Interleukins-6 (IL-6)/GP130 signaling pathway represents a promising target for cancer therapy due to its critical role in survival and progression of multiple types of cancer. We have identified Bazedoxifene, a Food and Drug Administration (FDA)-approved drug used for the prevention of postmenopausal osteoporosis, with novel function as inhibitor of IL-6/GP130 interaction. In this study, we investigate the effect of Bazedoxifene in rhabdomyosarcoma and evaluate whether inhibiting IL-6/GP130 signaling is an effective therapeutic strategy for rhabdomyosarcoma. The inhibitory effect of Bazedoxifene was assessed in rhabdomyosarcoma cell lines in vitro and RH30 xenograft model was used to further examine the suppressive efficacy of Bazedoxifene on tumor growth in vivo. Rhabdomyosarcoma cells showed their sensitivity to GP130 inhibition using gene knockdown or neutralized antibody, suggesting IL-6/GP130 as therapeutic target in rhabdomyosarcoma cells. Bazedoxifene decreased the signal transducer and activator of transcription3 (STAT3) phosphorylation, blocked STAT3 DNA binding, and down-regulated the expression of STAT3 downstream genes. Bazedoxifene also induced cell apoptosis, reduced cell viability, and inhibited colony formation in rhabdomyosarcoma cells. The inhibition of colony formation, STAT3 phosphorylation, or cell viability following Bazedoxifene treatment was partially reversed by addition of excess IL-6 or overexpression of constitutive STAT3, respectively, supporting Bazedoxifene acted through IL-6/GP130 signaling. In addition, Bazedoxifene repressed cell invasion and angiogenesis in vitro. Furthermore, oral administration of Bazedoxifene significantly suppressed tumor growth and expression of STAT3 phosphorylation in nude mice bearing established human rhabdomyosarcoma xenograft. Taken together, these findings validate IL-6/GP130 signaling as therapeutic target in rhabdomyosarcoma and provide first evidence that Bazedoxifene may serve as a novel promising drug targeting IL-6/GP130 for treatment of rhabdomyosarcoma.

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect. Because the spindle checkpoint pathway is highly conserved between yeast and humans, we performed a synthetic genetic array analysis in yeast, which revealed that Mad2 overexpression induced lethality in 13 gene deletions. Among the human homologs of candidate genes, knockdown of PPP2R1A, a gene encoding a constant regulatory subunit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 phosphorylation and suppressed Mad2 protein levels. Depletion of PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa cells but not of unphosphorylated Mad2 mutant-overexpressing cells, suggesting that the lethality induced by PP2A depletion in Mad2-overexpressing cells is dependent on Mad2 phosphorylation. Also, the PP2A inhibitor cantharidin induced Mad2 phosphorylation and inhibited the growth of Mad2-overexpressing cancer cells. Aurora B knockdown inhibited Mad2 phosphorylation in mitosis, resulting in the blocking of PPP2R1A inhibition-induced cell death. Taken together, our results strongly suggest that PP2A is a good therapeutic target in Mad2-overexpressing tumors.

Abstract The IL-6/GP130 signaling pathway is critical for the survival and progression of cancer cells of multiple cancer types. We report here the new discovery of Raloxifene (Marketed as Evista by Eli Lilly and Company) and Bazedoxifene [Marketed as Duavee (conjugated estrogens / bazedoxifene) by Pfizer] as novel inhibitors of IL-6/GP130 protein-protein interactions using multiple ligand simultaneous docking and drug repositioning approaches. Multiple drug scaffolds were simultaneously docked into binding hot spots of GP130 D1 domain by multiple ligand simultaneous docking to compete with the key interacting residues of IL-6, followed by tethering to generate virtual hit compounds. Similarity searches of virtual hits on drug databases identified Raloxifene and Bazedoxifene as potential inhibitors of IL-6/GP130 interaction. Both Raloxifene and Bazedoxifene are known as selective estrogen receptor modulators and we identified their novel function as GP130 inhibitors. Raloxifene and Bazedoxifene inhibited IL-6 mediated induction of STAT3 phosphorylation in cancer cells, which confirm their ability to inhibit IL-6/GP130 signaling. In contrast, Raloxifene and Bazedoxifene did not inhibit the induction of STAT1 phosphorylation by IFN-γ in cancer cells indicating their selectivity. Raloxifene and Bazedoxifene also inhibited STAT3 phosphorylation and resulted in apoptosis in cancer cells expressing elevated IL-6 levels. In addition, addition of excess IL-6 reversed the expression level of STAT3 phosphorylation which was decreased by Raloxifene or Bazedoxifene supporting that both FDA-approved drugs act through IL-6/GP130 signaling. Compared with an original GP130 inhibitor MDL-A whose IC50 values were 108-216 µM in cancer cell lines, Raloxifene and Bazedoxifene were found more potent in inhibiting cell viability which the IC50 values for Raloxifene and Bazedoxifene were 6.9-8.9 µM and 3.4-4.4 µM respectively in the same cancer cell lines expressing elevated IL-6 levels. Furthermore, both Raloxifene and Bazedoxifene significantly suppressed the tumor growth of human RH30 sarcoma cells in mouse xenograft model in vivo. These findings suggest that FDA-approved drugs Raloxifene and Bazedoxifene may serve as novel GP130-targeting therapeutic drugs in IL-6 dependent cancers. Citation Format: Hui Xiao, Yang Bian, Chengguang Zhao, Li Lin, David Jou, Huameng Li, Chenglong Li, Jiayuh Lin. GP130 as a novel therapeutic target in il-6-dependent cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2014-4598