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NRF2/ARE signaling pathway is a principal regulator of cellular redox homoeostasis. The stress-induced transcription factor, NRF2, can shield cells from the oxidative damages via binding to the consensus antioxidant-responsive element (ARE) and driving several cyto-protective genes expression. Increasing evidence indicated that aberrant activation of NRF2 in malignant cells may support their survival through various pathways to detoxify chemotherapy drugs, attenuate drug-induced oxidative stress, or induce drug efflux, all of which are crucial in developing drug resistance. Accordingly, NRF2 is a potential drug target for improving the effectiveness of chemotherapy and to reverse drug resistance in cancer cells. A stable ARE-driven reporter human head and neck squamous cell carcinoma (HNSCC) cell line, HSC3-ARE9, was established and utilized to screen novel NRF2 inhibitors from a compound library. The cotton plant derived phenolic aldehyde-gossypol was selected for further analyses. The effects of gossypol in cancer cells were determined by western blotting, RT-qPCR, clonogenic assay, and cell viability assays. The gossypol-responsive gene expression levels were assessed in the Oncomine database. The effects of gossypol on conferring chemo-sensitization were evaluated in etoposide-resistant and cisplatin-resistant cancer cells. Our study is the first to identify that gossypol is effective to reduce both basal and NRF2 activator tert-butylhydroquinone (t-BHQ)-induced ARE-luciferase activity. Gossypol diminishes NRF2 protein stability and thereby leads to the suppression of NRF2/ARE pathway, which resulted in decreasing the expression levels of NRF2 downstream genes in both time- and dose-dependent manners. Inhibition of NRF2 by gossypol significantly decreases cell viabilities in human cancer cells. In addition, we find that gossypol re-sensitizes topoisomerase II poison treatment in etoposide-resistant cancer cells via suppression of NRF2/ABCC1 axis. Moreover, gossypol suppresses NRF2-mediated G6PD expression thereby leads to induce synthetic lethality with cisplatin not only in parental cancer cells but also in cisplatin-resistant cancer cells. These findings suggest that gossypol is a novel NRF2/ARE inhibitor, and can be a potential adjuvant chemotherapeutic agent for treatment of chemo-refractory tumor.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear. Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2. Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.

A major challenge in personalized cancer medicine is to establish a systematic approach to translate huge oncogenomic datasets to clinical situations and facilitate drug discovery for cancers such as endometrial carcinoma. We performed a genome-wide somatic mutation-expression association study in a total of 219 endometrial cancer patients from TCGA database, by evaluating the correlation between ∼5,800 somatic mutations to ∼13,500 gene expression levels (in total, ∼78, 500, 000 pairs). A bioinformatics pipeline was devised to identify expression-associated single nucleotide variations (eSNVs) which are crucial for endometrial cancer progression and patient prognoses. We further prioritized 394 biologically risky mutational candidates which mapped to 275 gene loci and demonstrated that these genes collaborated with expression features were significantly enriched in targets of drugs approved for solid tumors, suggesting the plausibility of drug repurposing. Taken together, we integrated a fundamental endometrial cancer genomic profile into clinical circumstances, further shedding light for clinical implementation of genomic-based therapies and guidance for drug discovery.

Objective We report a rare case of vaginal cuff dehiscence with small bowel evisceration at 7 months post robotic-staging surgery. Case Report A 41-year-old woman was sent to the emergency room with sudden onset of abdominal pain, vaginal bleeding, and vaginal protruding mass after sexual activity. She had a history of synchronous uterine and ovarian cancer treated with robotic-staging surgery 7 months before. Then she received six courses of postoperative adjuvant chemotherapy, and the last chemotherapy ended 1 month ago. At the operation room, some small bowel loops were noted in the vaginal tip with cuff dehiscence and bleeding. After repositioning of the small bowel, a 2.5-cm vaginal cuff dehiscence was repaired transvaginally. The patient recovered well, and is free of disease and has normal sexual activity 2 months after repairs. Conclusion Unusual delayed-type vaginal cuff dehiscence hints the possibility that a combination of robotic surgery and postoperative chemotherapy might result in delayed healing of the vaginal cuff.

