Myron K. Evans

Mn porphyrin in combination with ascorbate acts as a pro-oxidant and mediates caspase-independent cancer cell death.

Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP(5+) and MnTnBuOE-2-PyP(5+)) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy.

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.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Abstract 1007: Elucidating a role for the translation initiation factor, eIF4G1, in resistance to therapy in inflammatory breast cancer (IBC)

Inflammatory breast cancer (IBC) is a highly invasive and aggressive breast cancer with relatively low survival rates compared with other locally advanced breast cancers. IBC is characterized by its rapid development of resistance to radio- and chemotherapies, which may in part be due to altered translational control driven by overexpression of the translation initiation factor, eIF4G1. This protein has been shown to enhance translation of eukaryotic mRNAs with internal ribosome entry sites (IRES) and enhanced translation in IBC may play a role in the survival of tumor cells during cellular stress, when translation would normally be suppressed. Some inhibitors of apoptosis proteins have 5′-untranslated regions (UTRs) that allow for IRES-mediated translation, and inhibition of apoptosis is one mechanism by which IBC cells have previously been shown to become resistant to therapy. Translational up-regulation of an inhibitor of apoptosis protein in therapy-resistant clones of IBC cell lines has been observed, which is of importance as the 5′UTR of such proteins contain highly active IRES elements. Regulation of such protein expression by eIF4G1-mediated IRES translation may be one reason for this observed therapeutic resistance. We knocked down eIF4G1 in a resistant IBC cell line (rSUM149) and assessed cellular viability after treatment with a powerful apoptosis inducer, TRAIL to evaluate the effect of targeted eIF4G1 knockdown on drug resistance in IBC. We observed that targeted knockdown of eIF4G1 sensitizes rSUM149 cells to TRAIL, significantly decreasing cellular viability. Additionally, using a novel culture system we show that inhibition of eIF4G1 expression decreases both the number of size of tumor emboli, a characteristic feature of IBC progression. These findings demonstrate a vital role for eIF4G1 for the development of therapeutic resistance and ultimately the aggressive pathobiology of IBC. Citation Format: Arianna Price, Courtney M. Edwards, Myron K. Evans, Gayathri Devi. Elucidating a role for the translation initiation factor, eIF4G1, in resistance to therapy in inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1007. doi:10.1158/1538-7445.AM2015-1007

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.

XIAP Regulation by MNK Links MAPK and NFκB Signaling to Determine an Aggressive Breast Cancer Phenotype

Hyperactivation of the NFκB pathway is a distinct feature of inflammatory breast cancer (IBC), a highly proliferative and lethal disease. Gene expression studies in IBC patient tissue have linked EGFR (EGFR/HER2)-mediated MAPK signaling to NFκB hyperactivity, but the mechanism(s) by which this occurs remain unclear. Here, we report that the X-linked inhibitor of apoptosis protein (XIAP) plays a central role in linking these two pathways. XIAP overexpression correlated with poor prognoses in breast cancer patients and was frequently observed in untreated IBC patient primary tumors. XIAP drove constitutive NFκB transcriptional activity, which mediated ALDH positivity (a marker of stem-like cells), in vivo tumor growth, and an IBC expression signature in patient-derived IBC cells. Using pathway inhibitors and mathematical models, we defined a new role for the MAPK interacting (Ser/Thr)-kinase (MNK) in enhancing XIAP expression and downstream NFκB signaling. Furthermore, targeted XIAP knockdown and treatment with a MNK inhibitor decreased tumor cell migration in a dorsal skin fold window chamber murine model that allowed for intravital imaging of local tumor growth and migration. Together, our results indicate a novel role for XIAP in the molecular cross-talk between MAPK and NFκB pathways in aggressive tumor growth, which has the potential to be therapeutically exploited.Significance: Signaling by the MNK kinase is essential in inflammatory breast cancer, and it can be targeted to inhibit XIAP-NFκB signaling and the aggressive phenotype of this malignancy. Cancer Res; 78(7); 1726-38. ©2018 AACR.

Abstract LB-015: XIAP induction by the MAPK-eIF4G1 pathway drives NFκB activation in inflammatory breast cancer growth and therapeutic resistance

Inflammatory breast cancer (IBC) is the most lethal, distinct form of breast cancer, however, the basis for its aggressiveness and rapid acquisition of drug resistance is not fully understood. Using immunohistochemical analysis, we identified a strong correlation between high grade, high stage, and triple-negative status and elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) in IBC. Molecular profiling of multiple IBC cell lines revealed that modulating XIAP expression can significantly alter the expression pattern of a 79-gene, characteristic IBC profile that was previously obtained from clinical samples. Using specific antagonists and RNAi, we determined that the mitogen activated protein kinase (MAPK) pathway and its interaction with the protein synthesis initiation factor eIF4G1, both of which are elevated in IBC, cooperate to drive XIAP induction and resistance to therapeutic apoptosis. Further, we found that XIAP expression directly correlates with activation of the transcription factor NFκB, a molecular component defining IBC. Mutational analysis revealed that the BIR1 domain of XIAP is essential for subsequent TAB1:IKKβ-dependent NFκB activation. After determining this association between XIAP and TAB1, we tested a peptide-mediated strategy used to block the BIR1:TAB1 interaction antagonized NFκB activity which led to decreased anchorage independent growth and reversed resistance to an EGFR tyrosine kinase inhibitor. Most significantly, orthotopic implantation of XIAP silenced IBC cells revealed the necessity of expression for IBC tumor growth, while overexpression of XIAP enhanced tumor growth. Our findings establish that XIAP augments the malignant properties in IBC by enhancing NFκB function, identify a druggable pathway with multiple targets, and provide feasibility for the development of novel therapeutics targeting the BIR1 domain of XIAP in IBC. Grant Support: Supported by American Cancer Society, the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236, the Duke Department of Surgery, and DOD grant W81XWH-13-1-0047. Citation Format: Myron K. Evans, Joseph Geradts, Courtney Edwards, Arianna Price, Arjun J. Arora, Amy J. Aldrich, Adrian Ramirez, Timothy J. Robinson, Peter B. Vermeulen, Steven van Laere, Gayathri R. Devi. XIAP induction by the MAPK-eIF4G1 pathway drives NFκB activation in inflammatory breast cancer growth and therapeutic resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-015.

