Overexpression Of Survivin Research Articles

Gravin and survivin gene expression levels as possible therapeutic targets for acute myeloid leukemia in adult Egyptian patients

Background Despite the recent progress in diagnosis and management, acute myeloid leukemia (AML) still remains a fatal hematologic malignancy, which invites the need for accurate predictors of clinical outcome. Objective The aim of the present study was to explore the possible prognostic importance of gravin and survivin gene expression in adult patients with de-novo AML by correlating their expression levels with response to induction therapy, disease-free survival (DFS), and overall survival (OS). Patients and methods This study was conducted on 105 patients with de-novo AML, and 45 age-matched and sex-matched patients were selected as a control group. RNA isolation from bone marrow aspirates or peripheral blood and cDNA preparation followed by quantitative real-time reverse transcription-PCR were done to assess expression of gravin and survivin. Results Gravin expression was markedly downregulated whereas survivin gene showed an overexpression in AML cases. There was a significant association between gravin and survivin expression levels with poor clinical outcome. OS and DFS were significantly lower in patients with low gravin and high survivin expression levels. Conclusion Survivin overexpression and gravin downregulation were significantly associated with adverse clinical outcome and tendency to chemoresistance in AML. The degree of their expression derangement has been found to be correlated with a lower complete remission rate and shorter OS and disease-DFS which renders them as future candidates for target through adjuvant immunotherapy.

Abstract 08: Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies

Abstract The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, IBR responses are incomplete in majority of cases, and not durable in MCL and high-risk CLL. We hypothesize that de novo resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be cotargeted with BTK to achieve deeper and durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generates synergistic cytotoxicity with IBR in MCL lines (Axelrod et al. Leukemia 2014;28:407-10). Recently, we extended these findings to circulating leukemic B cells in CLL, MCL (Jayappa et al. ASH 2016), and follicular lymphoma (FL) patient PBMCs treated ex vivo. Based on these results we initiated a phase I/Ib trial for IBR+VEN combination in MCL. Our ex vivo cytotoxicity studies showed a surprisingly high frequency of de novo resistance to IBR+VEN. Resistance did not correlate with previous therapy or clinical characteristics of patients. However, FACS analysis of primary CLL cells showed that cells displaying an “activation” phenotype (CD5+/CD19+/CD69+) were less sensitive to cytotoxicity of IBR+VEN, suggesting that cellular activation by extrinsic factors could generate drug resistance. To test this, we cocultured or preincubated CLL patient PBMCs with various stromal cells, cytokines, and other agonists and found that soluble CD40L, IL10, and CpG oligodeoxynucleotides (ODN) generated variable resistance to IBR+VEN in CLL samples, with CpG ODN, a specific TLR9 agonist, being the most effective. The combination of CpG ODN, sCD40L, and IL10 (“agonist mix”) generated near-complete resistance to IBR+VEN in most CLL(N=22)/MCL(N=7)/FL(N=4) patient PBMCs. The agonist mix treatment significantly upregulated nuclear localization of RelA and RelB proteins in CLL/MCL/FL cells analyzed by ImageStream, suggesting activation of NF-kB signaling. Agonist mix treatment also induced the NF-kB dependent overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, and inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame this drug resistance (Jayappa et al. ASH 2016). To further explore the role of TLR9 and other innate immune receptors in resistance, we performed IBR+VEN cytotoxicity analysis in CLL/MCL patient PBMCs cocultured with agonists of various TLRs and NOD1/2 receptors. Preliminary results showed that TLR9 agonist CpG ODN generated significant resistance to IBR+VEN in most CLL/MCL samples, and agonists of TLR1/2, TLR7, and NOD1/2 induced a moderate level of resistance in a MCL sample. The CpG ODN treatment induced a significant increase in NF-kB signaling measured by increased nuclear localization of RelB in CLL patient samples. To determine if soluble agonists in the extranodal macroenvironment of patients could generate NF-kB dependent resistance to IBR+VEN, we analyzed drug cytotoxicity in CLL(N=6) and MCL(N=1) patient PBMCs cultured with autologous plasma. We found that preincubation with autologous plasma induced variable levels of resistance to IBR+VEN in CLL/MCL samples, but did not induce resistance to a NF-kB inhibitor BMS-345541. In conclusion, soluble factors in the patient nodal/extranodal macroenvironment, particularly a TLR9 agonist, generate resistance to IBR+VEN by activating NF-kB signaling and overexpression of multiple anti-apoptotic proteins. The same resistance mechanism, based on innate immune signaling, occurs in three mature B-cell malignancies, CLL, MCL and FL, that differ in etiology and clinical course. The signaling pathway responsible for resistance contains new targets for therapeutic intervention with potential to prevent or reverse resistance to IBR and VEN in these and perhaps other cancers as well. Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 08.