OBJECTIVE: To review and evaluate the potential adverse effects of these oral contraceptives (OCP) to overweight women. CASE REPORT: A 19-year-old college student with a body mass index (BMI) of 35.2 kg/m(2) who received 2 months of OCP containing cyproterone and ethinyl estradiol for polycystic ovary syndrome (PCOS)-related menstrual problems was complicated with a thromboembolism-related life-threatened disease. After intensive care including the use of an extracorporeal membrane oxygenation system thrombolytic treatment anticoagulant and inferior vena filter she recovered well without significant sequelae. CONCLUSION: This case illustrates the risk of using OCPs especially for those containing cyproterone and ethinyl estradiol components as a treatment for menstrual problems in young women with PCOS and a high BMI. Copyright (c) 2016. Published by Elsevier B.V.

Background: To explore the relationship between overactive bladder (OAB) symptoms and paravaginal defects (PVDs), and to identify the necessity of PVD repair by transvaginal mesh (TVM) for the treatment of OAB symptoms. Methods: A retrospective clinical study of 30 women with advanced cystocele with limited apical and posterior vaginal wall prolapse was conducted to identify any changes in OAB symptoms following a single Perigee procedure. Prolapse was assessed using the pelvic organ prolapse quantification (POP-Q) system, and paravaginal defects were identified by sonography. Complete urodynamic examination was performed prior to and one year after operation. All patients completed the overactive bladder questionnaire pre- and postoperatively for a quantitative assessment of OAB symptoms. Results: All patients showed a significant improvement at points Aa and Ba in the POP-Q system. The results of the administered questionnaire revealed statistically significant improvement postoperatively. The difference of OAB symptoms between the group with PVDs and that with central defects was not statistically significant (p = 0.67). Moreover, no statistically significant improvement of OAB symptoms in the group with repaired PVDs was observed postoperatively (p = 0.42). Conclusion: Statistical improvements of symptoms exist after Aa and Ba points recovery as evaluated by POP-Q system regardless of PVD existence identified by sonography. Repairing PVD did not show significantly improve the severity of OAB symptoms in objective urodynamic data or subjective questionnaire data. The superiority of TVM in PVD repair to manage OAB symptoms seems not manifest. Keywords: Cystocele, Overactive bladder, Overactive detrusor, Surgical mesh, Urinary bladder prolapse, Vaginal prolapse

We have previously reported a gene Tdpoz1 (previously called 2cpoz56) that is temporally expressed in unfertilized eggs and in early embryos of the mouse. The putative TDPOZ1 protein carries a tumor necrosis factor receptor-associated factor (TRAF) domain (TD) and a POZ/BTB domain. On the analysis of nine bacterial artificial chromosome (BAC) clones, we have uncovered four more Tdpoz1 homologs in the mouse genome, designated Tdpoz2 through Tdpoz5. Tdpoz1 and Tdpoz2 are found 30 kb apart in a fully sequenced BAC clone (GenBank accession number AF545858 ). The genes are intronless in the coding region and each carries an intron in the 5′-untranslated region as in other early embryonic genes. The Tdpoz gene cluster is mapped on chromosome 3 at 3F2.1–2.2. RT-PCR experiments and a search of expressed sequence tag (EST) databases show that the Tdpoz1–5 genes are transcribed in early embryos, particularly at the two-cell stage. Exhaustive database searches have further uncovered three more mouse Tdpoz homologs in chromosomes 3 and 11 and 25 other Tdpoz-like orthologs in the genomes of other animal and plant species including human, rat, C. elegans, Drosophila, Arabidopsis and rice. In the rat genome, eight rat Tdpoz genes are found as a cluster in chromosome 2. Hence, TDPOZ proteins form a new protein family on the basis of similar protein domain organization. Based on reported characteristics of known TD- and POZ-bearing proteins, we speculate that TDPOZ proteins may be nuclear scaffold proteins probably involved in transcription regulation in early development and other cellular processes.

