Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) Stability Contributes to YM155 Resistance in Human Gastric Cancer Cells

YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.     

Akap200 suppresses the effects of Dv-cbl expression in the Drosophila eye

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.     

YM155 sensitizes non-small cell lung cancer cells to EGFR-tyrosine kinase inhibitors through the mechanism of autophagy induction

Resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib and gefitinib, is a major clinical problem in the treatment of patients with non-small cell lung cancer (NSCLC). YM155 is a survivin small molecule inhibitor and has been demonstrated to induce cancer cell apoptosis and autophagy. EGFR-TKIs have been known to induce cancer cell autophagy. In this study, we showed that YM155 markedly enhanced the sensitivity of erlotinib to EGFR-TKI resistant NSCLC cell lines H1650 (EGFR exon 19 deletion and PTEN loss) and A549 (EGFR wild type and KRAS mutation) through inducing autophagy-dependent apoptosis and autophagic cell death. The effects of YM155 combined with erlotinib on apoptosis and autophagy inductions were more obvious than those of YM155 in combination with survivin knockdown by siRNA transfection, suggesting that YM155 induced autophagy and apoptosis in the NSCLC cells partially depend on survivin downregulation. Meanwhile, we found that the AKT/mTOR pathway is involved in modulation of survivin downregulation and autophagy induction caused by YM155. In addition, YM155 can induce DNA damage in H1650 and A549 cell lines. Moreover, combining erlotinib further augmented DNA damage by YM155, which were retarded by autophagy inhibitor 3MA, or knockdown of autophagy-related protein Beclin 1, revealing that YM155 induced DNA damage is autophagy-dependent. Similar results were also observed in vivo xenograft experiments. Therefore, combination of YM155 and erlotinib offers a promising therapeutic strategy in NSCLC with EGFR-TKI resistant phenotype.     

Survivin and YM155: How faithful is the liaison?

Survivin belongs to the family of apoptosis inhibitors (IAPs), which antagonizes the induction of cell death. Dysregulated expression of IAPs is frequently observed in cancers, and the high levels of survivin in tumors compared to normal adult tissues make it an attractive target for pharmacological interventions. The small imidazolium-based compound YM155 has recently been reported to block the expression of survivin via inhibition of the survivin promoter. Recent data, however, question that this is the sole and main effect of this drug, which is already being tested in ongoing clinical studies. Here, we critically review the current data on YM155 and other new experimental agents supposed to antagonize survivin. We summarize how cells from various tumor entities and with differential expression of the tumor suppressor p53 respond to this agent in vitro and as murine xenografts. Additionally, we recapitulate clinical trials conducted with YM155. Our article further considers the potency of YM155 in combination with other anti-cancer agents and epigenetic modulators. We also assess state-of-the-art data on the sometimes very promiscuous molecular mechanisms affected by YM155 in cancer cells.     

Response of Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Xenografts to a Survivin Inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.     

Targeting Survivin Enhances Chemosensitivity in Retinoblastoma Cells and Orthotopic Tumors

Treatments for retinoblastoma (Rb) vary depending on the size and location of the intraocular lesions and include chemotherapy and radiation therapy. We examined whether agents used to treat Rb induce a pro-survival phenotype associated with increased expression of survivin, a member of the inhibitor of apoptosis family of proteins. We document that exposure to carboplatin, topotecan or radiation resulted in elevated expression of survivin in two human Rb cell lines but not in normal retinal pigmented epithelial (RPE) cells. Cellular levels of survivin were attenuated in Rb cells exposed to an imidazolium-based survivin suppressant, Sepantronium bromide (YM155). Protein expression patterns of survivin in RPE cells were not altered following treatment protocols involving exposure to YM155. Including YM155 with chemotherapy or radiation increased levels of apoptosis in Rb cells but not in RPE cells. Intraocular luciferase expressing Rb tumors were generated from the Rb cell lines and used to evaluate the effects of carboplatin and YM155 on in-vivo survivin expression and tumor growth. Carboplatin induced expression of survivin while carboplatin combined with YM155 reduced survivin expression in tumor bearing eyes. The combination protocol was also most effective in reducing the rate of tumor regrowth. These results indicate that targeted inhibition of the anti-apoptotic protein survivin provides a therapeutic advantage for Rb cells and tumors treated with chemotherapy.     

