M.J. Szpunar

β-Adrenergic receptors (β-AR) regulate VEGF and IL-6 production by divergent pathways in high β-AR-expressing breast cancer cell lines

Activation of β-adrenergic receptors (β-AR) drives proangiogenic factor production in several types of cancers. To examine β-AR regulation of breast cancer pathogenesis, β-AR density, signaling capacity, and functional responses to β-AR stimulation were studied in four human breast adenocarcinoma cell lines. β-AR density ranged from very low in MCF7 and MB-361 to very high in MB-231 and in a brain-seeking variant of MB-231, MB-231BR. Consistent with β-AR density, β-AR activation elevated cAMP in MCF7 and MB-361 much less than in MB-231 and MB-231BR. Functionally, β-AR stimulation did not markedly alter vascular endothelial growth factor (VEGF) production by MCF7 or MB-361. In the two high β-AR-expressing cell lines MB-231 and MB-231BR, β-AR-induced cAMP and VEGF production differed considerably, despite similar β-AR density. The β2-AR-selective agonist terbutaline and the endogenous neurotransmitter norepinephrine decreased VEGF production by MB-231, but increased VEGF production by MB-231BR. Moreover, β2-AR activation increased IL-6 production by both MB-231 and MB-231BR. These functional alterations were driven by elevated cAMP, as direct activation of adenylate cyclase by forskolin elicited similar alterations in VEGF and IL-6 production. The protein kinase A antagonist KT5720 prevented β-AR-induced alterations in MB-231 and MB-231BR VEGF production, but not IL-6 production. Conclusions β-AR expression and signaling is heterogeneous in human breast cancer cell lines. In cells with high β-AR density, β-AR stimulation regulates VEGF production through the classical β-AR-cAMP-PKA pathway, but this pathway can elicit directionally opposite outcomes. Furthermore, in the same cells, β-AR activate a cAMP-dependent, PKA-independent pathway to increase IL-6 production. The complexity of breast cancer cell β-AR expression and functional responses must be taken into account when considering β-AR as a therapeutic target for breast cancer treatment.

The antidepressant desipramine and α2-adrenergic receptor activation promote breast tumor progression in association with altered collagen structure.

Emotional stress activates the sympathetic nervous system (SNS) and release of the neurotransmitter norepinephrine to promote breast tumor pathogenesis. We demonstrate here that the metastatic mammary adenocarcinoma cell line 4T1 does not express functional adrenergic receptors (AR), the receptors activated by norepinephrine, yet stimulation of adrenergic receptor in vivo altered 4T1 tumor progression in vivo. Chronic treatment with the antidepressant desipramine (DMI) to inhibit norepinephrine reuptake increased 4T1 tumor growth but not metastasis. Treatment with a highly selective α2-adrenergic receptor agonist, dexmedetomidine (DEX), increased tumor growth and metastasis. Neither isoproterenol (ISO), a β-AR agonist, nor phenylephrine, an α1-AR agonist, altered tumor growth or metastasis. Neither DMI- nor DEX-induced tumor growth was associated with increased angiogenesis. In DMI-treated mice, tumor VEGF, IL-6, and the prometastatic chemokines RANTES, M-CSF, and MIP-2 were reduced. Tumor collagen microstructure was examined using second harmonic generation (SHG), a nonabsorptive optical scattering process to highlight fibrillar collagen. In DMI- and DEX-treated mice, but not ISO-treated mice, tumor SHG was significantly altered without changing fibrillar collagen content, as detected by immunofluorescence. These results demonstrate that α2-AR activation can promote tumor progression in the absence of direct sympathetic input to breast tumor cells. The results also suggest that SNS activation may regulate tumor progression through alterations in the extracellular matrix, with outcome dependent on the combination of adrenergic receptor activated. These results underscore the complexities underlying SNS regulation of breast tumor pathogenesis, and suggest that the therapeutic use of adrenergic receptor blockers, tricyclic antidepressants, and adrenergic receptor agonists must be approached cautiously in patients with breast cancer. Cancer Prev Res; 6(12); 1262–72. ©2013 AACR.

Early impact of social isolation and breast tumor progression in mice

Evidence from cancer patients and animal models of cancer indicates that exposure to psychosocial stress can promote tumor growth and metastasis, but the pathways underlying stress-induced cancer pathogenesis are not fully understood. Social isolation has been shown to promote tumor progression. We examined the impact of social isolation on breast cancer pathogenesis in adult female severe combined immunodeficiency (SCID) mice using the human breast cancer cell line, MDA-MB-231, a high β-adrenergic receptor (AR) expressing line. When group-adapted mice were transferred into single housing (social isolation) one week prior to MB-231 tumor cell injection into a mammary fat pad (orthotopic), no alterations in tumor growth or metastasis were detected compared to group-housed mice. When social isolation was delayed until tumors were palpable, tumor growth was transiently increased in singly-housed mice. To determine if sympathetic nervous system activation was associated with increased tumor growth, spleen and tumor norepinephrine (NE) was measured after social isolation, in conjunction with tumor-promoting macrophage populations. Three days after transfer to single housing, spleen weight was transiently increased in tumor-bearing and non-tumor-bearing mice in conjunction with reduced splenic NE concentration and elevated CD11b+Gr-1+ macrophages. At day 10 after social isolation, no changes in spleen CD11b+ populations or NE were detected in singly-housed mice. In the tumors, social isolation increased CD11b+Gr-1+, CD11b+Gr-1-, and F4/80+ macrophage populations, with no change in tumor NE. The results indicate that a psychological stressor, social isolation, elicits dynamic but transient effects on macrophage populations that may facilitate tumor growth. The transiency of the changes in peripheral NE suggest that homeostatic mechanisms may mitigate the impact of social isolation over time. Studies are underway to define the neuroendocrine mechanisms underlying the tumor-promoting effects of social isolation, and to determine the contributions of increased tumor macrophages to tumor pathogenesis.

