Kirk C. Hansen

Collagen architecture in pregnancy-induced protection from breast cancer

Summary The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of &bgr;1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell–cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.

A TDO2-AhR Signaling Axis Facilitates Anoikis Resistance and Metastasis in Triple-Negative Breast Cancer

The ability of a cancer cell to develop resistance to anoikis, a programmed cell death process triggered by substratum detachment, is a critical step in the metastatic cascade. Triple-negative breast cancers (TNBC) exhibit higher rates of metastasis after diagnosis, relative to estrogen-positive breast cancers, but while TNBC cells are relatively more resistant to anoikis, the mechanisms involved are unclear. Through gene expression and metabolomic profiling of TNBC cells in forced suspension culture, we identified a molecular pathway critical for anchorage-independent cell survival. TNBC cells in suspension upregulated multiple genes in the kynurenine pathway of tryptophan catabolism, including the enzyme tryptophan 2,3-dioxygenase (TDO2), in an NF-κB-dependent manner. Kynurenine production mediated by TDO2 in TNBC cells was sufficient to activate aryl hydrocarbon receptor (AhR), an endogenous kynurenine receptor. Notably, pharmacologic inhibition or genetic attenuation of TDO2 or AhR increased cellular sensitivity to anoikis, and also reduced proliferation, migration, and invasion of TNBC cells. In vivo, TDO2 inhibitor-treated TNBC cells inhibited colonization of the lung, suggesting that TDO2 enhanced metastatic capacity. In clinical specimens of TNBC, elevated expression of TDO2 was associated with increased disease grade, estrogen receptor-negative status, and shorter overall survival. Our results define an NF-κB-regulated signaling axis that promotes anoikis resistance, suggest functional connections with inflammatory modulation by the kynurenine pathway, and highlight TDO2 as an attractive target for treatment of this aggressive breast cancer subtype.

Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments

Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

Trauma/hemorrhagic shock instigates aberrant metabolic flux through glycolytic pathways, as revealed by preliminary 13C-glucose labeling metabolomics

BackgroundMetabolic derangement is a key hallmark of major traumatic injury. The recent introduction of mass spectrometry-based metabolomics technologies in the field of trauma shed new light on metabolic aberrations in plasma that are triggered by trauma and hemorrhagic shock. Alteration in metabolites associated with catabolism, acidosis and hyperglycemia have been identified. However, the mechanisms underlying fluxes driving such metabolic adaptations remain elusive.MethodsA bolus of U-13C-glucose was injected in Sprague–Dawley rats at different time points. Plasma extracts were analyzed via ultra-high performance liquid chromatography-mass spectrometry to detect quantitative fluctuations in metabolite levels as well as to trace the distribution of heavy labeled carbon isotopologues.ResultsRats experiencing trauma did not show major plasma metabolic aberrations. However, trauma/hemorrhagic shock triggered severe metabolic derangement, resulting in increased glucose levels, lactate and carboxylic acid accumulation. Isotopologue distributions in late Krebs cycle metabolites (especially succinate) suggested a blockade at complex I and II of the electron transport chain, likely due to mitochondrial uncoupling. Urate increased after trauma and hemorrhage. Increased levels of unlabeled mannitol and citramalate, metabolites of potential bacterial origin, were also observed in trauma/hemorrhagic shock rats, but not trauma alone or controls.ConclusionsThese preliminary results are consistent with observations we have recently obtained in humans, and expand upon our early results on rodent models of trauma and hemorrhagic shock by providing the kinetics of glucose fluxes after trauma and hemorrhage. Despite the preliminary nature of this study, owing to the limited number of biological replicates, results highlight a role for shock, rather than trauma alone, in eliciting systemic metabolic aberrations. This study provides the foundation for tracing experiments in rat models of trauma. The goal is to improve our understanding of substrate specific metabolic derangements in trauma/hemorrhagic shock, so as to design resuscitative strategies tailored toward metabolic alterations and the severity of trauma.

Skeletal muscle phosphatidylcholine and phosphatidylethanolamine are related to insulin sensitivity and respond to acute exercise in humans

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.

Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia

Acute myeloid leukemia (AML) is a blood cancer that is poorly responsive to conventional cytotoxic chemotherapy and a diagnosis of AML is usually fatal. More effective and better-tolerated therapies for AML are desperately needed. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most frequently observed genetic defects in AML. FLT3 inhibitors have shown impressive anti-leukemic activity in clinical trials; however, sustained remissions using these inhibitors as monotherapy have not been achieved. Our previous studies have implicated impaired glutamine metabolism in response to FLT3 inhibitors as a dominant factor causing AML cell death. In this study, we have employed metabolic flux analysis to examine the effects of FLT3 inhibition on glutamine utilization in FLT3-mutated AML cells using stable isotope tracers. We found that the FLT3 inhibitor AC220 inhibited glutamine flux into the antioxidant factor glutathione profoundly due to defective glutamine import. We also found that the glutaminase inhibitor CB-839 similarly impaired glutathione production by effectively blocking flux of glutamine into glutamate. Moreover, the combination of AC220 with CB-839 synergized to deplete glutathione, induce mitochondrial reactive oxygen species, and cause loss of viability through apoptotic cell death. In vivo, glutaminase inhibition with CB-839 facilitated leukemic cell elimination by AC220 and improved survival significantly in a patient-derived xenograft AML mouse model. Therefore, targeting glutaminase in combination with FLT3 may represent an effective therapeutic strategy for improving treatment of FLT3-mutated AML.

Abstract C78: ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common adult acute leukemia and accounts for approximately 20% of childhood leukemias. Although frontline treatment of AML with cytotoxic chemotherapy is capable of achieving high remission rates, 75-80% of patients will either not respond to or will relapse after initial therapy, and most patients will die of their disease. More effective and better-tolerated therapies for AML are required. Activating mutations in fms-like tyrosine kinase 3 (FLT3) are the most frequently observed genetic defect in AML and drive leukemic cell growth and survival. FLT3 tyrosine kinase inhibitors have shown impressive anti-leukemic activity in clinical trials, however, sustained remissions using these inhibitors as monotherapy have not been achieved. In order to identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a large-scale RNA interference-based screen. The screen identified several genes involved in metabolic regulation, including ataxia telangiectasia mutated (ATM), as being synthetic lethal with FLT3 inhibition in FLT3 mutated AML. Genetic or pharmacological inactivation of ATM or its downstream effector glucose-6-phosphate dehydrogenase (G6PD) sensitized AML cells to FLT3 inhibition through enhancing apoptosis. Whole metabolome profiling revealed that FLT3 inhibition causes severe and widespread metabolic deficiencies, including depletion of the antioxidant factor glutathione. Inactivation of either ATM or G6PD exacerbated glutathione depletion upon FLT3 inhibition. Subsequent analyses revealed that FLT3 inhibition elicits severe mitochondrial oxidative stress that is causative in apoptosis and accentuated by ATM or G6PD inhibition. The use of a drug that promotes the production of mitochondrial reactive oxygen species (ROS) in combination with a FLT3 inhibitor augmented elimination of AML cells both in vitro and in vivo. Our data support the hypothesis that FLT3 mutated AML cells are highly dependent on FLT3 activity to maintain glutathione levels and sufficient mitochondrial antioxidant capacity to sustain cell survival. Moreover, these data support the novel strategy of employing mitochondrial ROS inducing agents as adjuvant to FLT3 inhibitor therapy to more effectively treat FLT3 mutated AML. Citation Format: Mark A. Gregory, Angelo D9Alessandro, Francesca Alvarez-Calderon, Jihye Kim, Travis Nemkov, Aik Choon Tan, Kirk C. Hansen, James DeGregori. ATM/G6PD-dependent metabolic pathways promote mitochondrial redox homeostasis and resistance to FLT3 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C78.

