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Most cancer cells exhibit increased glycolysis and use this metabolic pathway to generate ATP (Adenosine triphosphate) as the main source of their energy supply. This phenomenon is known as the Warburg effect and is considered a fundamental metabolic alteration during malignant transformation. Increased glycolysis results in elevated production of lactate, which leads to acidification of tumor tissue and provides a microenvironment that promotes and selects cellular resistance to drugs. Thus, inhibition of glycolysis may lead to more effective treatment strategies for dealing with drug resistance. 3-Bromopyruvate (3-BrPA), an inhibitor of hexokinase II and an inhibitor of glycolysis, has been shown to be a potent chemotherapeutic agent. Despite many studies showing the efficacy of 3-BrPA against various cancer cells, there have been relatively few studies examining the effects of 3-BrPA on the anti-cancer effects of doxorubicin (Dox) in doxorubicin-resistant (DoxR) human neuroblastoma. The purpose of this study is to examine the effects of 3-BrPA upon DoxR human neuroblastoma cells to treatment with doxorubicin. In present study, we have found that 3-BrPA enhances the response of DoxR neuroblastoma to treatment with Dox and this enhancement exists in both normoxic conditions as well as hypoxic conditions. The results also suggested that this increased response of DoxR cells to treatment with Dox is mediated through decreased intracellular levels of ATP. Additionally the lactate assay revealed that intracellular lactate levels were decreased in neuroblastoma lines when glycolysis was inhibited with 3-BrPA. In order to determine if 3-BrPA, either alone or in combination with cytotoxic drugs, will lead to maximal efficacy of treating of drug-resistant tumor xenografts in vivo. Animal experiments were carried out to study the effects of 3-BrPA in drug-resistant neuroblastoma in nude mice. We have found that 3-BrPA combined with doxorubicin provided significant reduction in tumor size and the rate of tumor growth in mice. The efficacy of doxorubicin itself (88% reduction in tumor volume) overshadowed any effect of cotreatment with 3-BrPA on the Wild type tumors. However, in the DoxR tumors, doxorubicin alone had no efficacy as expected, but co-treatment with 3-BrPA suppressed tumor growth with an 80% reduction in tumor volume. These studies pave the way to determine whether inhibition of glycolysis may be an effective strategy to kill neuroblastoma cells and overcome drug resistance associated with hypoxic conditions. This novel approach may have broad applications in cancer treatment, considering the prevalent Warburg effect observed in a wide array of human cancers. Citation Format: Fei Chu, Yiyong Qiu, Sandra Clark, Kathryn Solka, Mary Beth Madonna. Glycolysis inhibition and its effect in doxorubicin resistance in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3531.

Introduction: Neuroblastoma is the most common extracranial solid tumor in childhood and accounts for ~15% of all pediatric cancer mortality. Approximately 650 new cases are reported annually in the USA, with 90% of cases diagnosed by 5 years of age. Novel therapies are needed as ~50% of cases are high-risk neuroblastoma with a poor ( Aim: Determine if inhibition of the Notch signaling pathway sensitizes chemoresistant neuroblastoma cells to doxorubicin. Methods: Doxorubicin-resistant (DoxR) cells were generated by incubating doxorubicin-sensitive, wild-type (WT) patient-derived cell lines with incremental doxorubicin concentrations (1 x 10-9 to 1 x 10-6 M) over six months. Cells were considered chemoresistant after surviving five consecutive passages in 1 µM doxorubicin (MTT assay). Differentially expressed genes were identified by microarray (Illumina HumanHT-12 v4 Expression BeadChip) and validated by RT-qPCR. Cells treated with doxorubicin and/or DAPT (γ-secretase inhibitor) were evaluated for cell viability (MTT assay), proliferation (PCNA immunofluorescence and Western blot), senescence (SA-β-Gal immunocytochemistry and p21 Western blot) and proteomic profiling (2D-DIGE). Results: Compared to WT cells, DoxR cells differentially expressed the following Notch pathway genes: downregulation of DLK1 and upregulation of HEY1 and SNAI2, as determined by microarray and RT-qPCR. Compared to WT cells, DoxR cells treated with both doxorubicin and DAPT exhibited significantly decreased cell viability, decreased proliferation and increased senescence. DAPT-treated DoxR cells differentially expressed nine proteins using 2D-DIGE: MCM3, MCM6, CAPN10, ACTN4, TUBA1B, HNRNPK, PDP1, NPM1 and BARD1. Conclusion: The γ-secretase inhibitor DAPT sensitizes DoxR neuroblastoma cells to doxorubicin. Mechanisms underlying the synergistic effect of doxorubicin and DAPT in DoxR cells warrant further investigation. Citation Format: Kathryn A. Solka, Fei Chu, Yi-Yong Qiu, Sandra H. Clark, Mary Beth Madonna. Inhibition of γ-secretase sensitizes drug-resistant neuroblastoma cells to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4901.