Sympathetic innervation, norepinephrine content, and norepinephrine turnover in orthotopic and spontaneous models of breast cancer.

Activation of the sympathetic nervous system (SNS) drives breast cancer progression in preclinical breast cancer models, but it has yet to be established if neoplastic and stromal cells residing in the tumor are directly targeted by locally released norepinephrine (NE). In murine orthotopic and spontaneous mammary tumors, tyrosine hydroxylase (TH)+ sympathetic nerves were limited to the periphery of the tumor. No TH+ staining was detected deeper within these tumors, even in regions with a high density of blood vessels. NE concentration was much lower in tumors compared to the more densely innervated spleen, reflecting the relative paucity of tumor TH+ innervation. Tumor and spleen NE concentration decreased with increased tissue mass. In mice treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to selectively destroy sympathetic nerves, tumor NE concentration was reduced approximately 50%, suggesting that the majority of tumor NE is derived from local sympathetic nerves. To evaluate NE utilization, NE turnover in orthotopic 4T1 mammary tumors was compared to spleen under baseline and stress conditions. In non-stressed mice, NE turnover was equivalent between tumor and spleen. In mice exposed to a stressor, tumor NE turnover was increased compared to spleen NE turnover, and compared to non-stressed tumor NE turnover. Together, these results demonstrate that NE in mammary tumors is derived from local sympathetic nerves that synthesize and metabolize NE. However, differences between spleen and tumor NE turnover with stressor exposure suggest that sympathetic NE release is regulated differently within the tumor microenvironment compared to the spleen. Local mammary tumor sympathetic innervation, despite its limited distribution, is responsive to stressor exposure and therefore can contribute to stress-induced tumor progression.

151. Evidence for sympathetic nervous system modulation of mammary tumor pathogenesis via tumor collagen

Our laboratory uses multiphoton laser scanning microscopy to study tumor collagen structure using an optical technique known as second harmonic generation (SHG). SHG is an endogenous optical signal produced when two excitation photons combine to produce one emission photon, “catalyzed” by a non-centrosymmetric structure such as ordered collagen triple helices. Manipulating collagen structure, as indicated by changes in SHG, also alters tumor growth and metastasis. We also study the role of sympathetic nervous system (SNS) activation and the neurotransmitter norepinephrine (NE) in breast cancer. To mimic SNS activation, mice were implanted with slow-release pellets containing desipramine (DMI), a NE uptake blocker. DMI elevated NE and its metabolite normetanephrine in spleen and in 4T1 tumors implanted orthotopically (in the mammary fat pad). Furthermore, DMI increased 4T1 mammary tumor growth, compared to placebo pellet controls. Tumor sections from DMI-implanted mice exhibited elevated SHG intensity. In these same images, fluorescent intensity generated by immunofluorescent staining with anti-collagen type 1 antibody was not altered by DMI treatment, indicating no alterations in total collagen (both SHG generating and non-SHG generating collagen). These results demonstrate that NE-induced tumor growth is associated with altered tumor collagen microstructure, and suggest that SNS activation may modulate tumor pathogenesis through a novel pathway, i.e., modulation of tumor extracellular matrix. The mechanisms linking SNS activation to collagen structure and tumor pathogenesis are currently under investigation.

Beta-adrenergic receptors, cAMP and proangiogenic factor production by breast cancer cell lines

381 BDNF expression in response to academic stress in asthmatic and healthy subjects C. Kemi , M. Lekander , J. Grunewald , J. Axén , S. Jernelöv , C. Müller-Suur , Y. Smith , R. Grönneberg , A. Eklund , P. Stierna , C. Olgart Höglund a,e,f,g a Dept. of Medicine Solna, Karolinska Institutet, Stockholm, Sweden b Dept. of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden c Stress Research Institute, Stockholm University, Stockholm, Sweden d Dept. of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden e Dept. of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden f Osher Center for Integrative Medicine, Karolinska Institutet, Stockholm, Sweden g Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden Stress may modulate immune responses and has been shown to regulate allergic inflammation in an unfavourable way. The neurotrophin brain-derived neurotrophic factor (BDNF) was originally discovered as a neuronal growth factor, but is today also recognized as a critical mediator of allergic inflammation and asthma. Recent studies have suggested a role for BDNF as a survival factor for eosinophils and inducer of bronchial hyperreactivity in asthma. Stress has been shown to regulate neurotrophins in brain and blood. However, if stress can regulate BDNF release from immune cells and if this regulation differs between asthmatic and healthy subjects is unknown. The aim of the study was to determine the regulation of BDNF secretion from white blood cells in asthmatic and healthy subjects, in parallel to studies of inflammatory parameters and bronchial hyperreactivity, in response to stress. Thirtyfive medical students were examined during a low-stress and stressful (academic exams) study period. ELISA analysis of white blood cells revealed generally higher secreted levels of BDNF in asthmatic compared to healthy subjects. At the stress period, BDNF secretion increased in healthy subjects only and correlated positively to IL-5 and CD4+ T-cells in asthmatics. A possible role for BDNF in the pathogenesis of asthma and stress-mediated neuroimmune regulation is therefore suggested. doi:10.1016/j.bbi.2010.07.135

