Erin M. Dauchy

Circadian Gating of Epithelial-to-Mesenchymal Transition in Breast Cancer Cells Via Melatonin-Regulation of GSK3β

Disturbed sleep-wake cycle and circadian rhythmicity are associated with cancer, but the underlying mechanisms are unknown. Employing a tissue-isolated human breast xenograft tumor nude rat model, we observed that glycogen synthase kinase 3β (GSK3β), an enzyme critical in metabolism and cell proliferation/survival, exhibits a circadian rhythm of phosphorylation in human breast tumors. Exposure to light-at-night suppresses the nocturnal pineal melatonin synthesis, disrupting the circadian rhythm of GSK3β phosphorylation. Melatonin activates GSK3β by inhibiting the serine-threonine kinase Akt phosphorylation, inducing β-catenin degradation and inhibiting epithelial-to-mesenchymal transition, a fundamental process underlying cancer metastasis. Thus, chronic circadian disruption by light-at-night via occupational exposure or age-related sleep disturbances may contribute to cancer incidence and the metastatic spread of breast cancer by inhibiting GSK3β activity and driving epithelial-to-mesenchymal transition in breast cancer patients.

Circadian regulation of molecular, dietary, and metabolic signaling mechanisms of human breast cancer growth by the nocturnal melatonin signal and the consequences of its disruption by light at night

Abstract:  This review article discusses recent work on the melatonin‐mediated circadian regulation and integration of molecular, dietary, and metabolic signaling mechanisms involved in human breast cancer growth and the consequences of circadian disruption by exposure to light at night (LAN). The antiproliferative effects of the circadian melatonin signal are mediated through a major mechanism involving the activation of MT1 melatonin receptors expressed in human breast cancer cell lines and xenografts. In estrogen receptor (ERα+) human breast cancer cells, melatonin suppresses both ERα mRNA expression and estrogen‐induced transcriptional activity of the ERα via MT1‐induced activation of Gαi2 signaling and reduction of 3′,5′‐cyclic adenosine monophosphate (cAMP) levels. Melatonin also regulates the transactivation of additional members of the steroid hormone/nuclear receptor super‐family, enzymes involved in estrogen metabolism, expression/activation of telomerase, and the expression of core clock and clock‐related genes. The anti‐invasive/anti‐metastatic actions of melatonin involve the blockade of p38 phosphorylation and the expression of matrix metalloproteinases. Melatonin also inhibits the growth of human breast cancer xenografts via another critical pathway involving MT1‐mediated suppression of cAMP leading to blockade of linoleic acid uptake and its metabolism to the mitogenic signaling molecule 13‐hydroxyoctadecadienoic acid (13‐HODE). Down‐regulation of 13‐HODE reduces the activation of growth factor pathways supporting cell proliferation and survival. Experimental evidence in rats and humans indicating that LAN‐induced circadian disruption of the nocturnal melatonin signal activates human breast cancer growth, metabolism, and signaling provides the strongest mechanistic support, thus far, for population and ecological studies demonstrating elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LAN.

Melatonin and Associated Signaling Pathways that Control Normal Breast Epithelium and Breast Cancer

This review article discusses recent work on the melatonin-mediated circadian regulation and integration of molecular and metabolic signaling mechanisms involved in human breast cancer growth and the associated consequences of circadian disruption by exposure to light-at-night (LAN). The anti-proliferative effects of the circadian melatonin signal are, in general, mediated through mechanisms involving the activation of MT1 melatonin receptors expressed in human breast cancer cell lines and xenografts. In estrogen receptor-positive (ERα+) human breast cancer cells, melatonin suppresses both ERα mRNA expression and estrogen-induced transcriptional activity of the ERα via MT1-induced activation of Gαi2 signaling and reduction of cAMP levels. Melatonin also regulates the transcriptional activity of additional members of the nuclear receptor super-family, enzymes involved in estrogen metabolism, and the expression of core clock and clock-related genes. The anti-invasive/anti-metastatic actions of melatonin involve the blockade of p38 phosphorylation and matrix metalloproteinase expression. Melatonin also inhibits the growth of human breast cancer xenografts via MT1-mediated suppression of cAMP leading to a blockade of linoleic acid (LA) uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Down-regulation of 13-HODE reduces the activation of growth factor pathways supporting cell proliferation and survival. Finally, studies in both rats and humans indicate that light-at-night (LAN) induced circadian disruption of the nocturnal melatonin signal activates human breast cancer growth, metabolism, and signaling, providing the strongest mechanistic support, thus far, for epidemiological studies demonstrating the elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LAN.

