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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. Cancer Res; 71(7); 2622–31. ©2011 AACR.

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

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [3H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.

Light and lighting protocols of animal research facilities are critically important to the outcomes of biomedical research that uses animals. Previous studies from our laboratory showed that the wavelength (color) of light in animal housing areas affects the nocturnal melatonin signal that temporally coordinates circadian rhythms in rodents. Here, we tested the hypothesis that exposure to LED light enriched in the blue-appearing portion (460-480 nm) of the visible spectrum during the light phase (bLAD) influences circadian concentrations of select neuroendocrine hormones in adolescent Sprague–Dawley rats. Male and female rats (4 to 5 wk old) were housed on a novel IVC system under a 12L:12D in either cool-white fluorescent (control, n = 72) or bLAD (experimental, n = 72) lighting. Every third day, body weight and food and water consumption were measured. On Day 30, rats were anesthetized with ketamine/xylazine and terminal collection of arterial blood was performed to quantify serum concentrations of melatonin, corticosterone, insulin, and glucose at 6 circadian time points (0400, 0800, 1200, 1600, 2000, 2400). As compared with male and female rats housed under cool white fluorescent (CWF) lighting, rats in bLAD lighting showed a 6-fold higher peak in dark phase serum melatonin (P < 0.05). Effects on serum corticosterone were sex dependent, as CWF and bLAD females had significantly higher corticosterone levels than did CWF and bLAD males, respectively. CWF and bLAD females had significantly higher serum glucose overall as compared with males. However, serum insulin was not affected by sex (M or F) or lighting conditions (CWF or bLAD). These data show that housing Sprague–Dawley rats under bLAD lighting conditions increases circadian peaks of melatonin without increasing serum levels of corticosterone, glucose or insulin, indicating less variation of circadian cycling of key neuroendocrine hormones in bLAD-exposed rats.

The retinoic acid-related orphan receptors alpha (RORa) are members of the steroid/thyroid nuclear receptor super-family and core components of the circadian timing system. In the present study, we continue to investigate the role of RORas in human breast cancer. Assays using the RORa response element (RORE)-tk-luciferase reporter demonstrate the functionality of the RORa1 in MCF-7 breast cancer cells and that over-expression of RORa1 stimulates MCF-7 human breast cancer cell proliferation. Genomic analysis revealed that RORα1 over-expression regulated the transcription of numerous genes in MCF-7 breast cancer cells including increasing the expression of connexin 43 (CX43), aldo-keto reductases 1C1 (AKR1C1), and AKR1C3. Furthermore, administration of the pineal hormone melatonin represses RORa1 induction of CX43, AKR1C1, and AKR1C3 in MCF-7 cells. AKR1C3 has been reported to impact in intra-tumoral production of androgens and estrogens and thus, might promote Tamoxifen resistance in breast cancer. Over-expression of RORa1 and subsequently AKR1C3 does promote Tamoxifen resistance, which can be inhibited by melatonin administration.

Light is a potent biologic force that profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in animals. Previously we examined the effects of light-phase exposure of rats to white light-emitting diodes (LED), which emit more light in the blue-appearing portion of the visible spectrum (465 to 485 nm) than do broad-spectrum cool white fluorescent (CWF) light, on the nighttime melatonin amplitude and circadian regulation of metabolism and physiology. In the current studies, we tested the hypothesis that exposure to blue-enriched LED light at day (bLAD), compared with CWF, promotes the circadian regulation of neuroendocrine, metabolic, and physiologic parameters that are associated with optimizing homeostatic regulation of health and wellbeing in 3 mouse strains commonly used in biomedical research (C3H [melatonin-producing], C57BL/6, and BALB/c [melatonin-non-producing]). Compared with male and female mice housed for 12 wk under 12:12-h light:dark (LD) cycles in CWF light, C3H mice in bLAD evinced 6-fold higher peak plasma melatonin levels at the middark phase; in addition, high melatonin levels were prolonged 2 to 3 h into the light phase. C57BL/6 and BALB/c strains did not produce nighttime pineal melatonin. Body growth rates; dietary and water intakes; circadian rhythms of arterial blood corticosterone, insulin, leptin, glucose, and lactic acid; pO2 and pCO2; fatty acids; and metabolic indicators (cAMP, DNA, tissue DNA 3H-thymidine incorporation, fat content) in major organ systems were significantly lower and activation of major metabolic signaling pathways (mTOR, GSK3β, and SIRT1) in skeletal muscle and liver were higher only in C3H mice in bLAD compared with CWF. These data show that exposure of C3H mice to bLAD compared with CWF has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters associated with the promotion of animal health and wellbeing that may influence scientific outcomes. The absence of enhancement in amelatonic strains suggests hyperproduction of nighttime melatonin may be a key component of the physiology.