Evidence for sympathetic nervous system regulation of breast cancer pathogenesis

373 The impact of acute hypoxia on learning and memory G. Chiu , D. Chatterjee , R.W. Johnson , G.G. Freund a,b a University of Illinois at Urbana-Champaign, Division of Nutritional Sciences, Urbana, IL 61801, United States b University of Illinois at Urbana-Champaign, College of Medicine, Department of Pathology, Urbana, IL 61801, United States c University of Illinois at Urbana-Champaign, Department of Animal Sciences, Urbana, IL 61801, United States Acute hypoxia is known to have many negative side effects, including but not limited to lethargy, anorexia, and anhedonia. In order to further investigate the biobehaviors impacted by acute hypoxia, we tested its effects on learning and memory. In this study, we exposed adult mice (male C57BL/6J, 8–12 weeks old) to 5% oxygen/95% nitrogen for 2 h and tested their memory by using a novel object recognition test. We separated memory into two components: recollection (the ability to remember past information), and formation (the ability to generate new memories). First, we investigated hypoxia’s impact on memory recollection by testing the animals on objects introduced prior to hypoxia at 0–6 h post exposure. Then, we explored memory formation by exposing mice to hypoxia first, then introducing them to the test 3–8 h post hypoxia. We found that hypoxia significantly impaired memory recollection for up to 3 h post-exposure, and significantly decreased new memory formation for up to 5 h post-hypoxia exposure. These results show that acute hypoxia impairs memory recollection and formation and suggests that more than one biological mechanism is involved. doi:10.1016/j.bbi.2010.07.131

82. Beta-adrenergic receptor (beta-AR) signaling differs between the human breast cancer cell line MB-231 and its brain-metastasizing variant MB-231BR

male C57BL/6J mice were randomized to one of four groups: (1) MA-treated + 24 h withdrawal (w/d), (2) saline (SAL)-treated + 24 h w/d, (3) MA-treated + 3wk w/d, and (4) SAL-treated + 3wk w/d. MA (1 mg/kg) or SAL was administered subcutaneously to mice for 7 consecutive days. Following cognitive testing using the novel object recognition task (NORT), blood and brain samples were collected for multiplex determination of cytokine and intracellular adhesion molecule (ICAM-1) expression levels. A number of significant MAinduced changes in peripheral and brain region-specific cytokines were observed. Of particular interest were the pro-inflammatory cytokines interleukin (IL)-2, IL-6 and IL-1beta and the cellular adhesion molecule ICAM-1, which were significantly increased in brain regions (i.e., hippocampus and neostriatum) of MA-treated mice, as compared to controls (p < 0.05). Since ICAM-1 is induced by proinflammatory cytokines and recruits activated leukocytes to tissue injury, MA-induced increases in ICAM-1 could facilitate the adhesion of leukocytes that produce toxic mediators thereby perpetuating the inflammatory response and potentially contributing to cognitive impairment.

120. Heterogeneity of β-adrenergic receptor (β-AR) signal transduction and vascular endothelial growth factor (VEGF) production by breast cancer cell lines

alters many aspects of acute stressor exposure, we hypothesized that acute stressor exposure would alter inflammatory protein content in WAT and prior voluntary exercise would modify this response. The effect of acute tail shock stress on WAT of male F344 sedentary rats was compared to that of rats allowed access to a running wheel for 6 weeks prior to stressor exposure (n = 6–7/group). Following 50 unpredictable tail shocks, rats were sacrificed and protein levels were examined in blood plasma, subcutaneous (SQ), visceral epididymal (VE), and visceral perirenal (VP) WAT depots using multiplex ELISA. Following stressor exposure, IL-1Beta was increased in the circulation and WAT (SQ, VP and VE) of the sedentary rats. Exercise reduced basal leptin in all WAT depots (SQ, VP and VE), increased basal levels of IL-1Beta the visceral depots, and increased basal TNFalpha in the VE depot. Exercise attenuated the stress induced increase in IL-1Beta in the circulation, VP and VE but not the SQ WAT. These data suggest that WAT may contribute to circulating cytokines following stressor exposure and indicate that exercise alters WAT responses to stress in a depot dependent fashion. Supported by RO1AIO57797.