Abstract P1-07-12: Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer

Background: Anoikis resistance is thought to be a critical trait of metastatic cancer cells, enabling them to leave the primary tumor and travel through extracellular matrix, intravasate, and survive in the vasculature or lymphatics in transit to a metastatic site. This is particularly important for the triple-negative breast cancer (TNBC) subtype, which has a peak risk of recurrence within the first three years post-diagnosis and an increased mortality rate in the first five years as compared to other subtypes. We performed global profiling of TNBC cells in attached versus forced suspension culture conditions (using poly-HEMA coated plates) for 24 hours. These data revealed that TNBC cells surviving in suspension upregulate multiple genes involved in tryptophan catabolism, also known as the kynurenine pathway (KP), including the rate limiting enzyme tryptophan 2,3,-dioxygenase (TDO) and kynureninase (KYNU). A key metabolite of this pathway has been found to activate the aryl hydrocarbon receptor (AhR), which was also up-regulated in suspended cells. Hypothesis: We hypothesize that the ability to upregulate the kynurenine pathway (KP) facilitates TNBC cell survival in suspension and mediates the migratory/invasive potential of TNBC. Methods: We assessed mRNA and protein levels of TDO2, KYNU and AhR by RT-qPCR and western blot. AhR luciferase reporter activity, as well as known AhR regulated genes, were measured in suspension compared to adherent conditions. TNBC cells were treated with small molecule inhibitors of AhR and TDO2. Additionally, secretion of endogenous kynurenine was measured by high performance liquid chromatography (HPLC). Purified kynurenine was added to rescue AhR activity following TDO2 inhibition. Finally, anoikis sensitivity and migratory potential were measured following pharmacological inhibition of TDO2 and AhR. Results: Relative mRNA levels of TDO2, KYNU, and AhR increase 9, 7, and 2 fold respectively in suspended TNBC cells compared to adherent conditions (P Conclusions: Collectively, these results suggest that the kynurenine pathway may play a critical role in metastatic TNBC. Further mechanistic studies will focus on how the kynurenine pathway is mediating these tumorigenic properties either through the de novo synthesis of NAD+ and/or activation of AhR by kynurenine. Targeting the kynurenine pathway in the clinic may provide a therapeutic strategy to reduce TNBC mortality rates. Citation Format: Thomas Rogers, Nicholas D9Amato, Travis Nemkov, Michael Gordon, Kirk Hansen, Jennifer Richer. Defining the role of the kynurenine pathway in mediating anoikis resistance in triple negative breast cancer [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 P1-07-12.

Abstract B090: Collagen organization implicated in tumor dormancy

It is known that tumor cells can reside in a dormant state for decades. Data from numerous studies strongly suggest that extracellular matrix (ECM) proteins can impart a dormant phenotype, however little is known regarding how ECM regulates tumor cell dormancy. Given the technical difficulties in studying tumor cell dormancy in the context of secondary lesions (i.e., the searching for a needle in a haystack problem), here we use tumor suppression at the primary site as a model to investigate the role of ECM in breast cancer dormancy. Specifically, we hypothesize that insight into tumor dormancy can be obtained by studying the response of the mammary ECM to tamoxifen treatment and parity, two conditions that protect the mammary epithelium from tumorigenesis. Utilizing breast cancer cell lines capable of transitioning from indolent to proliferative/invasive states, we demonstrate that ECM isolated from tamoxifen treated or parous rat mammary glands decreases tumor growth and impedes invasive phenotypes compared to nulliparous mammary matrix, using both in vitro and in vivo models. Collagen stained tissues and proteomic analysis identified increased abundance of collagen I in tamoxifen treated and parous mammary stroma, an observation extended to breast tissue of parous women. These results are unexpected given the known pro-tumorigenic attributes of fibrillar collagen. Second harmonic generation imaging and atomic force microscopy reveals that the abundant collagen observed in the mammary glands of parous rats is less linearized and has decreased stromal stiffness compared to nulliparous mammary glands. These data implicate collagen organization rather than density in tumor suppression. Using 3D cell culture models, we demonstrate that linearized fibrillar collagen I induces an invasive phenotype in mammary tumor cells through a mechanism dependent on β1 integrin endocytic recycling. Conversely, high density, non-fibrillar collagen I decreases β1 endocytic integrin recycling, increases junctional E-cadherin staining, upregulates adherens and tight junction genes, and downregulates EMT transcription factors and metalloproteinase genes, all attributes consistent with tumor suppression. These data show that collagen organization, rather than collagen density, is a key contributor to the tumor cell invasive phenotype. In conclusion, we demonstrate that collagen | organization is dominant over density in driving tumor cell invasion, an observation that may have implications in understanding how mammographic breast density determines breast cancer risk, as well as how collagen organization contributes to tumor cell dormancy at distant sites. Citation Format: Ori Maller, Kirk C. Hansen, Valerie Weaver, Rytis Prekeris, Pepper Schedin. Collagen organization implicated in tumor dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B090.