Light is a key extrinsic factor to be considered in operations and design of animal room facilities. Over the past four decades, many studies on typical laboratory animal populations have demonstrated impacts on neuroendocrine, neurobehavioral, and circadian physiology. These effects are regulated independently from the defined physiology for the visual system. The range of physiological responses that oscillate with the 24 hour rhythm of the day include sleep and wakefulness, body temperature, hormonal secretion, and a wide range of other physiological parameters. Melatonin has been the chief neuroendocrine hormone studied, but acute light-induced effects on corticosterone as well as other hormones have also been observed. Within the last two decades, a new photosensory system in the mammalian eye has been discovered. A small set of retinal ganglion cells, previously thought to function as a visual output neuron, have been shown to be directly photosensitive and act differently from the classic photoreceptors of the visual system. Understanding the effects of light on mammalian physiology and behavior must take into account how the classical visual photoreceptors and the newly discovered ipRGC photoreceptor systems interact. Scientists and facility managers need to appreciate lighting impacts on circadian, neuroendocrine, and neurobehavioral regulation in order to improve lighting of laboratory facilities to foster optimum health and well-being of animals.

Over 248,000 men in the U.S. alone this year will be diagnosed with prostate cancer, and over 33,300 will die from the disease. The World Health Organization, in the case of breast cancer, has classified night-shift work involving light at night (LAN)-induced circadian disruption to be a probable carcinogen (Class 2A). Exposure to LAN suppresses nighttime pineal melatonin (MLT) production that influences normal and neoplastic tissue metabolism, physiology, and proliferation. Previously, we showed in rodent and human tumors in vivo that MLT inhibits linoleic acid (LA)-uptake and conversion to 13-hydroxyoctadecadienoic acid (13-HODE), a lipoxygenase product that enhances epidermal growth factor and insulin-like growth factor-I-induced mitogenesis. Recently, we developed a tissue-isolated, castration-sensitive VCaP human prostate cancer xenograft model to test the hypothesis that suppression of the nocturnal MLT signal due to exposure to dim LAN (dLAN) accelerates tumor LA-uptake and metabolism, aerobic glycolysis (Warburg Effect), and proliferative activity. Male nude rats (Crl:NIHFoxn1rnu; n=6/group) bearing castration-sensitive VCaP human prostate cancer xenografts were maintained on either a control L(300 lux)D(0 lux),12:12 or experimental LD,12:12dLAN (300 lux light phase followed by dLAN 0.2 lux dark phase intensity) light/dark cycle. Results revealed (Mean ± S.D.) plasma MLT levels in controls peaked in the mid-dark phase (183.4 ± 12.8 pg/mL) and were lowest (2.2 ± 0.4 pg/mL) in the mid-light phase, and low (< 10 pg/mL) throughout the 24-hr period in dLAN rats. Tumors in rats exposed to dLAN exhibited a significantly shorter latency to onset and a two-fold faster growth rate than controls. Harvested control group tumors revealed elevated cAMP levels, LA uptake, 13-HODE production, the Warburg effect, and [3H]thymidine incorporation into DNA, as well as elevated patterns of expression of signaling pathways phospho-ERK1/2, -AKT, -STAT3, -glycogen synthase kinase-3β (GSK3β), and -NF-κβ, and full-length androgen receptor and aldo-keto reductase (AKR1C3), during the light phase and markedly suppressed during the dark phase. In the dLAN group, these measures were markedly elevated throughout the 24-hr period. This is the first evidence in vivo, that dLAN-induced disruption of integrated circadian rhythms of signaling, metabolism, and proliferation results in accelerated growth of castration-sensitive human prostate cancer xenografts. Thus, lighting design strategies to minimize human exposure to LAN that preserve the integrity of the circadian MLT signal may offer a novel approach to suppress the growth progression of human castration-sensitive prostate cancer in our increasingly 24-hour society. Citation Format: Erin M. Dauchy, Robert T. Dauchy, Steven M. Hill, Yan Dong, Victoria P. Belancio, Shulin Xiang, George C. Brainard, John P. Hanifin, David E. Blask. Exposure to dim light at night disrupts the nocturnal melatonin signal in male nude rats bearing tissue-isolated castration-sensitive VCaP human prostate cancer: Impact on tumor circadian dynamics of the Warburg effect, lipid signaling and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 222.