cis-Diaminedichloroplatinum (II) (cisplatin) is routinely used to treat various types of cancers; however, a significant number develop resistance. One of the underlying factors that contribute to cisplatin resistance is the elevated level of BCL-2 and/or BCL-XL, which promotes cell survival. A potential method of overcoming such resistance is to use a potentiator that is capable of neutralizing the antiapoptotic effects of BCL-2/BCL-XL, such as Siva-1. We previously cloned the proapoptotic protein Siva-1 and showed a possible role for it in both extrinsic and intrinsic apoptosis. Using an adenovirus-based expression system, we now show that Siva-1 can synergize with cisplatin in inducing apoptosis in MCF7 and MDA-MB-231 breast cancer cells. In an anchorage-independent clonogenicity assay, MCF7/caspase-3 cells stably expressing Siva-1, but not the control cells, showed a dramatic decrease in the number of colonies formed on one-time cisplatin treatment. Further, we show that the unique putative amphipathic helical region (SAH) in Siva-1 (amino acid residues 36-55) is necessary and sufficient for the observed enhancement in cisplatin-induced apoptosis by Siva-1. Although cisplatin treatment results in significant elevation in the expression of Fas ligand and intracellular p21 levels, expression of Siva-1 has no additional benefit. Instead, the enhancement in apoptosis seems to be due to activation of intrinsic pathway that involves caspase-9 activation. Moreover, Siva-1 augments cisplatin-mediated cell death in MCF7 cells stably expressing BCL-2. We therefore propose that Siva-1 or its SAH region can be used as a potentiator of cisplatin-based chemotherapy.

Irreversible growth arrest (also called senescence) has emerged recently as a tumor suppressor mechanism and a key determinant of cancer chemotherapy outcome. Previous work from our laboratory suggested that the cellular ability to undergo or to escape senescence dictates its fate to become drug-sensitive or drug-resistant, respectively. In the present study, we made the hypothesis that longevity genes, by virtue of their ability to inhibit senescence, may contribute to the onset of drug resistance. We report that expression of the longevity gene sirt1 increased both at the RNA and protein levels in all the five drug-resistant cell lines tested when compared with their drug-sensitive counterparts. In addition, biopsies from cancer patients treated with chemotherapeutic agents also expressed high levels of this molecule. These changes were specific for sirt1 because the expression of other members of its family was not affected. More importantly, small interfering RNA–mediated down-regulation of sirt1 significantly reversed the resistance phenotype and reduced expression of the multidrug resistance molecule P-glycoprotein. This was further confirmed by ectopic overexpression of sirt1, which induced expression of P-glycoprotein and rendered cells resistant to doxorubicin. Collectively, these findings uncovered a novel function for the longevity gene sirt1 as a potential target for diagnosis and/or treatment of cancer resistance to chemotherapy. They also describe a proof of principle that signaling pathways implicated in longevity may share similarities with those leading to development of drug resistance in cancer.

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 781. doi:1538-7445.AM2012-781