HDAC2 and HDAC5 Up-Regulations Modulate Survivin and miR-125a-5p Expressions and Promote Hormone Therapy Resistance in Estrogen Receptor Positive Breast Cancer Cells.

Intrinsic or acquired resistance to hormone therapy is frequently reported in estrogen receptor positive (ER+) breast cancer patients. Even though dysregulations of histone deacetylases (HDACs) are known to promote cancer cells survival, the role of different HDACs in the induction of hormone therapy resistance in ER+ breast cancer remains unclear. Survivin is a well-known pro-tumor survival molecule and miR-125a-5p is a recently discovered tumor suppressor. In this study, we found that ER+, hormone-independent, tamoxifen-resistant MCF7-TamC3 cells exhibit increased expression of HDAC2, HDAC5, and survivin, but show decreased expression of miR-125a-5p, as compared to the parental tamoxifen-sensitive MCF7 breast cancer cells. Molecular down-regulations of HDAC2, HDAC5, and survivin, and ectopic over-expression of miR-125a-5p, increased the sensitivity of MCF7-TamC3 cells to estrogen deprivation and restored the sensitivity to tamoxifen. The same treatments also further increased the sensitivity to estrogen-deprivation in the ER+ hormone-dependent ZR-75-1 breast cancer cells in vitro. Kaplan–Meier analysis and receiver operating characteristic curve analysis of expression cohorts of breast tumor showed that high HDAC2 and survivin, and low miR-125a-5p, expression levels correlate with poor relapse-free survival in endocrine therapy and tamoxifen-treated ER+ breast cancer patients. Further molecular analysis revealed that HDAC2 and HDAC5 positively modulates the expression of survivin, and negatively regulates the expression miR-125a-5p, in ER+ MCF7, MCF7-TamC3, and ZR-75-1 breast cancer cells. These findings indicate that dysregulations of HDAC2 and HDAC5 promote the development of hormone independency and tamoxifen resistance in ERC breast cancer cells in part through expression regulation of survivin and miR-125a-5p.

Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive degeneration of spinal-cord motor neurons, leading to atrophy of skeletal muscles. However, accumulating evidence indicates that it is a multi-system disorder, particularly in its severe forms. Several studies delineated structural and functional cardiac abnormalities in SMA patients and mouse models, yet the abnormalities have been primarily attributed to autonomic dysfunction. Here, we show in a severe mouse model that its cardiomyocytes undergo G0/G1 cell-cycle arrest and enhanced apoptosis during postnatal development. Microarray and real-time RT-PCR analyses revealed that a set of genes associated with cell cycle and apoptosis were dysregulated in newborn pups. Of particular interest, the Birc5 gene, which encodes Survivin, an essential protein for heart development, was down-regulated even on pre-symptomatic postnatal day 0. Interestingly, cultured cardiomyocytes depleted of SMN recapitulated the gene expression changes including downregulation of Survivin and abnormal cell-cycle progression; and overexpression of Survivin rescued the cell-cycle defect. Finally, increasing SMN in SMA mice with a therapeutic antisense oligonucleotide improved heart pathology and recovered expression of deregulated genes. Collectively, our data demonstrate that the cardiac malfunction of the severe SMA mouse model is mainly a cell-autonomous defect, caused by widespread gene deregulation in heart tissue, particularly of Birc5, resulting in developmental abnormalities through cell-cycle arrest and apoptosis.

Chromosomal instability induced by increased BIRC5/Survivin levels affects tumorigenicity of glioma cells

BackgroundSurvivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear.MethodsWe overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice.ResultsOverexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors.ConclusionsOur data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.

Up-regulation of 5-lipoxygenase by inhibition of cathepsin G enhances TRAIL-induced apoptosis through down-regulation of survivin.