Light has been a crucial part of everyday life since the beginning of time. Most recently, light-emitting diode (LED) light enriched in the blue-appearing portion of the visible spectrum (465 to 485 nm), which is more efficient in energy use, is becoming the normal lighting technology in facilities around the world. Previous reports revealed that blue-enriched LED light at day (bLAD) enhances animal health and wellbeing as compared with cool white fluorescent (CWF) lighting. We hypothesized that bLAD, compared with CWF light, has a positive influence on basic physiologic indices such as food consumption, water consumption, weight gain, nesting behavior, complete blood count, and blood chemistry profile. To test this, we allocated 360 mice into equal-sized groups by sex, strain (C3H/HeNCrl, C57BL/6NCrl, BALB/cAnNCrl), lighting conditions, and 6 blood collection time points (n = 5 mice/sex/strain/lighting condition/time point). Food consumption, water consumption, body weight, nest location, and nest type were recorded every 3 d. At the end of the study, all mice were anesthetized over a period of 1 wk and blood was collected via cardiocentesis at 6 different time points. Overall, male C3H/HeNCrl consumed more food under bLAD conditions as compared with CWF conditions; male C3H/HeNCrl had lower cholesterol levels under bLAD conditions than under CWF conditions; female BALB/cAnNCrl mice had higher serum total protein under bLAD conditions than under CWF conditions; female C57BL/6NCrl mice had higher phosphorus levels under bLAD conditions than under CWF conditions, and female C3H/HeNCrl mice had a higher neutrophil count under bLAD conditions as compared with CWF conditions. Although sex and strain differences were found in various physiologic parameters under bLAD as compared with CWF lighting conditions, the differences were minimal. Thus, this study suggests that for these strains of mice, bLAD and CWF are largely equivalent with regard to indices of health and wellbeing, although some differences could affect research outcomes.

Artificial light at night (ALAN) is a pervasive phenomenon. Although initially assumed to be innocuous, recent research has demonstrated its deleterious effects on physiology and behavior. Exposure to ALAN is associated with disruptions to sleep/wake cycles, development of mood disorders, metabolic disorders, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. We hypothesize that exposure to ALAN accelerates mammary tumor growth and predict that ALAN exacerbates negative affective behaviors in tumor-bearing mice. Adult (&gt, 8 weeks) female C3H mice received a unilateral orthotropic injection of FM3A mouse mammary carcinoma cells (1.0 × 105 in 100 μL) into the fourth inguinal mammary gland. Nineteen days after tumor inoculation, mice were tested for sucrose preference (anhedonia-like behavior). The following day, mice were subjected to an open field test (anxiety-like behavior), followed by forced swim testing (depressive-like behavior). Regardless of tumor status, mice housed in ALAN increased body mass through the first ten days. Tumor-bearing ALAN-housed mice demonstrated reduced latency to tumor onset (day 5) and increased terminal tumor volume (day 21). Exposure to ALAN reduced sucrose preference independent of tumor status. Additionally, tumor-bearing mice housed in dark nights demonstrated significantly increased anxiety-like behavior that was normalized via housing in ALAN. Together, these data reaffirm the negative effects of ALAN on tumorigenesis and demonstrate the potential anxiolytic effect of ALAN in the presence of mammary tumors.