Histone deacetylase inhibitors (HDACi) have been developed as anti-tumor agents and they may play a role in reversing drug resistance by combination treatment with other chemotherapeutic drugs. However, it has also been reported that HDACi can upregulate the cancer stem surface marker expression and promote stem cell self-renewal, which may cause inherit resistance to chemotherapeutic drugs. We investigated the effect of HDACi treatment on the stemness gene expression in drug resistant cells. Using SK-N-Be2C, high aggressive I type human of neuroblastoma cells, and SKN-SH, low aggressive mixed N and S type of human neuroblastoma cells, our laboratory generated doxorubicin drug resistant cell lines with and without intermittent histone deacetylase inhibitor Vorinostat treatment over one year. For each cell line, four groups were generated: wild type (WT), doxorubicin resistant (DoxR), vorinostat treated WT (WT-v), and vorinostat - treated doxorubicin resistant (DoxR-v). The cells’ sensitivity to chemotherapeutic drugs, the ability of forming tumorspheres and in vitro invasion were examined. Cell surface markers and side populations were analyzed by flow cytometry. The differential expressed stemness genes were identified by whole genome analysis and confirmed by real-time PCR. Vorinostat increased the sensitivity of SK-N-Be2C resistant cells to chemotherapy, made the cells lose the ability to form tumorspheres, and reduced in vitro invasion ability and the percentage of side population. In contrary, vorinostat decreased the sensitivity of SK-N-SH resistant cells to drugs, enhanced the cell's ability to form tumorshperes and had little effect on the cells’ invasion and side population. The putative neuroblastoma marker, CD133, was not enriched in either long term or short term vorinostat/doxorubicin treatment. Nine stemness linked genes (ABCB1, ABCC4, LMO2, SOX2, ERCC5, S100A10, IGFBP3, TCF3 and VIM) were upregulated in doxorubicin resistant SK-N-Be2C cells, compared to vorinostat - treated resistant cells. Vorinostat did not have significant effect on stemness gene expression in SK-N-SH cells, except for ABCB1 and ABCC4 genes, which were deregulated in vorinostat - treated resistant cells. In conclusion, Histone deacetylase inhibitor differentially regulates stemness gene expression in neuroblastoma drug resistant cells. It has better effect on reversing drug resistance in high aggressive neuroblastoma and this may be associated with downregulated stemness gene expression. This work may be valuable for clinicians to design treatment protocols specific for different neuroblastoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3365. doi:1538-7445.AM2012-3365

Background: Hypoxia inducible factor 1-alpha (HIF1-alpha) is overexpressed in doxorubicin-resistant neuroblastoma cell lines relative to drug sensitive parental cells lines, and has been implicated in inducing epithelial to mesenchymal transition (EMT). We hypothesized that the transition of human neuroblastoma cells from a drug-sensitive to drug-resistant phenotype involves mesenchymal change. Methods: A doxorubicin resistant (DoxR) human neuroblastoma cell line was generated by incubating parental wild type (WT) SK-N-Be(2)C cells in stepwise concentrations of doxorubicin. Cells were deemed resistant after surviving in a concentration of drug approximately 2 Log above the wild type IC50, confirmed by MTT colorimetric survival assay. Cell migration and capacity for invasion were assayed using the scratch test and Matrigel invasion culture system. EMT pathway related genes differentially expressed in the DoxR cells relative to the parental (WT) lines were analyzed using the Ilumina Human HT-12 v4 Expression Beadchip. Genes of interest with a fold change in expression >1.5 and an adjusted p < 0.1 are reported. Quantitative RT-PCR and western blot analysis were used to confirm the results. Results: Western blot confirmed that HIF1-alpha was up-regulated in BE(2)C/DoxR cells relative to WT. DoxR cells also demonstrated increased migration and invasion typical of mesenchymal cells. Up-regulation of TGF-beta dependent induction pathways of EMT via RhoA, PI3K, and ILK and via SMADs was seen on microarray pathway analysis in BE(2)C DoxR cells when compared to WT cells. Genes involved in these EMT pathways that were up-regulated in DoxR cells included TGFBRII, the gene encoding TGF beta-receptor II, TGFB3, the gene for TGF beta 3, as well as other genes implicated in TGF-beta signaling, including PAK1, LEF1, SMAD3, ZEB2, and NET1. EMT markers including vimentin (VIM), and slug (SNAI2) were also up-regulated. Interestingly, CDH2, the gene for N-cadherin, and TWIST1 were down regulated in DoxR cells. CDH1, the gene for E-cadherin, and SNAI1, the gene for Snail-1, were not differentially expressed. Conclusions: As SK-BE(2)C human neuroblastoma cell lines acquire drug-resistance, they undergo mesenchymal change via TGF-beta mediated pathways, displaying increased invasion and migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 335. doi:1538-7445.AM2012-335

It has become evident that the biological processes which allow malignant cells to survive in the presence of cytotoxic drugs are extraordinarily complex, and recent findings indicate that a multitude of cellular defense mechanisms may be involved in the development of drug resistance. In order to dissect the dynamic changes in gene expression during the process of acquiring drug resistance, four human neuroblastoma cell lines (IMR 32, BE(2)-C, SK-N-SH and SH-SY5Y) were used to generate drug-resistant cell lines using clinically appropriate chemotherapeutic agents (Doxorubicin and Busulfan). Additionally, in order to understand the physiological role of histone deacetylase (HDACs) in the development of drug resistance in human neuroblastoma and to determine if this development can be delayed or totally suppressed by the HDACs inhibition, the cells were separated into those that were pretreated with a histone deacetylase (HDAC) inhibitor: suberoylanilide hydroxamic acid (SAHA), and those that were not pretreated. Cells were deemed drug-resistant when the cells were able to grow efficiently in drug concentrations of two log concentrations higher than the IC50 level. Neuroblastoma cells were collected at each log phase increase of cytotoxic agent to compare changes in P-glycoprotein and midkine expression; cross-resistance between chemotherapeutic agents, and presence of survival proteins in each group. The creation of these resistant human neuroblastoma cell lines and the determination of the transcriptional changes occurring as the cells become resistant may have important implications for clinical treatment of human neuroblastoma. 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 3636.

