Christine Dethlefsen

The role of intratumoral and systemic IL-6 in breast cancer.

Chronic low-grade inflammation plays an important role in the pathogenesis of several cancer forms including breast cancer. The pleiotropic cytokine IL-6 is a key player in systemic inflammation, regulating both the inflammatory response and tissue metabolism during acute stimulations. Here, we review the associations between IL-6 and breast cancer ranging from in vitro cell culture studies to clinical studies, covering the role of IL-6 in controlling breast cancer cell growth, regulation of cancer stem cell renewal, as well as breast cancer cell migration. Moreover, associations between circulating IL-6 and risk of breast cancer, prognosis for patients with prevalent disease, adverse effects and interventions to control systemic IL-6 levels in patients are discussed. In summary, direct application of IL-6 on breast cancer cells inhibits proliferation in estrogen receptor positive cells, while high circulating IL-6 levels are correlated with a poor prognosis in breast cancer patients. This discrepancy reflects distinct roles of IL-6, with elevated systemic levels being a biomarker for tumor burden, physical inactivity, and impaired metabolism, while local intratumoral IL-6 signaling is important for controlling breast cancer cell growth, metastasis, and self renewal of cancer stem cells.

Voluntary Running Suppresses Tumor Growth through Epinephrine- and IL-6-Dependent NK Cell Mobilization and Redistribution

Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed over 60% reduction in tumor incidence and growth across five different tumor models. Microarray analysis revealed training-induced upregulation of pathways associated with immune function. NK cell infiltration was significantly increased in tumors from running mice, whereas depletion of NK cells enhanced tumor growth and blunted the beneficial effects of exercise. Mechanistic analyses showed that NK cells were mobilized by epinephrine, and blockade of β-adrenergic signaling blunted training-dependent tumor inhibition. Moreover, epinephrine induced a selective mobilization of IL-6-sensitive NK cells, and IL-6-blocking antibodies blunted training-induced tumor suppression, intratumoral NK cell infiltration, and NK cell activation. Together, these results link exercise, epinephrine, and IL-6 to NK cell mobilization and redistribution, and ultimately to control of tumor growth.

Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth

Regular physical activity protects against the development of breast and colon cancer, since it reduces the risk of developing these by 25-30%. During exercise, humoral factors are released from the working muscles for endocrinal signaling to other organs. We hypothesized that these myokines mediate some of the inhibitory effects of exercise on mammary cancer cell proliferation. Serum and muscles were collected from mice after an exercise bout. Incubation with exercise-conditioned serum inhibited MCF-7 cell proliferation by 52% and increased caspase activity by 54%. A similar increase in caspase activity was found after incubation of MCF-7 cells with conditioned media from electrically stimulated myotubes. PCR array analysis (CAPM-0838E; SABiosciences) revealed that seven genes were upregulated in the muscles after exercise, and of these oncostatin M (OSM) proved to inhibit MCF-7 proliferation by 42%, increase caspase activity by 46%, and induce apoptosis. Blocking OSM signaling with anti-OSM antibodies reduced the induction of caspase activity by 51%. To verify that OSM was a myokine, we showed that it was significantly upregulated in serum and in three muscles, tibialis cranialis, gastronemius, and soleus, after an exercise bout. In contrast, OSM expression remained unchanged in subcutaneous and visceral adipose tissue, liver, and spleen (mononuclear cells). We conclude that postexercise serum inhibits mammary cancer cell proliferation and induces apoptosis of these cells. We suggest that one or more myokines secreted from working muscles may be mediating this effect and that OSM is a possible candidate. These findings emphasize that role of physical activity in cancer treatment, showing a direct link between exercise-induced humoral factors and decreased tumor cell growth.