Breast cancer metastasis to bone is a major source of morbidity and mortality in women with advanced metastatic breast cancer. Morbidity from metastasis to bone is compounded by the fact that they cannot be surgically removed and can only be treated with chemotherapy and/or radiation therapy. Thus, there is critical need to develop new treatment strategies that kill bone metastatic tumors and reduce osteolytic lesions to improve patient quality of life and extend patient survival. Circadian rhythms are daily cycles of ~24 h that control many if not most physiologic processes and their disruption by exposure to light at night (LAN) or jet lag has been shown to be strongly associated with the development of cancer, particularly breast cancer. We have found that disruption of the anti-cancer circadian hormone melatonin (MLT) by light at night can significantly enhance the metastatic potential in breast cancer cells. Our work supports the report of the International Agency for Research on Cancer that shift work is a “probable human carcinogen” and highlights the association between exposure to light at night and invasive breast cancer. We recently reported that human breast tumor xenografts grown in athymic nude female rats housed in a photoperiod of 12h light at day: 12h dim light at night (dLAN, 0.2 lux - blocks the nighttime circadian MLT signal), display resistance to doxorubicin (Dox). More importantly, tumor growth and drug resistance could be blocked by the administration of Dox in circadian alignment with nocturnal MLT during dLAN. Our recent preliminary studies show that poorly invasive ERα positive MCF-7 breast cancer cells, when injected into the tibia (to mimic bone metastatic disease) of Foxn1nu athymic nude mice (which produce a strong circadian nighttime melatonin signal) housed in a dLAN photoperiod (suppressed nocturnal MLT production) developed full blown breast cancer tumors in bone (P

Disruption of circadian time structure and suppression of circadian nocturnal melatonin (MLT) production by exposure to dim light at night (dLAN), as occurs with night shift work and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of breast cancer and resistance to tamoxifen and doxorubicin. Melatonin inhibition of human breast cancer chemo-resistance involves mechanisms including suppression of tumor metabolism and inhibition of kinases and transcription factors which are often activated in drug-resistant breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3), frequently overexpressed and activated in Paclitaxel (PTX)-resistant breast cancer, promotes the expression of DNA methyltransferase one (DNMT1) to epigenetically suppresses the transcription of tumor suppressor Aplasia Ras homolog one (ARHI) which can sequester STAT3 in the cytoplasm to block PTX-resistance. We demonstrate that breast tumor xenografts in rats exposed to dLAN and circadian MLT disrupted express elevated levels of phosphorylated and acetylated STAT3, increased DNMT1, but reduced Sirtuin 1 (SIRT1) and ARHI. Furthermore, MLT and/or SIRT1 administration blocked/reversed Interleukin 6 (IL-6)-induced acetylation of STAT3 and its methylation of ARH1 to increase ARH1 mRNA expression in MCF-7 breast cancer cells. Finally, analyses of the I-SPY 1 trial demonstrates that elevated MT(1) receptor expression is significantly correlated with pathologic complete response following neo-adjuvant therapy in breast cancer patients. This is the first study to demonstrate circadian disruption of MLT by dLAN driving intrinsic resistance to PTX via epigenetic mechanisms increasing STAT3 expression and that MLT administration can reestablish sensitivity of breast tumors to PTX and drive tumor regression.

Liver cancer is the second leading cause of cancer death worldwide. Metabolic pathways within the liver and liver cancers are highly regulated by the central circadian clock in the suprachiasmatic nuclei (SCN). Daily light and dark cycles regulate the SCN-driven pineal production of the circadian anticancer hormone melatonin and temporally coordinate circadian rhythms of metabolism and physiology in mammals. In previous studies, we demonstrated that melatonin suppresses linoleic acid metabolism and the Warburg effect (aerobic glycolysis)in human breast cancer xenografts and that blue-enriched light (465-485 nm) from light-emitting diode lighting at daytime (bLAD) amplifies nighttime circadian melatonin levels in rats by 7-fold over cool white fluorescent (CWF) lighting. Here we tested the hypothesis that daytime exposure of tissue-isolated Morris hepatoma 7288CTC-bearing male rats to bLAD amplifies the nighttime melatonin signal to enhance the inhibition of tumor growth. Compared with rats housed under a 12:12-h light:dark cycle in CWF light, rats in bLAD light evinced a 7-fold higher peak plasma melatonin level at the mid-dark phase; in addition, high melatonin levels were prolonged until 4 h into the light phase. After implantation of tissue-isolated hepatoma 7288CTC xenografts, tumor growth rates were markedly delayed, and tumor cAMP levels, LA metabolism, the Warburg effect, and growth signaling activities were decreased in rats in bLAD compared with CWF daytime lighting. These data show that the increased nighttime circadian melatonin levels due to bLAD exposure decreases hepatoma metabolic, signaling, and proliferative activities beyond what occurs after normal melatonin signaling under CWF light.