The diverse biological role of nerve growth factor (NGF) as a necessary modulator of neuroectodermal cell survival, proliferation and differentiation has been previously well described in both normal and neoplastic cell lines. However, recent studies demonstrate that neuroectodermal tumors, such as neuroblastoma, contain genetic alterations that result in limited secretion of active NGF, as well as expression abnormalities of its receptors TrkA and p75. To better define the differential effects of exogenous and endogenously synthesized NGF, the in vitro response of wild-type and genetically engineered neuroblastoma cells was investigated. Replication-defective NGF-expressing retroviral vectors were prepared using the calcium phosphate co-precipitation method and used to transfect SK-N-BE(2)C cells. Presence of the NGF cDNA was confirmed by RT-PCR, and secretion of NGF into the conditioned media by western immunoblotting. NGF transfection resulted in decreased cell proliferation as determined by nonradioactive cell proliferation assay. Wild type and NGF transfected (BE(2)C-NGF) cells were then incubated with exogenous NGF at 200ng/mL for varying time periods up to 24 hours. The expression of TrkA and p75, as well as proteins of their associated survival and apoptosis cascades were determined by Western immunoblotting of whole cell lysates. p75 expression in the NGF transfected cell line was equivalent to the parental wild type cell, but TrkA expression was significantly lower in BE(2)C-NGF. At baseline, BE(2)C-NGF cells demonstrate significant down-regulation of phospho-cRAF and its downstream effector phospho-p42/44 in comparison to the wild type cell line. Additionally, NGF stimulation further down-regulated phospho-c-RAF expression in the NGF transfected cell line, with maximal down-regulation occurring at 24 hours. BE(2)C-NGF cells also demonstrate significant baseline activation of phospho-p38 in comparison to the wild type cell line, with expression further up-regulated with NGF stimulation at 5 minutes and 1 hour. In conclusion, nerve growth factor cDNA gene transfer into wild type SK-N-BE(2)C neuroblastoma results in decreased activation of the p-42/44 survival pathway and concomitant up-regulation of the p-38 apoptotic pathway, leading to a overall decreased rate of proliferation. This effect is likely the result of a decreased TrkA to p75 receptor ratio, and further understanding of these pathways may facilitate the discovery of novel therapeutic targets for neuroblastoma treatment. 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 5261.

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that mediates the downstream expression of genes controlling the cellular response to hypoxic conditions. Overexpression of HIF-1α has been implicated in promoting angiogenesis and a more invasive phenotype in a variety of cancer cells. HIF-1α expression may also promote chemoresistance and increased expression of the MDR gene. Previous work from our laboratory has implicated class II histone deacetylases (HDACs) as key mediators of chemoresistance, however the mechanism for this observation remains unclear. It is known that HDACs decrease HIF-1α acetylation, preventing proteosomal degradation and increasing HIF-1α activity. We hypothesized that class II HDAC modulation of HIF-1α activity may contribute to the development of resistance to anti-cancer therapy. In the present study, HDAC4 and HDAC6 expression were upregulated in doxorubicin-resistant human breast (MCF-7-DoxR), human osteosarcoma (SaOS2-DoxR) and human neuroblastoma (SK-N-SH-DoxR) cells compared to their parental cells by western blot and RT-PCR. Likewise, after treatment with deferoxamine mesylate (DFO) to prevent HIF-1α degradation under normoxic conditions, HIF-1α expression was found to be upregulated in MCF-7-DoxR and SaOS2-DoxR cells. Inhibition of HDACs with suberoylanilide hydroxamic acid (SAHA), sodium butyrate (NaB), or siRNA against HDAC4 or HDAC6 resulted in decreased HIF-1α expression and restoration of sensitivity to anti-cancer agents in both drug resistant cell lines. The percentage of viable drug-resistant cells was lower after treatment with doxorubicin plus SAHA or NaB, compared to doxorubicin, SAHA or NaB alone. In summary, HDAC regulation of HIF-1α expression may be an important mediator of tumor resistance to anti-cancer agents. 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 2549.