Jennifer L. Allensworth

XIAP Inhibition and Generation of Reactive Oxygen Species Enhances TRAIL Sensitivity in Inflammatory Breast Cancer Cells

We recently identified superoxide dismutase (SOD) overexpression and decreased induction of reactive oxygen species (ROS)-mediated apoptosis in models of inflammatory breast cancer (IBC) cells with acquired therapeutic resistance. This population of cells has high expression of X-linked inhibitor of apoptosis protein (XIAP), which inhibits both extrinsic and intrinsic apoptosis pathways. We therefore wanted to evaluate the effect of classical apoptosis-inducing agent TRAIL, a proapoptotic receptor agonist that selectively triggers death receptor (DR)-mediated apoptosis in cancer cells, in the IBC acquired resistance model. XIAP levels and subsequent inhibition of caspase activity inversely correlated with TRAIL sensitivity in our models of IBC. These include SUM149, a basal-type cell line isolated from primary IBC tumors and isogenic SUM149-derived lines rSUM149 and SUM149 wtXIAP, models of acquired therapeutic resistance with endogenous and exogenous XIAP overexpression, respectively. Inhibition of XIAP function using embelin, a plant-derived cell permeable small molecule, in combination with TRAIL caused a synergistic decrease in cell viability. Embelin treatment resulted in activation of extracellular signal–regulated kinase (ERK)1/2 and ROS accumulation, which correlated with downregulation of antioxidant protein SOD1 and consumption of redox modulator reduced glutathione in the XIAP-overexpressing cells. Simultaneous treatment with an SOD mimic, which protects against ROS accumulation, reversed the decrease in cell viability caused by embelin + TRAIL treatment. Embelin primes IBC cells for TRAIL-mediated apoptosis by its direct action on the anti-caspase activity of XIAP and by shifting the cellular redox balance toward oxidative stress–mediated apoptosis. Thus, ROS modulators represent a novel approach to enhance efficacy of TRAIL-based treatment protocols in IBC. Mol Cancer Ther; 11(7); 1518–27. ©2012 AACR.

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate

Abstract Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O2 ·−, reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP5+, MnTnHex-2-PyP5+, and a meta isomer MnTnHex-3-PyP5+, which differ greatly with regard to their metal-centered reduction potential, E1/2 (MnIIIP/MnIIP) and lipophilicity, were explored. Employing MnIIIP/MnIIP redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP5+ was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP5+ is most prone to oxidative degradation with H2 , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.

ErbB1/2 tyrosine kinase inhibitor mediates oxidative stress-induced apoptosis in inflammatory breast cancer cells

Overexpression of epidermal growth factor receptors (ErbB) is frequently seen in inflammatory breast cancer (IBC). Treatment with ErbB1/2-targeting agents (lapatinib) mediates tumor apoptosis by downregulating ErbB1/2 phosphorylation and downstream survival signaling. In this study, using carboxy-H2DCFDA, DHE, and MitoSOX Red to examine changes in hydrogen peroxide radicals, cytoplasmic and mitochondrial superoxide, respectively, we observed that GW583340 (a lapatinib-analog) increases reactive oxygen species (ROS) in two models of IBC (SUM149, SUM190) that are sensitive to ErbB1/2 blockade. This significant increase in ROS levels was similar to those generated by classical oxidative agents H2O2 and paraquat. In contrast, minimal to basal levels of ROS were measured in a clonal population of GW583340-resistant IBC cells (rSUM149 and rSUM190). The GW583340-resistant IBC cells displayed increased SOD1, SOD2, and glutathione expression, which correlated with decreased sensitivity to the apoptotic-inducing effects of GW583340, H2O2, and paraquat. The ROS increase and cell death in the GW583340-sensitive cells was reversed by simultaneous treatment with a superoxide dismutase (SOD) mimic. Additionally, overcoming the high levels of antioxidants using redox modulators induced apoptosis in the GW583340-resistant cells. Taken together, these data demonstrate a novel mechanism of lapatinib-analog-induced apoptosis and indicate that resistant cells have increased antioxidant potential, which can be overcome by treatment with SOD modulators.