Cathepsin G is a serine protease secreted from activated neutrophils, it has important roles in inflammation and immune response. Moreover, cathepsin G promotes tumor cell-cell adhesion and migration in cancer cells. In this study, we investigated whether inhibition of cathepsin G could sensitize TRAIL-mediated apoptosis in cancer cells. An inhibitor of cathepsin G [Cathepsin G inhibitor I (Cat GI); CAS 429676-93-7] markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), lung cancer (A549) and cervical cancer (Hela) cells. In contrast, combined treatment with Cat GI and TRAIL had no effect on apoptosis in normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. Cat GI induced down-regulation of survivin expression at the post-translational level, and overexpression of survivin markedly blocked apoptosis induced by combined treatment with Cat GI plus TRAIL. Interestingly, Cat GI induced down-regulation of survivin via 5-lipoxygenase (5-LOX)-mediated reactive oxygen species (ROS) production. Inhibition of 5-LOX by gene silencing (siRNA) or a pharmacological inhibitor of 5-LOX (zileuton) markedly attenuated combined treatment-induced apoptosis. Taken together, our results indicate that inhibition of cathepsin G sensitizes TRAIL-induced apoptosis through 5-LOX-mediated down-regulation of survivin expression.

Survivin overexpression is potentially associated with pituitary adenoma invasiveness.

Background and objectiveSurvivin is an inhibitor of apoptosis. Its role in guiding the treatment of neoplasms, making diagnosis and predicting prognosis has been reported. However, there is little information on the implications and uses of survivin in predicting pituitary adenoma (PA) invasiveness. Existing information is unclear and controversial. We thus conducted this meta-analysis to explore whether the surviving expression levels in invasive PAs (IPA) and regular PAs are different or not. We considered both non-secreting and secreting tumors together.MethodsA global search strategy was systematically applied among five databases including Cochrane Library, Embase, PubMed, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) up to June 18th, 2017. With a specially designed form including PAs’ invasive features, etc., data was collected. The included studies should present the data representing the surviving levels in IPA groups and regular PA groups, respectively. Differences were expressed as standard mean differences (SMDs) or odds ratios (ORs) with 95% confidence interval (CI). To estimate the heterogeneities, I2 test, Cochran's Q-test and Galbr figure were all conducted. A sensitivity-analysis and potential-publication bias were also performed.ResultsIn the present meta-analysis, 9 studies containing 489 patients were included. Seven studies with dichotomous-data showed that survivin over-expression in PA tissue was closely associated with a high invasive tendency (OR 6.226, 95% CI 3.970, 9.765; P<0.001), but 2 continuous-data studies revealed that there was no significant association (SMD −5.043, 95% CI-10.965, 0.878; p=0.095). A sensitivity-analysis suggested a statistically stable result. We did not find publication bias.ConclusionWe suggest that survivin overexpression is potentially associated with PA invasiveness. More research based on medical big data is needed to confirm this finding.

Survivin Is Required for Mouse and Human Bone Marrow Mesenchymal Stromal Cell Function.

Although mesenchymal stromal cells (MSCs) have significant potential in cell-based therapies, little is known about the factors that regulate their functions. While exploring regulatory molecules potentially involved in MSC activities, we found that the endogenous multifunctional factor Survivin is essential for MSC survival, expansion, lineage commitment, and migration. Pharmacological or genetic blockade of Survivin expression in mouse and human bone marrow MSC enhances caspase 3 and 7 expression and reduces proliferation resulting in fewer MSC and clonogenic colony-forming unit-fibroblasts (CFU-F), whereas ectopic Survivin overexpression in MSC results in their expansion. Survivin is also required for the MSC proliferative responses to basic fibroblast growth factor and platelet derived growth factor. In a wound healing model, Survivin inhibition results in suppression of MSC migration to the wound site. In addition, loss of Survivin in MSCs compromises their hematopoiesis-supporting capacity. These results demonstrate that Survivin is a key regulator of mouse and human MSC function, and suggest that targeted modulation of Survivin in MSCs may have clinical utility to enhance MSC recovery and activity following insult or stress. Stem Cells 2018;36:123-129.