Abstract P6-14-05: A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology

Background: Inflammatory breast cancer (IBC) has the highest lethality amongst all subtypes of breast cancer and develops rapid therapeutic resistance. High NFκB activation has been identified as a distinct molecular determinant in IBC pathobiology; however, the precise sequence of its activation and functional consequence in IBC remains unknown. Our previous work identified increased expression of the X-linked inhibitor of apoptosis protein (XIAP) due to altered translation in IBC, while other studies have noted a crosstalk between XIAP and NFκB. We hypothesized that XIAP drives NFκB activation in IBC promoting therapeutic resistance and tumorigenesis. Methods: NFκB phosphorylation, nuclear translocation, and target gene expression were evaluated in triple-negative SUM149 IBC cells with targeted overexpression or knockdown of XIAP. Using specific point mutants, we assessed the domain and mechanism of XIAP-mediated NFκB activation in IBC. We evaluated proliferation and viability in 2D and 3D culture of SUM149 cells treated with JSH-23, a small molecule inhibitor of NFκB nuclear translocation. We monitored the effects of XIAP overexpression or knockdown on in vivo tumorigenicity in IBC xenograft models by measuring tumor growth and NFκB signaling. IHC analysis of XIAP and NFκB was performed on tumor microarrays containing both non-IBC and IBC. Results: Knockdown of XIAP significantly decreased NFκB activation in IBC cells. Domain analysis revealed the necessity of the BIR1 domain of XIAP and TAB1:IKKβ complex formation in activating NFκB. NFκB antagonism inhibited proliferation of cells and sensitized therapy-resistant, XIAP overexpressing cells to targeted therapy. Loss of XIAP inhibited tumor growth of SUM149 tumor cells, correlating with decreased ALDH activity and varied epithelial-mesenchymal characteristics in these cells, while overexpression of XIAP significantly enhanced tumor growth of SUM149 cells. Further analysis revealed altered SMAD7 expression in XIAP knockdown cells, revealing crosstalk between XIAP, NFκB, and TGFβ signaling in IBC. IHC analysis of XIAP expression in invasive non-IBC tumors correlated with triple-negative status as well as increased grade and stage of tumors. In IBC tumors, XIAP expression associated with increased NFκB. Conclusions: In summary, our studies reveal that XIAP expression is necessary for NFκB activation in IBC and is critical for IBC development and progression. This study provides a novel insight into how an anti-apoptotic protein may regulate survival signaling and disease progression and may guide further research into innovative inhibitors of this interaction. Citation Format: Myron K Evans, Scott J Sauer, Amy J Aldrich, Joseph Geradts, Peter Vermeulen, Luc Dirix, Steven Van Laere, Gayathri R Devi. A novel link between anti-apoptotic signaling, NFκB, and SMAD7 in IBC pathobiology [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-05.

Abstract 5552: Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death.

Resistance to therapy-mediated apoptosis in inflammatory breast cancer (IBC), an aggressive and distinct subtype of breast cancer was recently attributed to increased antioxidant expression (superoxide dismutase [SOD]; reduced glutathione) and decreased accumulation of reactive oxygen species (ROS) resulting in redox adaptation and increased cell survival. Mn porphyrins (MnPs) are amongst the most potent synthetic mimics of the antioxidant, superoxide dismutase (SOD). In the present study, we demonstrate the unique ability of two Mn porphyrin (MnP)-based SOD mimics (MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+) to act as pro-oxidants when combined with ascorbate (vitamin C) and enhance ROS-mediated cell death. We identified that the ethyl species, MnTE-2-PyP5+, was less stable than its butoxyethyl analog, MnTnBuOE-2-PyP5+, resulting in greater potency in inducing cytotoxicity via radical-driven oxidative degradation in cellular models of IBC isolated from patient tumors and an isogenic derivative with acquired resistance to therapeutic apoptosis. The combination of MnP and ascorbate induced accumulation of mitochondrial superoxide and H2O2-derived radicals in both therapy-sensitive and therapy-resistant cell lines. High intracellular ROS resulted in decreases in both pro-survival signaling (pNF-κB, pERK1/2) and expression of X-linked inhibitor of apoptosis protein (XIAP), the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and Annexin V staining, administration of a pan-caspase inhibitor (QVD-OPh) did not reverse the observed cytotoxicity. Nuclear translocation of apoptosis inducing factor (AIF) was observed, suggesting a caspase-independent cell death following mitochondrial outer membrane permeabilization after treatment with this combination. In conclusion, this study provides the molecular basis of a novel MnP-based anti-cancer strategy and suggests the feasibility of using MnPs to sensitize cancer cells to many chemotherapies and radiotherapy that are dependent on potent induction of the oxidative stress response. Citation Format: Myron Evans, Artak Tovmasyan, Ines Batinic-Haberle, Gayathri Devi. Manganese porphyrins in combination with ascorbate act as pro-oxidants and mediate caspase-independent cancer cell death. [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 5552. doi:10.1158/1538-7445.AM2013-5552 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.