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS‐mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non‐IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA‐approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF‐Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF‐Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress‐mediated apoptosis in multiple IBC cellular models. In vivo, DSF‐Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS‐inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo‐ and/or radio‐sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines

BackgroundCrosstalk between malignant hepatocytes and the surrounding peritumoral stroma is a key modulator of hepatocarcinogenesis and therapeutic resistance. To examine the chemotherapy resistance of these two cellular compartments in vitro, we evaluated a well-established hepatic tumor cell line, HepG2, and an adult hepatic stellate cell line, LX2. The aim was to compare the chemosensitization potential of arsenic trioxide (ATO) in combination with sorafenib or fluorouracil (5-FU), in both hepatic tumor cells and stromal cells.MethodsCytotoxicity of ATO, 5-FU, and sorafenib, alone and in combination against HepG2 cells and LX2 cells was measured by an automated high throughput cell-based proliferation assay. Changes in survival and apoptotic signaling pathways were analyzed by flow cytometry and western blot. Gene expression of the 5-FU metabolic enzyme, thymidylate synthase, was analyzed by real time PCR.ResultsBoth HepG2 and LX2 cell lines were susceptible to single agent sorafenib and ATO at 24 hr (ATO IC50: 5.3 μM in LX2; 32.7 μM in HepG2; Sorafenib IC50: 11.8 μM in LX2; 9.9 μM in HepG2). In contrast, 5-FU cytotoxicity required higher concentrations and prolonged (48–72 hr) drug exposure. Concurrent ATO and 5-FU treatment of HepG2 cells was synergistic, leading to increased cytotoxicity due in part to modulation of thymidylate synthase levels by ATO. Concurrent ATO and sorafenib treatment showed a trend towards increased HepG2 cytotoxicity, possibly due to a significant decrease in MAPK activation in comparison to treatment with ATO alone.ConclusionsATO differentially sensitizes hepatic tumor cells and adult hepatic stellate cells to 5-FU and sorafenib. Given the importance of both of these cell types in hepatocarcinogenesis, these data have implications for the rational development of anti-cancer therapy combinations for the treatment of hepatocellular carcinoma (HCC).

Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization.

Although there is no standard treatment protocol for inflammatory breast cancer (IBC), multi-modality treatment has improved survival. In this study we profiled the NCI approved oncology drug set in a qHTS format to identify those that are efficacious in basal type and ErbB2 overexpressing IBC models. Further, we characterized the sensitivity of an acquired therapeutic resistance model to the oncology drugs. We observed that lapatinib-induced acquired resistance in SUM149 cells led to cross-resistance to other targeted- and chemotherapeutic drugs. Removal of the primary drug to which the model was developed led to re-sensitization to multiple drugs to a degree comparable to the parental cell line; this coincided with the cells regaining the ability to accumulate ROS and reduced expression of anti-apoptotic factors and the antioxidant SOD2. We suggest that our findings provide a unique IBC model system for gaining an understanding of acquired therapeutic resistance and the effect of redox adaptation on anti-cancer drug efficacy.

The Role of Oxidative Stress in Breast Cancer

Abstract Breast cancer is the most common cancer in women worldwide and it accounts for the second highest morbidity and mortality. Disease etiology and progression is multifactorial and several risk factors associated with breast cancer exert their effects by modulation of oxidative stress status in the cells. Oxidative stress occurs due to an imbalance between reactive species and antioxidant defenses in the cells. Excess reactive species are deleterious in normal cells, while in cancer cells, they can lead to accelerated growth and survival correlating with an aggressive and therapy-resistant phenotype. Specifically, risk factors and their effect on the oxidative stress response are associated with breast cancer development, progression, and treatment outcome. This chapter provides a review of the accepted concepts, recent findings, and limitations in the understanding of the cross-talk between antioxidant capacity, redox-sensitive transcription factors, and cell survival/death signaling in oxidative stress response and redox adaptation in breast cancer. Addressing these matters and identifying pathway dysregulation is required for a rational basis to improve the design of redox-related therapeutics and clinical trials in breast cancer.

Abstract 951: Cross-resistance and re-sensitization to multiple drugs in an IBC model of lapatinib acquired resistance parallels redox adaptation.