Enhancement of chemosensitivity by simultaneously silencing of Mcl-1 and Survivin genes using small interfering RNA in human myelomonocytic leukaemia

Acute myeloid leukaemia (AML) is a genetically heterogeneous, severe and rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Overexpression of myeloid cell leukaemia-1 (Mcl-1) and Survivin is associated with drug resistance, tumour progression and inhibition of apoptotic mechanisms in leukaemia and several cancers. In the present study, we examined the combined effect of etoposide and dual siRNA-mediated silencing of Mcl-1 and Survivin on U-937 AML cells. The AML cells were co-transfected with Mcl-1 and Survivin-specific siRNAs and genes silencing were confirmed by quantitative real-time PCR and Western blotting. Subsequently, MTT assay was used for the evaluation of cytotoxic effects by dual siRNA and etoposide on their own and in combination. For the studying of apoptosis, DNA-histone ELISA and annexin-V/FITC assays were performed. Co-transfection of Mcl-1 and Survivin siRNA significantly blocked their expression at the mRNA and protein levels, leading to the induction of apoptosis and strong inhibition of growth (p < .05). Besides, combined treatment of etoposide with Mcl-1 and Survivin siRNAs co-transfection leads to synergistically enhance etoposide-induced cytotoxic and apoptotic effects (p < .05). The results showed that Mcl-1 and Survivin play a major role in the U937 cells survival and their resistance relative to etoposide. Thus, Mcl-1 and Survivin can be considered as promising molecular targets for the treatment of AML. The combination treatment with etoposide, and siRNA-mediated silencing of corresponding genes may be a novel strategy in chemoresistance AML treatment.

The kinesin Eg5 inhibitor K858 induces apoptosis and reverses the malignant invasive phenotype in human glioblastoma cells.

Glioblastoma multiforme is the most common primary malignant brain tumor and its current chemotherapeutic options are limited to temozolomide. Recently, some synthetic compounds acting as inhibitors of kinesin spindle protein Eg5 have shown pronounced antitumor activity. Our group has recently demonstrated that one of these kinesin Eg5 inhibitors, named K858, exerted important antiproliferative and apoptotic effects on breast cancer cells. Since glioblastoma cells usually express high levels of kinesin Eg5, we tested the effect of K858 on two human glioblastoma cell lines (U-251 and U-87) and found that K858 inhibited cell growth, induced apoptosis, reversed epithelial-mesenchymal transition and inhibited migration in both cell lines. We also detected that, at the same time, K858 increased the expression of survivin, an anti-apoptotic molecule, and that the forced down-regulation of survivin, obtained with the specific inhibitor YM155, boosted K858-dependent apoptosis. This indicated that the anti-tumor activity of K858 on glioblastoma cells is limited by the over-expression of survivin and that the negative regulation of this protein sensitizes tumor cells to K858. These data confirmed that kinesin Eg5 is an interesting target for new therapeutic approaches for glioblastoma. We showed that K858, specifically, was a potent inhibitor of replication, an inducer of apoptosis and a negative regulator of the invasive phenotype for glioblastoma cells.

Expression Analysis of Survivin and XIAP in Gallbladder Cancer: a Case-control Study in Indo-Gangetic Plain.

Gallbladder cancer is a highly mortal disease with poor prognosis because of late presentation of disease. Survivin and X-linked inhibitor of apoptosis (XIAP) are one of the two important members of inhibitors of apoptosis. Thus, this study aimed to look at the expression of Survivin and XIAP in gallbladder cancer patients. Survivin and XIAP expression were investigated in tissues of gallbladder cancer patients (40 cases) and compared with cholelithiasis as control (40 cases) by using immunohistochemistry. Their expression was correlated with clinicopathological parameters. Significantly higher (p<0.05), Survivin protein was expressed in gallbladder cancer (n=67.5%) than control (n=35%). But it did not show any significant association with any of the clinicopathological parameter while XIAP was not expressed in the GBC patients (p>0.05). Overexpression of Survivin in gallbladder cancer suggests its possible role and association with poor prognosis. But XIAP has not been found to be associated with gallbladder carcinogenesis.

MiR-338-5p enhances the radiosensitivity of esophageal squamous cell carcinoma by inducing apoptosis through targeting survivin

Radioresistance is a challenge in the treatment of esophageal squamous cell carcinoma (ESCC). MicroRNAs (miRNAs) are known to play an important role in the functional modification of cancer cells and recent studies have reported miRNA-mediated radiotherapy resistance. However, further research is necessary to reveal the regulation mechanisms, and treatment strategies using miRNA are yet to be established for ESCC. We compared the miRNA expression profiles of ESCC parental (TE-4) and acquired radioresistance (TE-4R) cell lines using a miRNA microarray and qRT-PCR. Our data showed that miR-338-5p, one of the target miRNA biomarkers, was significantly downregulated in TE-4R. Ectopic overexpression of miR-338-5p induced apoptosis and sensitivity to radiation treatment by interfering with survivin, which is a known inhibitor of apoptosis. Overexpression of survivin reversed miR-338-5p-induced apoptosis. Tumor xenograft experiments indicated that therapeutic delivery of the miR-338-5p mimics via direct injection into tumor mass increased sensitivity to radiation therapy. In conclusion, our findings suggest that miR-338-5p is a potential radiosensitizer and may be a therapeutic biomarker for radioresistant in ESCC.