Chemotherapeutic resistance, particularly to doxorubicin (Dox), represents a major impediment to successfully treating breast cancer and is linked to elevated tumor metabolism and tumor over-expression and/or activation of various families of receptor- and non-receptor-associated tyrosine kinases. Disruption of circadian time structure and suppression of nocturnal melatonin production by dim light exposure at night (dLEN), as occurs with shift work, and/or disturbed sleep-wake cycles, is associated with a significantly increased risk of an array of diseases, including breast cancer. Melatonin inhibits human breast cancer growth via mechanisms that include the suppression of tumor metabolism and inhibition of expression or phospho-activation of the receptor kinases AKT and ERK1/2 and various other kinases and transcription factors. We demonstrate in tissue-isolated estrogen receptor alpha-positive (ERα+) MCF-7 human breast cancer xenografts, grown in nude rats maintained on a light/dark cycle of LD 12:12 in which dLEN is present during the dark phase (suppressed endogenous nocturnal melatonin), a significant shortening of tumor latency-to-onset, increased tumor metabolism and growth, and complete intrinsic resistance to Dox therapy. Conversely, a LD 12:12 dLEN environment incorporating nocturnal melatonin replacement resulted in significantly lengthened tumor latency-to-onset, tumor regression, suppression of nighttime tumor metabolism, and kinase and transcription factor phosphorylation, while Dox sensitivity was completely restored. Melatonin acts as both a tumor metabolic inhibitor and circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to Dox and drive tumor regression, indicating that dLEN-induced circadian disruption of nocturnal melatonin production contributes to a complete loss of tumor sensitivity to Dox chemotherapy.

The present review discusses recent work on melatonin-mediated circadian regulation, the metabolic and molecular signaling mechanisms that are involved in human breast cancer growth, and the associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin suppresses ERα mRNA expression and ERα transcriptional activity via the MT1receptor. Melatonin also regulates the transactivation of other members of the nuclear receptor superfamily, estrogen-metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (the Warburg effect) and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type-specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways, including inhibition of p38 MAPK and repression of epithelial–mesenchymal transition (EMT). Studies have demonstrated that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models has indicated that LEN-induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer and drives breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms that underpin the epidemiologic demonstration of elevated breast cancer risk in night-shift workers and other individuals who are increasingly exposed to LEN.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.

Melatonin has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its antiproliferative effects have been well studied in MCF-7 human breast cancer cells and several other estrogen receptor α (ERα)-positive human breast cancer cell lines. However, the MDA-MB-231 breast cancer cell line, an ERα-negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin's growth-suppressive effect in vitro. Here, we examined the effect of melatonin on the cell proliferation of several ERα-negative breast cancer cell lines including MDA-MB-231, BT-20, and SK-BR-3 cells. Although the MT1 G-protein-coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK-BR-3 cells without having any significant effect on the growth of MDA-MB-231 and BT-20 cells. We confirmed that the MT1-associated Gα proteins are expressed in MDA-MB-231 cells. Further studies demonstrated that the melatonin unresponsiveness in MDA-MB-231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.

This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells.

To determine if melatonin, via its MT1 G protein-coupled receptor (GPCR), impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT1-Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud (TEB) formation. Elevated MT1 receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT1 expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha (ERα), progesterone receptors (PR) A and B, and milk proteins β-casein and whey acidic protein (WAP). Estrogen and progesterone stimulated mammary gland development was repressed by elevated MT1 receptor expression and exogenous melatonin administration. These studies demonstrate that the MT1 melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.

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 MT(1) 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 MT(1) -induced activation of G(αi2) 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 MT(1) -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.

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. Cancer Res; 71(7); 2622–31. ©2011 AACR.

The circadian production of melatonin by the pineal gland during the night provides an inhibitory signal to tissue-isolated steroid receptor SR+ and — MCF-7 human breast cancer xenografts in female nude rats. A pivotal mechanism for melatonin’s anticancer effects in vivo involves a melatonin receptor-mediated inhibition of linoleic acid (LA) uptake and its metabolism to mitogenically active 13-hydroxyoctadecadienoic acid (13-HODE). Exposure of (SR-) xenograft-bearing rats to increasing intensities of polychromatic white light at night suppresses melatonin while increasing tumor growth rates, DNA content, [3H]thymidine incorporation into DNA, LA uptake, 13-HODE formation, cAMP levels and ERK1/2 activation a dose-dependent manner. Similar effects occur in SR- human breast cancer xenografts perfused in situ with melatonin-depleted blood from healthy female subjects after their exposure to a single bright intensity (2800 lux) of polychromatic light at night. Additionally, SR- human breast cancer xenografts exhibit robust circadian rhythms of LA uptake, 13-HODE formation and proliferative activity. Exposure of xenograft-bearing rats to dim light at night results in the complete elimination of these rhythms which culminates in unfettered, high rates of tumor metabolism and growth. The organization of tumor metabolism and growth within circadian time structure by the oncostatic melatonin signal helps create a balance between the cancer and its host that is disrupted by host exposure to light at night. This biological mechanism may partially explain the higher risk of breast and other cancers in women working rotating night shifts and possibly others who also experience prolonged exposure to light at night.