Light at Night Activates IGF-1R/PDK1 Signaling and Accelerates Tumor Growth in Human Breast Cancer Xenografts

Regulation of diurnal and circadian rhythms and cell proliferation are coupled in all mammals, including humans. However, the molecular mechanisms by which diurnal and circadian rhythms regulate cell proliferation are relatively poorly understood. In this study, we report that tumor growth in nude rats bearing human steroid receptor-negative MCF-7 breast tumors can be significantly accelerated by exposing the rats to light at night (LAN). Under normal conditions of an alternating light/dark cycle, proliferating cell nuclear antigen (PCNA) levels in tumors were maximal in the early light phase but remained at very low levels throughout the daily 24-hour cycle period monitored. Surprisingly, PCNA was expressed in tumors continually at a high level throughout the entire 24-hour period in LAN-exposed nude rats. Daily fluctuations of Akt and mitogen activated protein kinase activation in tumors were also disrupted by LAN. These fluctuations did not track with PCNA changes, but we found that activation of the Akt stimulatory kinase phosphoinositide-dependent protein kinase 1 (PDK1) directly correlated with PCNA levels. Expression of insulin-like growth factor 1 receptor (IGF-1R), an upstream signaling molecule for PDK1, also correlated with fluctuations of PDK1/PCNA in the LAN group. In addition, circulating IGF-1 concentrations were elevated in LAN-exposed tumor-bearing nude rats. Finally, RNAi-mediated knockdown of PDK1 led to a reduction in PCNA expression and cell proliferation in vitro and tumor growth in vivo, indicating that PDK1 regulates breast cancer growth in a manner correlated with PCNA expression. Taken together, our findings demonstrate that LAN exposure can accelerate tumor growth in vivo, in part through continuous activation of IGF-1R/PDK1 signaling.

Light Exposure at Night Disrupts Host/Cancer Circadian Regulatory Dynamics: Impact on the Warburg Effect, Lipid Signaling and Tumor Growth Prevention

The central circadian clock within the suprachiasmatic nucleus (SCN) plays an important role in temporally organizing and coordinating many of the processes governing cancer cell proliferation and tumor growth in synchrony with the daily light/dark cycle which may contribute to endogenous cancer prevention. Bioenergetic substrates and molecular intermediates required for building tumor biomass each day are derived from both aerobic glycolysis (Warburg effect) and lipid metabolism. Using tissue-isolated human breast cancer xenografts grown in nude rats, we determined that circulating systemic factors in the host and the Warburg effect, linoleic acid uptake/metabolism and growth signaling activities in the tumor are dynamically regulated, coordinated and integrated within circadian time structure over a 24-hour light/dark cycle by SCN-driven nocturnal pineal production of the anticancer hormone melatonin. Dim light at night (LAN)-induced melatonin suppression disrupts this circadian-regulated host/cancer balance among several important cancer preventative signaling mechanisms, leading to hyperglycemia and hyperinsulinemia in the host and runaway aerobic glycolysis, lipid signaling and proliferative activity in the tumor.

Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma

Leiomyosarcoma (LMS) represents a highly malignant, rare soft tissue sarcoma with high rates of morbidity and mortality. Previously, we demonstrated that tissue‐isolated human LMS xenografts perfused in situ are highly sensitive to the direct anticancer effects of physiological nocturnal blood levels of melatonin which inhibited tumour cell proliferative activity, linoleic acid (LA) uptake and metabolism to 13‐hydroxyoctadecadienoic acid (13‐HODE). Here, we show the effects of low pharmacological blood concentrations of melatonin following oral ingestion of a melatonin supplement by healthy adult human female subjects on tumour proliferative activity, aerobic glycolysis (Warburg effect) and LA metabolic signalling in tissue‐isolated LMS xenografts perfused in situ with this blood. Melatonin markedly suppressed aerobic glycolysis and induced a complete inhibition of tumour LA uptake, 13‐HODE release, as well as significant reductions in tumour cAMP levels, DNA content and [3H]‐thymidine incorporation into DNA. Furthermore, melatonin completely suppressed the phospho‐activation of ERK 1/2, AKT, GSK3β and NF‐kB (p65). The addition of S20928, a nonselective melatonin antagonist, reversed these melatonin inhibitory effects. Moreover, in in vitro cell culture studies, physiological concentrations of melatonin repressed cell proliferation and cell invasion. These results demonstrate that nocturnal melatonin directly inhibited tumour growth and invasion of human LMS via suppression of the Warburg effect, LA uptake and other related signalling mechanisms. An understanding of these novel signalling pathway(s) and their association with aerobic glycolysis and LA metabolism in human LMS may lead to new circadian‐based therapies for the prevention and treatment of LMS and potentially other mesenchymally derived solid tumours.

Insulin and IGF1 enhance IL-17-induced chemokine expression through a GSK3B-dependent mechanism: a new target for melatonin's anti-inflammatory action.

Obesity is a chronic inflammation with increased serum levels of insulin, insulin‐like growth factor 1 (IGF1), and interleukin‐17 (IL‐17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL‐17‐induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)‐dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL‐17‐induced expression of C‐X‐C motif ligand 1 (Cxcl1) and C‐C motif ligand 20 (Ccl20) in the Gsk3b+/+, but not in Gsk3b−/− mouse embryonic fibroblast (MEF) cells. IL‐17 induced higher levels of Cxcl1 and Ccl20 in the Gsk3b−/− MEF cells, compared with the Gsk3b+/+ MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P‐GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL‐17 alone or by a combination of insulin and IL‐17. Melatonins inhibitory effects were only observed in the Gsk3b+/+, but in not Gsk3b−/− MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il‐6 that was induced by a combination of insulin and IL‐17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il‐6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL‐17‐mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.

Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor-mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue-isolated human leiomyosarcoma xenografts.

Abstract:  Melatonin provides a circadian signal that regulates linoleic acid (LA)‐dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth‐stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13‐hydroxyoctadecadienoic acid (13‐HODE). This study tested the hypothesis that both acute and long‐term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue‐isolated human leiomyosarcoma (LMS), a rare, mesenchymally‐derived smooth muscle tissue sarcoma, via melatonin receptor‐mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue‐isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 ± 0.02 g/day) versus control xenografts that continued to grow at 0.22 ± 0.03 g/day over a 10‐day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose‐dependent suppression of tumor cAMP production, LA uptake, 13‐HODE release, extracellular signal‐regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [3H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8‐Br‐cAMP. These results demonstrate that, as observed in epithelially‐derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long‐term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor‐mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.

Dietary Fish Oil Deactivates a Growth-Promoting Signaling Pathway in Hepatoma 7288CTC in Buffalo Rats

Abstract: Dietary fish oil decreases growth of solid tumors in rodents. Mechanisms for this effect are not well defined. In rat hepatoma 7288CTC, short-term (1–2 h) treatment with eicosapentaenoic acid during perfusion in situ reduced fatty acid uptake and [3H]thymidine incorporation. To determine if dietary fish oil had this effect in vivo, 48 male Buffalo rats were implanted with tissue-isolated hepatoma 7288CTC and were divided into three groups: Diet I (8% olive oil/2% corn oil), Diet II (6% olive oil/2% corn oil/2% fish oil), or Diet III (3% olive oil/3% corn oil/4% fish oil).When tumors weighed 4 to 6 g rats were anesthetized and tumor fatty acid uptake and 13-hydroxyoctadecadienoic acid release were measured in vivo by arterial minus venous differences. Tumors were analyzed for cyclic adenosine monophosphate (cAMP), DNA content, and [3H]thymidine incorporation. Fish oil feeding significantly (P < 0.05) reduced tumor growth, cAMP content, fatty acid uptake, 13-hydroxyoctadecadienoic acid formation, DNA content, and [3H]thymidine incorporation. Addition of either pertussis toxin or 8-bromoadenosine-cAMP to the arterial blood reversed the inhibitions in tumors in rats fed diet II. These results provide in vivo evidence that dietary fish oil suppressed a specific linoleic acid-dependent, inhibitory G protein-coupled, growth-promoting signaling pathway in rat hepatoma 7288CTC.

Abstract 1324: Melatonin-depleted blood from healthy adult men exposed to environmental light at night stimulates growth, signal transduction and metabolic activity of tissue-isolated human prostate cancer xenografts in nude rats

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Light at night (LAN), via its ability to suppress nocturnal circadian pineal melatonin production, has been associated with an increased risk of prostate, breast, and endometrial cancers reported in rotating night shift workers. In previous studies, we determined that melatonins tumor growth inhibitory mechanism occurs via an MT1 melatonin receptor-mediated suppression of tumor cAMP leading to an inhibition of tumor linoleic acid (LA) uptake and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). These events culminate in down-regulation of the epidermal growth factor and insulin-like growth factor-1 pathways. Tissue-isolated, androgen-independent PC3 human prostate tumor xenografts (n=3/group; 57 perfusions) perfused for 60 minutes in situ in male nude rats with melatonin-rich blood (> 100 pg/ml) collected from healthy adult, male subjects (n = 3) during the night were compared to those perfused with melatonin-deficient blood (< 10 pg/ml) collected during the daytime or nighttime following 90 minutes of ocular exposure to bright, white fluorescent light (2800 lux). Perfusion of tissue-isolated PC3 human prostate xenografts with human donor nighttime-collected, melatonin-rich blood samples resulted in substantial reductions (60 – 99%) in tumor cAMP levels, LA uptake, 13-HODE production, glucose uptake, O2 consumption and CO2 production, and [3H]thymidine incorporation into tumor DNA, compared to tumors perfused with daytime-collected or nighttime/light exposure-collected, melatonin-diminished blood samples. The activation of MEK, ERK 1/2, Akt, and glycogen synthase kinase-3β (GSK3β), was also markedly diminished in tumors perfused with melatonin-rich, nighttime-collected blood and stimulated in tumors perfused with melatonin-deficient blood collected after exposure to light at night. Tumor inhibitions by melatonin-rich blood were completely prevented by a non-selective MT1/MT2 melatonin receptor antagonist, forskolin, pertussis toxin, or 8-Bromo-cAMP. These results are the first to demonstrate that the nocturnal melatonin signal in blood collected from male human volunteers during the night suppresses signal transduction, metabolic and growth activity in tissue-isolated PC3 human prostate cancer xenografts via an MT1 melatonin receptor-mediated mechanism. These findings are also the first to show that blood collected from human subjects exposed to light at night markedly stimulates human prostate cancer growth, signal transduction and metabolic activity via suppression of the nocturnal circadian melatonin signal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2011-1324