Molecular events involved in gene expression in unfertilized eggs and pre-implantation embryos are beginning to be understood. In this work, we investigated the transcription and processing of two structurally juxtaposed mouse RING finger protein genes, Rnf33 and Rnf35. Transcripts of these genes are detected only in eggs and in pre-implantation embryos. Both genes are intronless except for a solitary intron in the 5'-untranslated region. Here, we showed by rapid amplification of cDNA ends (RACE) and reverse transcription experiments that Rnf35 transcription uses a single promoter and a terminating site. On the other hand, Rnf33 is transcribed using multiple promoters. At the four-cell stage, however, Rnf33 mRNA with a single transcription start site derived from the proximal promoter is detected, indicating that it is the major promoter. Sequences upstream of the Rnf35 and the major Rnf33 transcription start sites carry no TATA boxes but a putative transcription initiator (Inr) element is discernible in each case. The processing of the 3'-end of the Rnf33 mRNA is also in disarray with multiple 3'-ends, an event that may be related to the absence of the AAUAAA element and the utilization of AAUAAA-like proxies. The multiplicity of the 3'-untranslated region is partially amended at the four-cell stage when only two major 3'-ends are in use. This work demonstrates that expression of some maternal and early zygotic genes may be opportunistic until a stringent transcriptional regulation mechanism is imposed.

Rnf33 and Rnf35 are mouse RING finger protein genes that are transcribed temporally in the preimplantation mouse embryo, predominantly at the two-cell embryonic stage. The genes are juxtaposed in a 20-kb genomic region and are both intronless except for a single intron in the 5' untranslated region (5'-UTR). Based on analysis of the Rnf33/35 genomic sequence and cDNA sequences derived by in silico mining, we found that the Rnf33 and Rnf35 mRNAs are apparently transcribed from the same putative promoter and may be products of alternative splicing of the same pre-mRNA generated through differential 3' cleavage and polyadenylation. We also detected a second variant of Rnf35 in two-cell embryo generated through a second splicing event using an unconventional 5' splice junction. Our observations on the mode of transcription of Rnf33 and Rnf35 are consistent with the hypothesis that transcription of zygotic genes is promiscuous, and that the solo 5'-UTR intron may serve to facilitate efficient translation.

BACKGROUND: The aim of this study is to evaluate perioperative complications related to robotic-assisted laparoscopic surgery for management of gynaecologic disorders. MATERIALS AND METHODS: Eight hundred and fifty-one women who underwent robotic procedures between December 2011 and April 2015 were retrospectively included for analysis. Patient demographics, surgical outcomes and complications were evaluated. RESULTS: The overall complication rate was 5.5%, whereas the rate of complications for oncologic cases was 8.4%. Intra-operative complications (n = 7, 0.8%) consisted of five cases of bowel lacerations, one case of ureter laceration and one case of bladder injury. Early and late post-operative complications were 4.0% (n = 34) and 0.8% (n = 6), respectively. Six patients (0.7%) experienced Grade III complications based on the Clavien-Dindo classification and required further surgical intervention. CONCLUSION: Robotic-assisted laparoscopic surgery is a feasible approach for management of gynaecologic disorders; the complication rates for this type of procedure are acceptable.

Adlay has long been used in traditional Chinese medicine and as a nourishing food. The acetone extract of adlay hull had previously been demonstrated to possess potent antimutagenic activity. The aims of this study were to identify the antimutagenic constituents from adlay hull by using Ames antimutagenic activity-guide isolation procedures and to investigate their chemopreventive efficacies in cultured cells. The results demonstrated that six compounds showing great antimutagenic activity were identified by spectroscopic methods and by comparison with authentic samples to be p-hydroxybenzaldehyde, vanillin, syringaldehyde, trans-coniferylaldehyde, sinapaldehyde, and coixol. Two of them, trans-coniferylaldehyde and sinapaldehyde, exhibit relatively potent scavenging of DPPH radicals, inhibit TPA stimulated superoxide anion generation in neutrophil-like leukocytes, and induce Nrf2/ARE-driven luciferase activity in HSC-3 cells. Moreover, trans-coniferylaldehyde possesses cytoprotective efficacy against tert-butyl hydroperoxide-induced DNA double-strand breaks in cultured cells, and the chemopreventive potency induced by trans-coniferylaldehyde may be through the activation of kinase signals, including p38, ERK1/2, JNK, MEK1/2, and MSK1/2. In summary, we first identified six antimutagenic constituents from adlay hull. Among them, trans-coniferylaldehyde would be a highly promising agent for cancer chemoprevention and merits further investigation.

BackgroundSurvivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression.ResultsHere, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines.ConclusionsWe showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small molecule inhibitors will induce the over-expression of survivin in certain cancer cell lines and subsequently enhances the ability of cell survival in drug-treated situations. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted.

We have previously reported a mouse zinc-finger protein gene, Zfp352 (formerly 2czf48), that is expressed in early mouse embryos. Here, we report the genomic structure of Zfp352 and its lung-specific homolog, Zfp353. The two genes map on different chromosomes at 4C6 and 8B3.1. Both genes are intronless, except for the presence of a single 4.6-kb intron in the 5' untranslated region of Zfp352. The genes use different RNA start sites located 1.2 kb apart within the 5' homologous region. LINE1 sequences are structurally associated with the genes and form an integral part of Zfp353 transcripts, suggesting previous retrotransposition events. We propose a model of evolution of the genes. The main feature of the model is the presence of a fortuitous upstream promoter and an intron in the first retrotransposition site, creating a pre-Zfp352 gene with a 5' untranslated region intron. A second retrotransposition event copying from the pre-Zfp352 retroposon and removing the fortuitous intron resulted in the intronless Zfp353 at a different chromosomal location and with a different mode of expression. The model may be applicable to other genes with a similar structure with a single intron in the 5' untranslated region. The exact role of LINE1 in the retrotransposition events remains to be elucidated.

The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins, and has been considered as a potential target for cancer chemoprevention by eliminating harmful reactive oxygen species (ROS) or reactive intermediates generated from carcinogens. The objectives of this study were to identify a novel Nrf2/ARE activator and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen the potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (−) double cyclized type of lignan obtained from adlay (Coix lachrymal-jobi L. var. ma-yuen Stapf), more effectively activates ARE-driven luciferase activity than the classical ARE activator, tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme, and their protein products. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via up-regulation of HO-1. Inhibition of PI3K/AKT signal by chemical inhibitors or RNA interference suppressed Nrf2 activation and HO-1 up-regulation. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling. These results may have relevance to the clinical application of 4-KPR and its significance in cancer chemoprevention. The study was supported by grants of DOH100-TD-C-111-004, NSC98-2320-B-400-003-MY3, and NSC99-2323-B-400-006. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A83.

Etoposide (VP-16), a DNA topoisomerase II poison, is an important clinical used chemotherapeutic agent for human oral malignancies. An etoposide-resistant cell line, KB-7D, has been generated from human oral epidermoid carcinoma KB cells to investigate the mechanism of action of drug resistance in oral malignancies. Previous studies revealed that KB-7D cells were approximately 50-fold more resistant to etoposide as compared to parental KB cells. It also exhibited cross-resistant to chemotherapeutic agents such as doxorubicin. This multi-drug resistance may be caused by the over-expression of ABCC1, leading to the decrease in drug accumulation in KB-7D cells. Our current work continues the effort to investigate the mechanism of regulation of ABCC1 expression in the etoposide-derived drug resistant cells and subsequently find the molecular target that can be used to restore therapeutic efficacy in chemo-refratory cancers. Down-regulation of ABCC1 by RNA interference and a selective inhibitor, MK-571, significantly enhanced the chemosensitivity to etoposide and doxorubicin in KB and KB-7D cells. To further determine the possible transcriptional factors that regulate the ABCC1 transactivation, the promoter region of ABCC1 was examined. A NRF2 binding sequence, antioxidant responsive element (ARE), has been found locate in the ABCC1 promoter at -470 to -458 upstream from the transcription start site. Real-time RT-PCR and Western blot analyses showed that expression of NRF2 mRNA and protein in KB-7D cells was 1.4 to 1.8 folds higher than those expressed in the parental cells. In addition, Chromatin Immunoprecipitation (ChIP) analysis revealed that NRF2 directly targeted to the ARE sequence in the ABCC1 promoter region. Interestingly, down-regulation of NRF2 decreased the expression of ABCC1 and also increased the chemosensitivity of KB-7D cells against selected anti-cancer drugs. In addition, cells incubated with an NRF2 activator, tBHQ, induced nucleus accumulation of NRF2 and over-expression of ABCC1, resulting in the enhancement of drug-resistance against etoposide or doxorubcin in KB and KB-7D cells. In summary, constitutive activation of NRF2-dependent ABCC1 contributed to the causation of chemoresistance in etoposide-derived drug resistant cells. Blockage of ABCC1 expression by manipulation of the NRF2 signaling pathway enhanced the chemotherapeutic efficacy in cells. Therefore, targeting NRF2 may be able to reverse chemoresistance in chemo-refractory oral malignancies. In addition, development of NRF2 inhibitors may be a new strategy to overcome chemoresistance in human cancers. (The study was supported by grants of Department of Health DOH99-TD-C-111-004, Taiwan.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1528. doi:10.1158/1538-7445.AM2011-1528