Early studies on rodents showed that short-term exposure to high-intensity light (> 70 lx) above 600 nm (red-appearing) influences circadian neuroendocrine and metabolic physiology. Here we addressed the hypothesis that long-term, low-intensity red light exposure at night (rLEN) from a ‘safelight’ emitting no light below approximately 620 nm disrupts the nocturnal circadian melatonin signal as well as circadian rhythms in circulating metabolites, related regulatory hormones, and physiologic parameters. Male Sprague–Dawley rats (n = 12 per group) were maintained on control 12:12-h light:dark (300 lx; lights on, 0600) or experimental 12:12 rLEN (8.1 lx) lighting regimens. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-wk period to assess arterial plasma melatonin, total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin and corticosterone concentrations. Results revealed plasma melatonin levels (mean ± 1 SD) were high in the dark phase (197.5 ± 4.6 pg/mL) and low in the light phase (2.6 ± 1.2 pg/mL) of control conditions and significantly lower than controls under experimental conditions throughout the 24-h period (P < 0.001). Prominent circadian rhythms of plasma levels of total fatty acid, glucose, lactic acid, pO2, pCO2, insulin, leptin, and corticosterone were significantly (P < 0.05) disrupted under experimental conditions as compared with the corresponding entrained rhythms under control conditions. Therefore, chronic use of low-intensity rLEN from a common safelight disrupts the circadian organization of neuroendocrine, metabolic, and physiologic parameters indicative of animal health and wellbeing.

Conjugated linoleic acid (CLA) and some trans fatty acids (FA) decrease tumor growth and alter tumor and host lipid uptake and storage. The goal of this study was to test the hypothesis that the acute inhibitory effects of CLA isomers and trans FAs on FA transport in tumors and white adipose tissue are mediated via an inhibitory G-protein coupled (GPC), FFA receptor (FFAR). Experiments were performed in hepatoma 7288CTC and inguinal fat pads in Buffalo rats during perfusion in situ. CLA isomers and trans FAs (0.03-0.4 mmol/L, in plasma) were added to the arterial blood, and FA uptake or release was measured by arterial minus venous difference. In hepatoma 7288CTC, the CLA isomers, t10,c12-CLA > (±)-9-HODE [13-(S)-hydroxyoctadecadienoic acid] > t9,t11-CLA, and the trans FAs, linolelaidic = vaccenic > elaidic, decreased cAMP content and inhibited FA uptake, 13(S)-HODE release, extracellular signal-regulated kinase p44/p42 phosphorylation, and [ 3 H]thymidine incorporation. Other CLA isomers, c9,t11-CLA, 13-(S)-HODE, c9,c11-CLA, and c11,t13-CLA, had no effect. In inguinal fat pads, FA transport was inhibited by t10,c12-CLA = linolelaidic acid > trans vaccenic acid, whereas c9,t11-CLA had no effect. In both hepatoma 7288CTC and inguinal fat pad, addition of either pertussis toxin or 8-Br-cAMP to the arterial blood reversed the inhibitions of FA transport. These results support the idea that an inhibitory GPC FFAR reduces cAMP and controls FA transport by CLA isomers and trans FAs. Ligand activity is conferred by the presence of a trans double bond proximal to the carboxyl group. J. Nutr. 134: 1989-1997, 2004.

