Kelly Schoenfelt

Contextualised urinary biomarker analysis facilitates diagnosis of paediatric obstructive sleep apnoea

BACKGROUND Intrinsic variance of the urine proteome limits the discriminative power of proteomic analysis and complicates potential biomarker detection in the context of paediatric sleep disorders. METHODS AND RESULTS Using a rigorous workflow for proteomic analysis of urine, we demonstrate that gender and diurnal effects constitute two important sources of variability in healthy children. In the context of disease, complex pathophysiological perturbations magnify these proteomic differences and therefore require contextualised biomarker analysis. Indeed, by performing biomarker discovery in a gender- and diurnal-dependent manner, we identified ∼30-fold more candidate biomarkers of paediatric obstructive sleep apnoea (OSA), a highly prevalent condition in children characterised by repetitive episodes of intermittent hypoxia and hypercapnia, and sleep fragmentation in the context of recurrent upper airway obstructive events during sleep. Remarkably, biomarkers were highly specific for gender and sampling time as poor overlap (∼3%) was observed in the proteins identified in boys and girls across morning and bedtime samples. CONCLUSIONS As no clinical basis to explain gender-specific effects in OSA or healthy children is apparent, we propose that implementation of contextualised biomarker strategies will be applicable to a broad range of human diseases, and may be specifically applicable to paediatric OSA.

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic.

Metabolically activated macrophages in mammary adipose tissue link obesity to triple-negative breast cancer

Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Macrophages, which accumulate in adipose tissue and are activated during obesity, are an attractive mechanistic link. Here, we show that, during obesity, murine and human mammary adipose tissue macrophages adopt a pro-inflammatory, metabolically- activated (MMe) macrophage phenotype that promotes TNBC stem-like markers and functions, including increased tumorsphere growth in vitro and tumor-initiating potential in vivo. We demonstrate that MMe macrophages release cytokines in an NADPH oxidase 2 (NOX2)-dependent manner that signal through glycoprotein 130 (GP130) on TNBC cells to promote their stem-like properties. Accordingly, deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates the ability of obesity to drive TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue during obesity as a mechanism for promoting TNBC stemness and tumorigenesis. HIGHLIGHTS ⁘ Obesity promotes TNBC tumor formation and stemness. ⁘ Mammary adipose tissue macrophages are metabolically activated (MMe) in obese mice and humans. ⁘ MMe macrophages in mammary adipose tissue contribute to obesity-induced stemness. ⁘ MMe macrophages promote TNBC stemness through GP130 signaling.

Obesity and Insulin Resistance Promote Atherosclerosis through an IFNγ-Regulated Macrophage Protein Network

SUMMARY Type 2 diabetes (T2D) is associated with increased risk for atherosclerosis; however, the mechanisms underlying this relationship are poorly understood. Macrophages, which are activated in T2D and causatively linked to atherogenesis, are an attractive mechanistic link. Here, we use proteomics to show that diet-induced obesity and insulin resistance (obesity/IR) modulate a pro-atherogenic “macrophage-sterol-responsive-network” (MSRN), which, in turn, predisposes macrophages to cholesterol accumulation. We identify IFNγ as the mediator of obesity/IR-induced MSRN dysregulation and increased macrophage cholesterol accumulation and show that obesity/IR primes T cells to increase IFNγ production. Accordingly, myeloid cell-specific deletion of the IFNγ receptor (Ifngr1−/−) restores MSRN proteins, attenuates macrophage cholesterol accumulation and atherogenesis, and uncouples the strong relationship between hyperinsulinemia and aortic root lesion size in hypercholesterolemic Ldlr−/− mice with obesity/IR, but does not affect these parameters in Ldlr−/− mice without obesity/IR. Collectively, our findings identify an IFNγ-macrophage pathway as a mechanistic link between obesity/IR and accelerated atherogenesis.

Abstract 2888: Metabolically activated macrophages in obesity associated TNBC

Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC; obese women have a 35% higher risk of developing TNBC and 46% higher risk of developing distant metastases. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Mechanisms by which obesity worsens TNBC prognosis are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and macrophage infiltration into adipose tissue is a key mediator of this inflammation. Recent studies demonstrated that macrophages are enriched in breast adipose tissue of obese humans and mice. Moreover, depletion of macrophages in mice diminished the effects of obesity on TNBC growth and metastasis. Although anti-inflammatory ‘M2-like’ TAMs are key effector cells that promote tumorigenesis, it is well accepted that obesity stimulates macrophages to adopt a pro-inflammatory ‘M1-like’ state. However, M1 macrophages exhibit potent anti-tumor functions. From these discordant observations an important paradox emerges: How can obesity promote breast cancer if it elicits an M1, anti-tumor macrophage phenotype? Using a combination of proteomics, immunology, and cell biology, we recently identified a novel metabolically activated (MMe) macrophage phenotype produced by exposure to high levels of insulin, glucose, and palmitate, conditions characteristic of obese and diabetic patients. Here we show that gene expression patterns in MMe (but not M1) macrophages are strongly associated with pathways involved in breast cancer. Moreover, pre-treating BM1 cells with conditioned media derived from MMe macrophages promotes TNBC cell invasion by 2.3 fold, suggesting that MMe macrophages potentiate metastasis. We further show that breast adipose tissue macrophages isolated from obese women express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naive macrophages with media conditioned by human mammary adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. Thus, mammary adipose tissue from obese women supports metabolic activation of macrophages. Together, these observations suggest that obesity-induced changes to adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC. A comprehensive understanding of signaling mechanisms involved in metabolic activation would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Swati Kulkarni, Marsha Rosner, Lev Becker. Metabolically activated macrophages in obesity associated TNBC. [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 2888. doi:10.1158/1538-7445.AM2015-2888