]) were compared with compensated RV hypertrophy rats (pulmonary artery banding). Echocardiography and right heart catheterization were performed. Macrophages, atrial natriuretic peptides, and fibrosis were evaluated by microscopy or flow cytometry. NLRP3 inflammasome activation and cardiotoxicity were confirmed by immunoblot and in vitro strategies. MCT rats were treated with SC-144 (a GP130 antagonist) or MCC950 (an NLRP3 inhibitor). Macrophage-NLRP3 activity was evaluated in patients with PAH RVF. Measurements and Main Results: Macrophages, fibrosis, and atrial natriuretic peptides were increased in MCT and SuHx RVs but not in left ventricles or pulmonary artery banding rats. Although MCT RV macrophages were inflammatory, lung macrophages were antiinflammatory. CCR2+ macrophages (monocyte-derived) were increased in MCT and SuHx RVs and highly expressed NLRP3. The macrophage-NLRP3 pathway was upregulated in patients with PAH with decompensated RVs. Cultured MCT monocytes showed NLRP3 activation, and in coculture experiments resulted in cardiomyocyte mitochondrial damage, which MCC950 prevented. In vivo, MCC950 reduced NLRP3 activation and regressed pulmonary vascular disease and RVF. SC-144 reduced RV macrophages and NLRP3 content, prevented STAT3 (signal transducer and activator of transcription 3) activation, and improved RV function without regressing pulmonary vascular disease. Conclusions: NLRP3-macrophage activation occurs in the decompensated RV in preclinical PAH models and patients with PAH. Inhibiting GP130 or NLRP3 signaling improves RV function. The concept that PAH RVF results from RV inflammation rather than solely from elevated RV afterload suggests a new therapeutic paradigm.

Up-regulation of hypoxia-inducible factor antisense as a novel approach to treat ovarian cancer.

Ovarian cancer (OC) is estimated to kill ~14,000 women in the United States in 2019. Current chemotherapies to treat OC initially show therapeutic efficacy but frequently drug resistance develops, at which point therapies with alternative targets are needed. Herein, we are describing a novel approach to sensitize these tumors to standard chemotherapies by increasing the transcription of hypoxia-inducible factor antisense. Methods: Genome-wide Bru-seq analysis was performed to fully capture the nascent transcriptional signature of OC cells treated with the gp130 inhibitor, SC144. In vitro and in vivo analysis, including characterization of hypoxia and select protein expression, combination with standard of care chemotherapy and antitumor efficacy were performed to assess the biological activity of SC144 on induction of hypoxia in OC cells. Results: Bru-seq analysis of OVCAR8 cells treated with SC144 shows upregulation of hypoxia related genes. In addition, transcription of hypoxia-inducible factor antisense (HIF1A-AS2) was induced that in turn reduced expression of HIF-1α and simultaneously increased expression of NDRG1. Furthermore, we observed decreased protein levels of EGFR, Met, c-Myc, cyclin D1, MMP-2, MMP-9 and TF, and phosphorylation of Src and P130-cas. SC144-induced alterations of HIF-1α and NDRG1 were also confirmed in prostate cancer cells. Ciclopirox olamine (CPX) induces a cellular transcriptional profile comparable to SC144, suggesting a similar cellular mechanism of action between these two compounds. In addition, SC144 sensitized OC cells to olaparib, carboplatin and cisplatin, and shows better in vivo efficacy than CPX. Conclusion: Induction of hypoxic stress responses through inhibition of gp130 represents a novel approach to design effective anticancer treatments in combination with standard-of-care chemotherapy in OC and the efficacy reported here strongly supports their clinical development.

Synergistic inhibition of GP130 and ERK signaling blocks chemoresistant bladder cancer cell growth.

Multidrug resistance is a major treatment obstacle for recurrent and metastatic bladder cancer, which often leads to disease progression and poor clinical outcome. Although overexpression of interleukin-6 (IL-6) appears to play a critical role in the development of chemotherapy resistance, inhibitors for IL-6 alone have not improved clinical outcomes. Since the IL-6/IL-6R/GP130 complex is involved in multidrug resistance, another strategy would be to focus on glycoprotein-130 (GP130) since it dimerizes with IL-6R/CD26 as a membrane-bound signaling transducer receptor and initiates subsequent signaling activation and may be a potential therapeutic target. Currently, the role of GP130 in chemoresistant bladder cancer is unknown. In the present study, we demonstrate that GP130 is over-expressed in cisplatin and gemcitabine-resistant bladder cancer cells, and that the inhibition of GP130 expression significantly reduces cell viability, survival and migration. Downstream of GP130 is PI3K/AKT/mTOR signaling, which is inactivated by SC144, a GP130 inhibitor. However, Raf/MEK/ERK signaling, which also is downstream of GP130 is activated by SC144. This activation is likely based on a mTOR/S6K1/PI3K/ERK negative feedback loop, which is presumed to counteract the inhibitory effect of SC144 on tumor aggressiveness. Blocking both GP130 and pERK resulted in synergistic inhibition of cytotoxicity, clonal survival rates and cell migration in our chemotherapy resistant bladder cancer cells. This vertical inhibition offers a novel therapeutic strategy for targeting human chemoresistant bladder cancer.