Nasopharyngeal carcinoma (NPC) is one of the predominant cancers found in Southeastern China and Taiwan. Chemotherapy is one of the major treatment modalities, and the cisplatin-based regimen is the front-line treatment. However, NPC patients failing to cisplatin treatment will have a compromised survival. To explore the resistance spectrum and mechanisms of cisplatin resistance in NPC, two cisplatin-resistant sublines (cis6 and cis15) derived from the parental NPC cell line HONE-1 were obtained. Compared with HONE-1 cells, cis6 and cis15 cells showed upto 18-fold resistance to cisplatin in each, and also possessed a cross-resistance to oxaliplatin and arsenic trioxide. The level of platinum-DNA-adduct and γH2AX were significantly decreased in cis6 and cis15 cells. To unravel the behind details, the systems of DNA repair and cisplatin detoxification were examined and found to be more active in both resistant cells. In terms of the DNA repair proteins, the levels of p-DNA-PK and XRCC1 were increased; in terms of cellular localization of cisplatin, the level of copper transporter ATP7A was upregulated; in terms of Nrf2/antioxidant/detoxifizing enzyme, the resistant cells had a higher Nrf2 activity and an increased intracellular level of GSH, GR, NQO1, AKR1C1 and AKR1C2. Moreover, as compared with HONE-1 cells, our results showed that the resistant cells spared the cisplatin-induced cell death via the suppression of pro-apoptotic proteins and the enhancement of anti-apoptotic proteins. Since the members of ErbB family could be overexpressed in NPC and the ErbB/Akt/FoxO axis could involve in the actions of apoptosis, DNA damage repair and detoxification of reactive oxygen species, the ErB/Akt/FoxO were investigated. Indeed, the phosphorylations of Akt, FoxO1, and FoxO3 were upregulated in cis6 and cis15 cells. Thus, the resistance to cisplatin in cis6 and cis15 cells was partially explained by the Akt/FoxO pathway. Surprisingly, EGFR was downregulated in the resistant cells, whereas ErbB2 was upregulated. The elevations of the negative regulators of EGFR, including c-Cbl, GCF and LRIG1, were observed in cis6 and cis15 cells, indicating that the negative regulation of EGFR could be at the levels of transcription and protein degradation. Since LRIG1 could be upregulated and negatively feedback to control the level of ErbB after the ErbB was activated, it was plausible that the overexpression of ErbB2 upregulated the expression of LRIG1 and subsequently, caused the downregulation of EGFR. Nevertheless, the hypothesis which both overexpression of ErbB2 and LRIG1 contributes to the resistance to cisplatin in our cell model awaits experimental verification. Taken together, our results suggested that the mechanism responsible for cisplatin resistance in NPC, at least in part, through Akt/FoxO pathway. (The study was supported by grants of Department of Health DOH99-TD-C-111-004, Taiwan, R.O.C.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2011-1701

Survivin is a member of the inhibitors-of-apoptosis protein (IAPs) family. It promotes cell survival through interference with multiple cell cycle-related proteins such as INCENP and Aurora B kinase. Survivin also inhibits cell death through interference with both caspase-dependent and -independent cell apoptosis. Interestingly, recent evidence suggests that survivin may also play a role in the regulation of cancer cell autophagy. At the clinical level, studies on clinical specimens have shown that survivin expression is up-regulated in various human cancers and its up-regulation is associated with tumour resistance to both chemotherapy and radiation therapy. On the basis of these findings, survivin has been proposed as an attractive target for new anti-cancer interventions. However, despite the role that survivin plays in cancer cell survival and anti-drug response, the development of survivin inhibitors is relatively slow as compared to other therapeutic inhibitors for cancer treatment. In this review, the relationships between survivin expression and the causation of drug resistance in cancers are re-addressed. This review also summarizes the recent development of survivin inhibitors for clinical usage.