Muscle specific miRNAs are induced by testosterone and independently upregulated by age

Age dependent decline in skeletal muscle function leads to impaired metabolic flexibility in elderly individuals. Physical activity and testosterone treatment have proven efficient strategies for delaying this condition. However, a common molecular pathway has not been identified. Muscle specific miRNAs (myomiRs) regulate metabolic pathways in skeletal muscle, are regulated by physical activity, and have response elements for testosterone in their promoter region. We therefore hypothesized that myomiRs would be regulated in skeletal muscle during aging. We further investigated any potential gender-dependent regulation of these miRNAs. We found that the myomiRs miR-1, miR-133a, and miR-133b were increased in skeletal muscle of elderly men compared to younger men. In addition, miR-133a/133b expression was markedly higher in women compared to men. Elimination of circulating testosterone in men was associated with lower levels of miR-133a and miR-133b. A positive regulatory effect of testosterone on miR-133a/133b expression was confirmed in castrated male C57BL/6J mice and in a model of primary human myocytes. Yet, an improvement of fitness level in the testosterone depleted men resulted in a down-regulation of miR133a/b. In conclusion, alterations in fitness level and circulating testosterone seem to represent two independent regulatory events where testosterone is a specific regulator of miR-133a/b expression.

Voluntary Exercise Prevents Cisplatin-Induced Muscle Wasting during Chemotherapy in Mice

Loss of muscle mass related to anti-cancer therapy is a major concern in cancer patients, being associated with important clinical endpoints including survival, treatment toxicity and patient-related outcomes. We investigated effects of voluntary exercise during cisplatin treatment on body weight, food intake as well as muscle mass, strength and signalling. Mice were treated weekly with 4 mg/kg cisplatin or saline for 6 weeks, and randomized to voluntary wheel running or not. Cisplatin treatment induced loss of body weight (29.8%, P < 0.001), lean body mass (20.6%, P = 0.001), as well as anorexia, impaired muscle strength (22.5% decrease, P < 0.001) and decreased glucose tolerance. In addition, cisplatin impaired Akt-signalling, induced genes related to protein degradation and inflammation, and reduced muscle glycogen content. Voluntary wheel running during treatment attenuated body weight loss by 50% (P < 0.001), maintained lean body mass (P < 0.001) and muscle strength (P < 0.001), reversed anorexia and impairments in Akt and protein degradation signalling. Cisplatin-induced muscular inflammation was not prevented by voluntary wheel running, nor was glucose tolerance improved. Exercise training may preserve muscle mass in cancer patients receiving cisplatin treatment, potentially improving physical capacity, quality of life and overall survival.

Exercise-Induced Catecholamines Activate the Hippo Tumor Suppressor Pathway to Reduce Risks of Breast Cancer Development

Strong epidemiologic evidence documents the protective effect of physical activity on breast cancer risk, recurrence, and mortality, but the underlying mechanisms remain to be identified. Using human exercise-conditioned serum for breast cancer cell incubation studies and murine exercise interventions, we aimed to identify exercise factors and signaling pathways involved in the exercise-dependent suppression of breast cancer. Exercise-conditioned serum from both women with breast cancer (n = 20) and healthy women (n = 7) decreased MCF-7 (hormone-sensitive) and MDA-MB-231 (hormone-insensitive) breast cancer cell viability in vitro by 11% to 19% and reduced tumorigenesis by 50% when preincubated MCF-7 breast cancer cells were inoculated into NMRI-Foxn1nu mice. This exercise-mediated suppression of cell viability and tumor formation was completely blunted by blockade of β-adrenergic signaling in MCF-7 cells, indicating that catecholamines were the responsible exercise factors. Both epinephrine (EPI) and norepinephrine (NE) could directly inhibit breast cancer cell viability, as well as tumor growth in vivo EPI and NE activate the tumor suppressor Hippo signaling pathway, and the suppressive effect of exercise-conditioned serum was found to be mediated through phosphorylation and cytoplasmic retention of YAP and reduced expression of downstream target genes, for example, ANKRD1 and CTGF. In parallel, tumor-bearing mice with access to running wheels showed reduced growth of MCF-7 (-36%, P < 0.05) and MDA-MB-231 (-66%, P < 0.01) tumors and, for the MCF-7 tumor, increased regulation of the Hippo signaling pathway. Taken together, our findings offer a mechanistic explanation for exercise-dependent suppression of breast cancer cell growth. Cancer Res; 77(18); 4894-904. ©2017 AACR.