Schisandrin B reverses doxorubicin resistance through inhibiting P-glycoprotein and promoting proteasome-mediated degradation of survivin

Acquired drug resistance poses a great challenge in cancer therapy. Drug efflux and anti-apoptotic processes are the most two common mechanisms that confer cancer drug resistance. In this study, we found that Schisandrin B (Sch B), one of the major dibenzocyclooctadiene derivatives extracted from Chinese herbal medicine Schisandrae Chinensis Fructus, could significantly enhance the sensitivity of doxorubicin (DOX)-resistant breast cancer and ovarian cancer cells to DOX. Our results showed that Sch B increased the intracellular accumulation of DOX through inhibiting expression and activity of P-glycoprotein (P-gp). Meanwhile, Sch B could markedly downregulate the expression of anti-apoptotic protein survivin. Overexpression of survivin attenuated the sensitizing effects of Sch B, while silencing of survivin enhanced Sch B-mediated sensitizing effects. Furthermore, Sch B preferentially promoted chymotryptic activity of the proteasome in a concentration-dependent manner, and the proteasome inhibitor MG-132 prevented Sch B-induced survivin downregulation. Taken together, our findings suggest that Sch B could be a potential candidate for combating drug resistant cancer via modulating two key factors that responsible for cancer resistance.

Abstract 2329: TMEM33 induces apoptosis via UPR signaling and autophagy in breast cancer cells

Abstract TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR). It has been shown to activate the PERK and IRE1α branches of the unfolded protein response (UPR). However, the underlying mechanism of action of this EnR resident protein TMEM33 and the cellular functions that it regulates remain largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. Inhibition of the PERK/eIF2alpha pathway with eIF2alpha inhibitor ISRIB blocks the TMEM33 induced cell death. TMEM33 overexpression strongly activates UPR associated pro-death JNK-p53 signaling. We also observed a significant inhibition of the downstream survivin, which blocks cell death activation by binding to caspases and inhibiting their activation. We further show that the blockage of JNK activation with either an inhibitor or overexpression of survivin, protects cells against TMEM33 induced apoptosis. In addition, we show that TMEM33 overexpression induces autophagy in breast cancer cells. Inhibition of autophagy with using either the inhibitor chloroquine or knockdown of the Atg5 gene, further sensitizes breast cancer cells to the effects of TMEM33 overexpression. Cell death induced by TMEM33 is also decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating IRE1α-JNK-p53-survivin signaling in breast cancer cells. Concurrently, autophagy is also activated by TMEM33, and functions as a pro-survival mechanism. Cell fate reflects the balance between the pro-death and pro-survival activities as regulated by TMEM33. Citation Format: Rong Hu, Xiyuan Zhang, Leena Hilakivi-Clarke, Usha Kasid, Robert Clarke. TMEM33 induces apoptosis via UPR signaling and autophagy in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2329. doi:10.1158/1538-7445.AM2017-2329

