Scott J. Sauer

Asymmetric total synthesis of the antimalarial drug (+)-mefloquine hydrochloride via chiral N-amino cyclic carbamate hydrazones.

Mefloquine hydrochloride is an important antimalarial drug. It is currently manufactured and administered in racemic form; however there are indications regarding the biological activity of the two enantiomers that suggest the superiority of the (+)-form. The asymmetric total synthesis of the (+)-enantiomer of mefloquine hydrochloride is described. The key asymmetric transformation utilized is a novel asymmetric Darzens reaction of a chiral α-chloro-N-amino cyclic carbamate hydrazone derived from an N-amino cyclic carbamate (ACC) chiral auxiliary.

Direct carbon-carbon bond formation via reductive soft enolization: a kinetically controlled syn-aldol addition of α-halo thioesters and enolizable aldehydes.

The direct addition of enolizable aldehydes and α-halo thioesters to produce β-hydroxy thioesters enabled by reductive soft enolization is reported. The transformation is operationally simple and efficient and has the unusual feature of giving high syn-selectivity, which is the opposite of that produced for (thio)esters under conventional conditions. Moreover, excellent diastereoselectivity results when a chiral nonracemic α-hydroxy aldehyde derivative is used.

Bisphenol A activates EGFR and ERK promoting proliferation, tumor spheroid formation and resistance to EGFR pathway inhibition in estrogen receptor-negative inflammatory breast cancer cells

Emerging evidence from epidemiological studies suggests a link between environmental chemical exposure and progression of aggressive breast cancer subtypes. Of all clinically distinct types of breast cancers, the most lethal phenotypic variant is inflammatory breast cancer (IBC). Overexpression of epidermal growth factor receptors (EGFR/HER2) along with estrogen receptor (ER) negativity is common in IBC tumor cells, which instead of a solid mass present as rapidly proliferating diffuse tumor cell clusters. Our previous studies have demonstrated a role of an adaptive response of increased antioxidants in acquired resistance to EGFR-targeting drugs in IBC. Environmental chemicals are known to induce oxidative stress resulting in perturbations in signal transduction pathways. It is therefore of interest to identify chemicals that can potentiate EGFR mitogenic effects in IBC. Herein, we assessed in ER-negative IBC cells a subset of chemicals from the EPA ToxCast set for their effect on EGFR activation and in multiple cancer phenotypic assays. We demonstrated that endocrine-disrupting chemicals such as bisphenol A (BPA) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane can increase EGFR/ERK signaling. BPA also caused a corresponding increase in expression of SOD1 and anti-apoptotic Bcl-2, key markers of antioxidant and anti-apoptotic processes. BPA potentiated clonogenic growth and tumor spheroid formation in vitro, reflecting IBC-specific pathological characteristics. Furthermore, we identified that BPA was able to attenuate the inhibitory effect of an EGFR targeted drug in a longer-term anchorage-independent growth assay. These findings provide a potential mechanistic basis for environmental chemicals such as BPA in potentiating a hyperproliferative and death-resistant phenotype in cancer cells by activating mitogenic pathways to which the tumor cells are addicted for survival.

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.

Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies

Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy.

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 B49: Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth

Our objective was to assess GLI pathway antagonists for activity in inflammatory breast cancer (IBC) phenotypic models. Introduction: IBC is one of the most lethal forms of breast cancer and accounts for ~15% of all breast cancer deaths. IBC affects younger patients and is diagnosed in minorities at higher rates than in other racial and ethnic groups1. African-American women with IBC appear to have worse survival regardless of inflammatory status, stage, socioeconomic position, tumor, or treatment characteristics2,3. IBC is characterized by rapid progression, is highly invasive and is not usually detected by mammograms or ultrasounds. The presence of tumor emboli (TE) is a hallmark of the disease in which distinctive clusters of tumor cells migrate collectively and drive lymphatic invasion and metastasis. Activation of GLI1, the terminal effector of hedgehog (Hh) signaling, via gene amplification, has been linked to tumorigenesis, and invasiveness in many cancers including our studies on IBC4. Targeting at the level of GLI1 with small molecules has been effective with some demonstrating activity in vivo. Experimental procedures: In our studies we have used a tiered approach of phenotypic and functional assays to evaluate a panel of GLI antagonists for effects on IBC cell growth, in particular assessing their effects in models that more closely model or predict tumor growth in vivo including; migration, anchorage independent growth (AIG), mammosphere formation and TE assays. Results: Using our previously described quantitative cell proliferation assay5 we first identified a subset of GLI antagonists that effected IBC proliferation and down-regulated GLI1 transcriptional activity. We have now used high content imaging to simultaneously measure nuclear count, morphology, cell integrity, and mitochondrial health in live cells. We have identified GLI antagonists that show an anti-proliferative response but have no effect on nuclear size suggesting a lack of general cytotoxicity. In contrast, other antagonists had dramatic effects on nuclear area and nuclear texture indicative of cytotoxicity. We further examined their effects on colony formation using the AIG assay. SUM149 cells were treated with GLI inhibitors and effects on colony size and number noted. All three GLI antagonists tested reduced the size of the colonies with one also giving a significant reduction in number of colonies formed. Recently we have optimized an in vitro TE assay and are using this to screen GLI antagonists for effects on TE formation. Conclusion: we have identified a subset of GLI antagonists by phenotypic profiling for those with effects in cancer-specific functional assays that will be prioritized for testing in vivo. Funded in part by DOD/CDMRP IDEA (BC121850) (KPW). 1 Anderson, W. F., Schairer, C., Chen, B. E., Hance, K. W. & Levine, P. H. Epidemiology of inflammatory breast cancer (IBC). Breast disease 22, 9-23 (2006). 2 Schlichting, J. A., Soliman, A. S., Schairer, C., Schottenfeld, D. & Merajver, S. D. Inflammatory and non-inflammatory breast cancer survival by socioeconomic position in the Surveillance, Epidemiology, and End Results database, 1990-2008. Breast Cancer Research and Treatment 134, 1257-1268 (2012). 3 Yang, R. et al. A comprehensive evaluation of outcomes for inflammatory breast cancer. Breast cancer research and treatment 117, 631-641 (2009). 4 Thomas, Z. et al. Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration. British Journal of Cancer 104, 1575-1586, doi:10.1038/bjc.2011.133 (2011). 5 Williams, K. P. et al. Quantitative high-throughput efficacy profiling of approved oncology drugs in inflammatory breast cancer models of acquired drug resistance and re-sensitization. Cancer Letters 337, 77-89 (2013). Citation Format: Helen Oladapo, Scott Sauer, Michael Tarpley, Gayathri Devi, Kevin P. Williams. Profiling of GLI antagonists in phenotypic models for effects on inflammatory breast cancer cell growth. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B49.

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.