Exercise suppresses tumor growth through epinephrine- and IL-6-dependent mobilization and redistribution of NK cells

Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed significant exercise related reduction in tumor incidence and growth across several tumor models including transplantable tumors (Lewis lung and B16 melanoma), chemically (diethylnitrosamine (DEN) induced liver cancer, and a model of spontaneous melanoma (Tg(Grm1)EPv transgenic mice). Microarray analysis revealed exercise-induced up-regulation of pathways associated with immune function, prompting further investigations. NK cell infiltration was significantly increased in tumors from exercising mice, and depletion of NK cells by anti-asialo-GM1 administration increased tumor growth and blunted the exercise-dependent tumor suppression. Mechanistic analyses showed that NK cells were engaged through an epinephrine-dependent mobilization, and blockade of this response by s-adrenergic blockade blunted the exercise-dependent tumor inhibition. Moreover, exercise-induced IL-6 facilitated redistribution of NK cells to peripheral tissues and induced a shift towards more cytotoxic (CD11b-, CD27+) NK cells at the tumor site. Together these results link exercise, epinephrine and IL-6 to NK cell mobilization and activation, and ultimately to improved control of tumor growth.

Abstract 5400: Exercise activates AMP-activated protein kinase in breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Objective: Cancer cells are characterized by a changed cellular metabolism, favoring glycolysis. AMPK, a serine/threonine kinase regulates cellular metabolism, and induces fatty acid oxidation and has in regard to this, been shown to suppress tumor cell growth. During physical activity, which is associated with a reduced cancer risk, working muscles secrete myokines, known to activate AMPK. This could indicate a connection between AMPK activity, myokine release from the working muscle, and an anti-cancer response. We hypothesize that exercise-induced myokine secretion causes an anti-cancer response via AMPK activation. Methods: Exercise-induced AMPK activation was studied in two models, 1) A cell study using the two breast cancer cell lines, MDA-MB-231 and MCF-7, incubated with exercise-conditioned serum, representing a myokine pool. Serum was collected from 9 females during a 2 hours cycling bout, and during a subsequent 3 hours rest. Following incubation, cell viability and AMPK phosphorylation was determined in the cells. 2) An In Vivo model, where the two cell lines were injected into nude mice. When the tumors reached 6 mm in diameter, the mice were subjected to 1 hour forced swimming bout, after which the tumors were dissected and analyzed for pAMPK content. Results: 1) Cell study: Two hours of cycling induced a 6-fold increase in serum IL-6, as expected. Incubating cancer cells with this post-exercise serum resulted in a significant reduction in the viability of MCF-7 cells, while no effect on the viability of MDA-MB-231 cells were observed. Preliminary data show increased AMPK phosphorylation in both MDA-MB-231 and MCF-7 cells after incubation with 5% exercise-conditioned serum. 2) In vivo study: 1 hour of swimming resulted in significantly increased AMPK phosphorylation in the MCF-7 tumors. Conclusion: Our preliminary data indicate that exercise-induced factors can cause decreased viability and increased AMPK activity in MCF-7 cells, while MDA-MB-231 cells need further investigation. Acknowledgement: The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National research Foundation (#02-512-55). This study was further supported by the Danish Medical Research Council and the Lundbeck Foundation. Citation Format: Grith W. Hojfeldt, Christine Dethlefsen, Bente K. Pedersen, Pernille Hojman. Exercise activates AMP-activated protein kinase in breast cancer cells. [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 5400. doi:10.1158/1538-7445.AM2013-5400