Inflammatory breast cancer (IBC) is characterized by rapid progression from onset of disease, therefore an early and aggressive multimodal therapy is essential to improve outcome. IBC tumors are frequently ErbB2- and EGFR-positive, and the use of trastuzumab in combination with lapatinib has seen clinical success; however, de novo and acquired resistance to these agents due to interplay between different members of the ErbB family is a significant challenge. In addition, it is well recognized that there is paucity of IBC cellular models. We have characterized a novel isogenic-derived progression model of lapatinib drug resistance (rSUM149) and re-sensitization (rrSUM149). Four IBC cell lines representing either HER2 overexpression or basal-type were profiled to identify approved oncology drugs from that can act as potent inhibitors of IBC cell proliferation. In our present study, rSUM149 cells showed cross-resistance to a number of the drugs previously shown to act on the parental cells. We show that long term removal of lapatinib, the primary drug against which resistance was developed from rSUM149 cells led to isolation of a population of cells (rrSUM149) that are then re-sensitized to multiple drugs, behaving in a manner comparable to the parental SUM149 cell line. Recently, we identified that the lapatinib-resistant rSUM149 had increased levels of anti-apoptotic proteins, increased antioxidant expression (superoxide dismutase and GSH), and decreased ability to accumulate reactive oxygen species (ROS), all of which lead to inhibition of drug-induced apoptosis. We had previously validated this finding with the observations that: (i). Overexpressing XIAP in therapy-sensitive IBC cells renders them therapy-resistant and this corresponds to low ROS accumulation in response to oxidative stress; (ii). specific XIAP inhibition using siRNA or XIAP small molecule inhibitors reversed drug resistance, decreased the antioxidants involved in redox-adaptation and increased significant ROS accumulation leading to IBC cell death. In our results presented herein, re-sensitization was accompanied by a decrease in expression of the apoptosis inhibitor XIAP and antioxidant SOD2; assessment of ROS following challenge with lapatinib revealed that the ability to accumulate ROS was restored to the cells through those changes. This mechanistic phenotype supports our current observations of cross-resistance to multiple drugs, which is also commonly seen in patients. These results strengthen the need for novel strategies to modulate cellular redox to overcome drug resistance in IBC. *Joint corresponding authors (GRD & KPW). Supported in part by NIH grant CA137844 (KPW), American Cancer Society grant RSG-08-290-01-CCE (GRD), DCI Cancer and Environment Initiative award, Duke Uni (GRD). Additional funding; Golden LEAF Foundation and BIOIMPACT Initiative of the State of NC. Citation Format: Gayathri R. Devi, Jennifer L. Allensworth, Shalonda M. Ingram, Ginger R. Smith, Amy J. Aldrich, Kevin P. Williams. Cross-resistance and re-sensitization to multiple drugs in an IBC model of lapatinib acquired resistance parallels redox adaptation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2013-951

101 Smac Mimetic Induces Apoptosis and Synergizes with TRAIL in Inflammatory Breast Cancer Cells in an IAP-Dependent and TNF-a-Independent Mechanism

Burkitt’s lymphoma cell line BJAB-LexR that has been made naturally resistant to TRAIL through long-term culture in the presence of increasing concentrations of the drug Lexatumumab, an anti-DR5 antibody, as well as BJAB cells with Six1 overexpression. Performing experiments in both these contexts would allow us to distinguish between resistance genes that arise spontaneously versus resistance genes that are a direct effect of Six1 expression. We performed a genome-wide loss of function screen using the GeneNet lentiviral shRNA library containing 200 000 shRNAs with sequencing tags, allowing for high throughput deep sequencing. After selection for a particular phenotype, in this case TRAIL resistance, the shRNAs that promote or prevent this phenotype can be deduced from the frequency in which the shRNA is present in the selected population versus the unselected population. A library was constructed using sequence tag specific primers and deep sequencing was performed using Illumina sequencing technology. Bioinformatic analysis yielded a list of putative resistance genes that are currently being validated in the BJAB system and also in MDAMB231 breast cancer cells that have been made resistant to TRAIL in a similar fashion. With this unbiased approach, we aim to identify not only known components of the TRAIL pathway that may be deregulated but also completely novel pathways of TRAIL resistance that could be targeted for improved cancer therapy.