Survivin is a dual functioning protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Furthermore, over-expression of survivin has been shown to induce drug resistance to various chemotherapeutic compounds. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that positively regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently promote cancer progression under certain circumstances. Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced amount of 26S proteasome and decreased proteasomal activity while inhibition of 26S proteasome further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. In conclusion, we showed for the first time that targeting Hsp90 induced the over-expression of survivin through various mechanisms in cancer cells. These data indicate that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. The current study suggests that dual inhibition of Hsp90 and survivin may be warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4447.

Background Survivin is a dual function protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Over-expression of survivin has been demonstrated to induce drug-resistance to various chemo-therapeutic agents such as cisplatin (DNA damaging agent) and paclitaxel (microtubule stabilizer) in cancers. However, survivin-induced resistance to microtubule de-stabilizers such as Vinca alkaloids and Combretastatin A-4 (CA-4)-related compounds were seldom demonstrated in the past. Furthermore, the question remains as to whether survivin plays a dominant role in processing cytokinesis or inhibiting caspases activity in cells treated with anti-mitotic compounds. The purpose of this study is to evaluate the effect of survivin on the resistance and susceptibility of human cancer cells to microtubule de-stabilizer-induced cell death. Results BPR0L075 is a CA-4 analog that induces microtubule de-polymerization and subsequent caspase-dependent apoptosis. To study the relationship between the expression of survivin and the resistance to microtubule de-stabilizers, a KB-derived BPR0L075-resistant cancer cell line, KB-L30, was generated for this study. Here, we found that survivin was over-expressed in the KB-L30 cells. Down-regulation of survivin by siRNA induced hyper-sensitivity to BPR0L075 in KB cells and partially re-stored sensitivity to BPR0L075 in KB-L30 cells. Western blot analysis revealed that down-regulation of survivin induced microtubule de-stabilization in both KB and KB-L30 cells. However, the same treatment did not enhance the down-stream caspase-3/-7 activities in BPR0L075-treated KB cells. Translocation of a caspase-independent apoptosis-related molecule, apoptosis-inducing factor (AIF), from cytoplasm to the nucleus was observed in survivin-targeted KB cells under BPR0L075 treatment. Conclusion In this study, survivin plays an important role in the stability of microtubules, but not with caspases inhibition. Over-expression of survivin counteracts the therapeutic effect of microtubule de-stabilizer BPR0L075 probably by stabilizing tubulin polymers, instead of the inhibition of caspase activity in cancer cells. Besides microtubule-related caspase-dependent cell death, caspase-independent mitotic cell death could be initiated in survivin/BPR0L075 combination treatments. We suggest that combining microtubule de-stabilizers with a survivin inhibitor may attribute to a better clinical outcome than the use of anti-mitotic monotherapy in clinical situations.

Bone cells respond to mechanical stimulation via mechanoreceptors and convert biophysical stimulation into biochemical signals that alter gene expression and cellular adaptation. Pulsed acoustic energy treatment raises membrane potential and induces osteogenic activity. How membrane-bound osteoblast mechanoreceptors convert physical ultrasound (US) stimuli into osteogenic responses is not fully understood. We demonstrated that low-intensity pulsed US treatment (200-μs pulse, 1 kHz, 30 mW/cm2) elevated Cbfa1/Runx2 mRNA expression and progressively promoted osteocalcin mRNA expression in human osteoblasts. Pretreatment with pertussis toxin (PTX), but not with cholera toxin, suppressed US-augmented osteogenic transcription. This indicated that Gi proteins, but not Gs proteins, were involved in US promotion of osteogenic transcription. Further studies demonstrated US treatment could rapidly increase PTX-sensitive Gαi protein levels and subsequently enhanced phosphorylation of extracellular signal-regulated kinase (ERK). PTX pretreatment significantly reduced US promotion of ERK activation. Moreover, inhibition of ERK activity by PD98059 suppressed US augmentation of Cbfa1/Runx2 and osteocalcin mRNA expression. Membranous Gαi proteins and cytosolic ERK pathways acted as potent mechanosensitive signals in the response of osteoblasts to pulsed US stimulation.