Induction of autophagy-dependent cell death by the survivin suppressant YM155 in salivary adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is one of the most common malignancies of the major and minor salivary glands. However, the molecular mechanism underlying the aggressive growth of human salivary ACC remains unclear. In the present study, we showed that survivin, which belongs to the family of inhibitors of apoptosis, is closely related to the high expression of CDK4 and cyclin D1 in human ACC specimens. By employing the small-molecule drug YM155, we found that the inhibition of survivin in ACC cells caused significant cell death and induced autophagy. Chloroquine, an autophagy inhibitor, prevented cell death induced by YM155, suggesting YM155-induced autophagy contributed to the cell death effects in ACC cells. More importantly, evidence obtained from a xenograft model using ACC-2 cells proved the occurrence of YM155-induced autophagy and cell death in vivo was correlated with the suppression of Erk1/2 and S6 activation as well as increased TFEB nuclear translocation. Taken together, our results indicate YM155 is a novel inducer of autophagy-dependent cell death and possesses therapeutic potential in ACC.     

长非编码 RNA ILF3-AS1 通过调控 miR-130a-3p/PTEN轴抑制宫颈癌细胞的增殖

ILF3反义 RNA 1(ILF3 antisense RNA 1,ILF3-AS1)是一条定位于染色体 19p13.2的lncRNA,它是白介素增强子结合因子3(interleukin enhancer binding factor 3,ILF3)的反义 RNA.ILF3-AS1在多种肿瘤发生发展中发挥关键作用,但其...     

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.Head and neck squamous cell carcinoma (HNSCC), which occurs in the oral cavity, oropharynx, larynx and hypopharynx, is the sixth most common malignancy worldwide.¹ It affects 600 000 new patients each year, which accounts for over 90% of head and neck cancers.^{2, 3} The current preferred therapy for HNSCC is combined surgery, radiotherapy, chemotherapy and biotherapy; however, the 5-year survival rate is still <50%, and the long-term survival rate has only marginally improved.^{4, 5, 6} As an important hallmark of head and neck cancer, apoptosis resistance restricts the efficacy of traditional therapies.⁷ Survivin (also called BIRC5) inhibits apoptosis-related proteins, regulates cell division, relates to stress response and promotes tumor-associated angiogenesis in HNSCC.⁸ Survivin is also associated with high-grade and advanced HNSCC, poor survival, high recurrence rate and chemotherapy and radiation resistance. Therefore, targeting survivin is promisingly beneficial for head and neck cancer therapies.Sepantronium bromide (YM155) is a small imidazolium-based compound (1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide) that selectively suppresses survivin expression⁹ and displays potent anticancer activities against various types of cancer.^{10, 11, 12} Previous researches have focused on that YM155 induced the apoptosis by downregulating survivin in cancer cells.^{10, 13, 14} Recent studies including ours have demonstrated YM155 also triggered autophagy in cancer cells.^{15, 16, 17} Macroautophagy or autophagy is considered to be another type of programmed cell death wherein proteins are degraded by autophagosomes and lysosomes.¹⁸ Autophagy also has an important role in tumorigenesis.¹⁹ Autophagy shares several regulatory systems and common pathways with apoptosis; thus, autophagy is closely linked with apoptosis. Beclin1 (ATG6), an autophagy-specific gene that is essential for autophagosome induction and elongation, interacts with several apoptosis-related genes, such as bcl-2, bcl-xl and survivin.²⁰ Therefore, YM155 may not only induce the apoptosis but also affect the autophagy in HNSCC.The present study investigated the antitumor effects of YM155 on HNSCC in vitro and in vivo through dual induction of apoptotic and autophagic cell death. Although it specifically suppressed the expression of survivin, we here proved YM155 also targeted the mTOR signaling pathway, which was the principal regulator of cancer cell survival and autophagy. Most importantly, in an inducible tissue-specific spontaneous HNSCC mouse model with 100% penetrance by the combined deletion of Tgfbr1 and Pten (Tgfbr1/Pten 2cKO) in the oral mucosa²¹ with ubiquitous activation of the Akt/mTOR/survivin pathway,²² YM155 exerted significant therapeutic effects by delaying tumor onset and suppressing tumor growth. This finding coincided with the xenograft results. Finally, the effects of YM155 when combined with traditional chemotherapeutic agent were also determined.     