Abstract 696: Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam

Abstract Ewing sarcoma (ES) is the most common soft tissue and bone tumor in adolescents and young adults. The survival rate of patients with metastatic disease is poor suggesting an urgent need for the development of more effective therapeutic options. Our aim was to investigate novel targets and identify less toxic agents to improve therapeutic efficacy in ES patients. Specificity protein 1 (Sp1) transcription factor regulates critical genes involved in cell proliferation. Sp1 also mediates the expression of survivin, an inhibitor of apoptosis protein. Overexpression of Sp1 and survivin is linked to aggressiveness and poor prognosis in several cancers; however, their precise association is not adequately evaluated in ES. Previously, we showed that tolfenamic acid (TA) inhibits leukemia, medulloblastoma and neuroblastoma cell proliferation by targeting Sp1. In this study, we investigated the anti-cancer activity of this small molecule using human ES cell lines. CHLA-9 and TC-32 cells were treated with vehicle (DMSO) or TA (2.5-20 µg/ml) and cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter Glo kit. ES cells were treated with vehicle or TA (15 µg/ml) for 48 h. The mRNA and protein expression of Sp1 and survivin was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis respectively. Flow cytometry was used to measure apoptotic cell population using Annexin-V staining and cell cycle phase distribution with propidium iodide. Markers of apoptosis (caspase 3/7 activity using caspase Glo kit and cleaved PARP by Western blot) and cell cycle arrest (Cyclin D1 and p21 expression by Western blot) were also determined. Sp1 DNA-binding was evaluated by gel shift assay. TA treatment inhibited ES cell viability in a dose and time-dependent manner which is accompanied by the inhibition of Sp1 and survivin protein expression and a 2-3 fold increase in apoptotic markers, caspase 3/7 activity, Annexin-V staining, and c-PARP expression. Interestingly, TA inhibited mRNA expression of survivin but not Sp1, suggesting it has post-translational effects, perhaps proteasome-dependent degradation of Sp1. Gel shift assay revealed a decrease in Sp1 DNA-binding, indicating that TA could directly disrupt the binding of Sp1 with its consensus binding site in the promoter of target genes. Cyclin D1 is crucial for moving the cells from G0/G1 to next phases and induction of p21 is known to inhibit Cyclin D1. In this study, TA up-regulated p21, reduced Cyclin D1 expression and accumulated cells in G0/G1 phase. These results demonstrate that TA induces apoptosis and causes cell cycle arrest in ES cells by targeting Sp1 and survivin. Citation Format: Sagar Shelake, Umesh T. Sankpal, W. Paul Bowman, Matthew Wise, Anish Ray, Riyaz Basha. Inhibition of Ewing sarcoma cell growth by targeting Sp1 and survivin with the small molecule clotam [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2017-696

Co-delivery of paclitaxel and anti-survivin siRNA via redox-sensitive oligopeptide liposomes for the synergistic treatment of breast cancer and metastasis

The overexpression of survivin in breast cancer cells is an important factor of paclitaxel (PTX) resistance in breast cancer. To overcome PTX resistance and improve the antitumor effect of PTX, we developed a novel liposome-based nanosystem (PTX/siRNA/SS-L), composed of a redox-sensitive cationic oligopeptide lipid (LHSSG2C14) with a proton sponge effect, natural soybean phosphatidylcholine (SPC), and cholesterol for co-delivery of PTX and anti-survivin siRNA, which could specifically downregulate survivin overexpression. PTX/siRNA/SS-L exhibited high encapsulation efficiency and rapid redox-responsive release of both PTX and siRNA. Moreover, in vitro studies on the 4T1 breast cancer cells revealed that PTX/siRNA/SS-L offered significant advantages over other experimental groups, such as higher cellular uptake, successful endolysosomal escape, reduced survivin expression, the lowest cell viability and wound healing rate, as well as the highest apoptosis rate. In particular, in vivo evaluation of 4T1 tumor-bearing mice showed that PTX/siRNA/SS-L had lower toxicity and induced a synergistic inhibitory effect on tumor growth and pulmonary metastasis. Collectively, the collaboration of anti-survivin siRNA and PTX via redox-sensitive oligopeptide liposomes provides a promising strategy for the treatment of breast cancer and metastasis.

Expression of survivin, MUC2 and MUC5 in colorectal cancer and their association with clinicopathological characteristics.

Survivin is a bifunctional protein that suppresses apoptosis and regulates cell division, and is highly expressed in various cancer types. Mucins are high-molecular-weight, heavily glycosylated proteins. In the present study, the association between survivin, mucin 2 (MUC2) and MUC5 expression, and the clinicopathological features of colorectal cancer (CRC) were investigated. The immunohistochemistry and western blotting results demonstrated that survivin was highly expressed in CRC tissues and rarely expressed in normal colon tissues. Moreover, the overexpression of survivin and MUC5 was strongly associated with lymph node metastasis, poor cellular differentiation, advanced tumor stage and a poor prognosis in CRC. By contrast, low expression of MUC2 was significantly associated with lymph node metastasis, poor cellular differentiation and an advanced tumor stage in CRC. The results of the present study suggest that survivin, MUC2 and MUC5 levels may be associated with tumor progression and could be used to aid the early diagnosis and clinical characterization of CRC.