Solution structure of the DNA-binding domain of interleukin enhancer binding factor 1 (FOXK1a)

Interleukin enhancer binding factor (ILF) binds to the interleukin-2 (IL-2) promoter and regulates IL-2 gene expression. In this study, the 3D structure of the DNA-binding domain of ILF was determined by multidimensional NMR spectroscopy. NMR structure analysis revealed that the DNA-binding domain of ILF is a new member of the winged helix/forkhead family, and that its wing 2 contains an extra alpha-helix. This is the first study to report the presence of a C-terminal alpha-helix in place of a typical wing 2 in a member of this family. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix/forkhead proteins. Our deletion studies of the fragments of ILF also suggest that the C-terminal region plays a regulatory role in DNA binding.     

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins.

We have identified a novel human karyopherin (Kap) beta family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.     

Protein-arginine methyltransferase I, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3

Arginine methylation is a common post-translation modification found in many proteins. Protein-arginine methyltransferase I (PRMT1) contributes >90% of type I protein-arginine methyltransferase activity in cells and tissues. To expand our knowledge on the regulation and role of PRMT1 in cells, we used the yeast two-hybrid system to identify proteins that interact with PRMT1. One of the interacting proteins we cloned is interleukin enhancer-binding factor 3 (ILF3), also known as M phase phosphoprotein 4. ILF3 is closely related to nuclear factor 90 (NF90). Using an immunofluorescence analysis, we determined that ILF3 and PRMT1 co-localize in the nucleus. Moreover, PRMT1 and ILF3 co-precipitate in immunoprecipitation assays and can be isolated together in "pull-down" experiments using recombinant fusion proteins. ILF3 is a robust substrate for methylation by PRMT1 and can modulate PRMT1 activity in in vitro methylation assays. Deletion studies demonstrated that the COOH-terminal region of ILF3, which is rich in arginine, glycine, and serine, is responsible for the strong interaction between PRMT1 and ILF3 and is the site of ILF3 methylation by PRMT1. Although ILF3 and NF90 are highly similar, they differ in their carboxyl-terminal regions. Because of this difference, NF90 does not interact with PRMT1, is a much poorer substrate than ILF3 for PRMT1-dependent methylation, and does not modulate PRMT1 enzyme activity.     

Adhesion Molecule-Mediated Hippo Pathway Modulates Hemangioendothelioma Cell Behavior

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell''s proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.     

Survivin suppression heightens BZML-induced mitotic catastrophe to overcome multidrug resistance by removing therapy-induced senescent A549/Taxol cells

Mitotic catastrophe (MC) is a newly identified type of anticancer mechanism for multidrug resistance (MDR) prevention. However, the long cellular death process resulting from MC is not beneficial for anticancer treatment. BZML is a novel colchicine-binding site inhibitor which can overcome MDR by inducing MC; however, BZML-induced MC cells underwent a long cellular death process. Thus, to improve anticancer therapies based on drug-induced MC, BZML-induced MC was served as a model to further study the underlying molecular mechanisms in the process of MC. Here, BZML could induce p53-dependent senescence in A549/Taxol cells, a MDR cell line. This senescence was a secondary effect of MC in overcoming MDR. During MC, BZML-induced destruction of protein-degradation system contributed not only to an increase of p53 protein but also to the accumulation of survivin in nucleus of A549/Taxol cells. Importantly, the nuclear accumulation of survivin was not the inducer but the result of BZML-induced MC, and it promoted the survival of senescent cells. Moreover, it provided additional vulnerability and critical opportunities for sequentially applied therapies. Further, targeting survivin with YM155 accelerated the death of MC cells by timely eliminating therapy-induced senescent cells and strengthening the efficiency of BZML in overcoming MDR in A549/Taxol cells. Collectively, nuclear accumulation of survivin delayed cellular death during MC by promoting the survival of BZML-induced senescent A549/Taxol cells. Moreover, “one-two punch” approach to cancer treatment based on combination therapy with YM155 for survivin suppression might be a new strategy for potentiating MC to overcome MDR.