HSP90 inhibitor NVP-AUY922 induces cell apoptosis by disruption of the survivin in papillary thyroid carcinoma cells

Heat shock protein 90 (HSP90) is a molecular chaperone required for maintaining the stability and function of signal proteins that plays an important role in promoting the growth and survival of cancer cells. The incidence of papillary thyroid carcinoma (PTC) has been increasing in recent years. The effect of the novel non-geldanamycin HSP90 inhibitor NVP-AUY922 on apoptosis of papillary thyroid carcinoma cells has not been investigated. The influence of AUY922 on the survival of PTC cell lines K1 and IHH4 was evaluated. Cell viability was determined by cell counting kit method. Cell apoptosis was assessed by flow cytometry and western blotting and the potential mechanism was evaluated by western blotting and immunoprecipitation. Overexpression plasmid was transfected by Lipofectamine 2000 method. In K1 and IHH4 cell lines, after the treatment of AUY922, cell viability decreased, and the proportion of apoptosis cells increased. AUY922 caused the cleavage of PARP and caspase-3 proteins, and altered expression of survivin, which was a client protein of HSP90. In AUY922-treated cells, overexpression of survivin attenuated growth inhibition and cell apoptosis. The results indicate that AUY922 induces apoptotic cell death in PTC cells. Moreover, our findings demonstrate that AUY922 induced apoptosis by downregulating the expression of survivin protein in PTC cells.

Abstract P4-10-02: Transmembrane protein 33 (TMEM33) induces apoptosis via UPR signaling and autophagy in breast cancer cells

Abstract Breast cancer is the most common cancer diagnosed in women. Endoplasmic reticulum stress (EnR stress) and the related unfolded protein response (UPR) are activated in breast cancer cells and can promote cell survival and endocrine resistance. TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR) and has been shown to activate the PERK and IRE1α branches of the UPR. However, the underlying mechanism of action of this EnR resident protein TMEM33 and the cellular functions that it regulates remain largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. TMEM33 overexpression strongly activates UPR associated pro-death JNK-p53 signaling. We also observed a significant inhibition of the downstream survivin, which blocks cell death activation by binding to caspases and inhibiting their activation. We further show that the blockage of JNK activation with either an inhibitor or overexpression of survivin, protects cells against TMEM33 induced apoptosis. In addition, we show that TMEM33 overexpression induces autophagy in breast cancer cells. Inhibition of autophagy with using either the inhibitor chloroquine or knockdown of the Atg5 gene, further sensitizes breast cancer cells to the effects of TMEM33 overexpression. Cell death induced by TMEM33 is also decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating IRE1α-JNK-p53-survivin signaling in breast cancer cells. Concurrently, autophagy is also activated by TMEM33, and functions as a pro-survival mechanism. Cell fate reflects the balance between the pro-death and pro-survival activities as regulated by TMEM33. Citation Format: Clarke R, Hu R, Zhang X, Hilakivi-Clarke L, Kasid U. Transmembrane protein 33 (TMEM33) induces apoptosis via UPR signaling and autophagy in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-10-02.

EMODIN DOWNREGULATES CELL PROLIFERATION MARKERS DURING DMBA INDUCED ORAL CARCINOGENESIS IN GOLDEN SYRIAN HAMSTERS.

Background:Cell-cycle disruption is the major characteristic features of neoplastic transformation and the status of cell-cycle regulators can thus be utilized to assess the prognostic significance in patients with cancer. The PCNA, cyclin D1, CDK4, CDK6 and survivin expression in the buccal mucosa was utilized to evaluate the Emodin efficacy on abnormal cell proliferation during 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis in golden Syrian hamsters.Materials and methods:Topical application of DMBA, three times a week for 14 weeks, on the hamsters’ buccal pouches developed well differentiated squamous cell carcinoma.Results:Cyclin D1 and PCNA over-expression and up-regulation of CDK4, CDK6 and survivin were noticed in the buccal mucosa of hamsters treated with DMBA alone. Emodin administration (50mg/kg b.w) orally to hamsters treated with DMBA down-regulated the expression of cell proliferation markers in the buccal mucosa.Conclusions:The anti-cell proliferative role of Emodin is owing to its modulating efficacy on cell-cycle markers towards the tumor suppression during DMBA induced oral carcinogenesis.