Over 36,000 people in the United States will be diagnosed with hepatocellular carcinoma (HCC) in 2016, the second leading cause of cancer death worldwide. Metabolic pathways within the liver and in HCC are highly regulated by the central circadian clock in the suprachiastmatic nucleus (SCN). The SCN drives nighttime production of the circadian anti-cancer hormone melatonin by the pineal gland in rats and humans. We have shown that the nighttime circadian melatonin signal suppresses the Warburg effect (aerobic glycolysis) in human breast cancer xenografts and that blue-enriched light (460-480 nm) from LEDs at daytime (bLAD), amplifies the nighttime circadian melatonin signal by 7-fold in rats over cool white fluorescent (CWF) lighting. Here we tested whether daytime exposure of tissue-isolated HepG2 xenograft-bearing male nude rats to bLAD amplifies the nighttime melatonin signal to increase tumor sensitivity to nighttime administered sorafenib (FDA-approved multi-kinase inhibitor) treatment via enhancing suppression of the Warburg effect. Animals were randomized to 6 subgroups (n = 4): CWF Groups I, 12 h light:12 h dark (LD,12:12) (CWF + vehicle) and II (CWF + sorafenib); bLAD Groups III (LD,12bLAD:12) (bLAD + vehicle), IV (bLAD + sorafenib), V (bLAD + S20928 MT1/MT2 receptor blocker), and VI (bLAD + S20928 + sorafenib). Drug treatments began when tumors were 2.5 g estimated weight. Plasma nighttime melatonin levels were 7-fold higher in Groups III-VI, compared to Groups I and II. Tumor latency-to-onset of growth and growth rates were markedly delayed and decreased, respectively, in Group III compared to Group I. Sorafenib induced tumor regression at a rate that was 2-fold higher in the bLAD- vs CWF-exposed rats. Tumor glucose uptake and lactate production (Warburg effect) at the mid-dark phase were significantly reduced in CWF-exposed rats receiving sorafenib vs vehicle. In vehicle-treated bLAD-exposed rats, the Warburg effect was significantly decreased vs CWF-exposed rats receiving vehicle. In bLAD + sorafenib-treated rats, the Warburg effect was reduced by an additional 51% (glucose uptake) and 89% (lactate production), respectively, vs CWF + sorafenib-treated rats. Melatonin receptor blocker S20928 completely prevented the effects of bLAD and bLAD + sorafenib on the Warburg effect and made these tumors completely resistant to sorafenib-induced tumor regression. These findings are the first to show in vivo that: 1) nighttime circadian sorafenib therapy inhibited the Warburg effect and induced HepG2 tumor regression under CWF lighting conditions, and 2) the bLAD-amplified nocturnal circadian melatonin signal increased tumor sensitivity to sorafenib-induced regression by enhancing a melatonin receptor-mediated suppression of the Warburg effect. Citation Format: Robert T. Dauchy, David T. Pointer, Aaron E. Hoffman, Melissa A. Wren-Dail, Shulin Xiang, Lin Yuan, David E. Blask, Victoria P. Belancio, Steven M. Hill. Daytime blue-enriched LED light-induced circadian amplification of the nighttime melatonin signal increases sorafenib sensitivity in human hepatocellular carcinoma via enhanced suppression of the Warburg effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2017-3191

Cancer patients with disrupted 24-hour (circadian) rhythms are reported to have poorer survival as compared to those with normal rhythms. Severe alterations in circadian rhythms predict an increased risk of death in patients with colorectal and breast cancer, suggesting that circadian disruption may impact tumor progression and metastasis. We recently reported that circadian/melatonin (MLT) disruption by exposure to dim light at night (dLAN) resulted in constitutive phospho-activation of ERK1/2, CREB, NF-kB, and STAT3 in breast tumor xenografts promoting resistance to Tamoxifen and Doxorubicin therapy. Given that chemoresistant breast cancer is frequently metastatic, this study examined if dLAN-induced circadian/MLT disruption can promote epithelial-to- mesenchymal transition (EMT) of epithelial MCF-7 breast tumor xenografts leading to the development of metastatic foci in the lungs, livers, and brains of circadian complete (MLT-producing) athymic nude female rats and mice. Female nude rats with ERα+ MCF-7 or T47D human epithelial breast cancer xenografts were housed in LD,12:12 and LD,12:12dLAN (dLAN) photoperiods or in dLAN supplemented with nighttime MLT (0.05 µg/ml) in the drinking water, with lights on at 0600 hrs and off at 1800 hrs. Blood samples collected during the mid-dark phase (2400 hrs) showed elevated nocturnal melatonin (118.4 pg/ml) in the LD,12:12 group, but significantly suppressed melatonin (10.0 pg/ml) in the dLAN group. Tumor xenografts from rats housed in dLAN showed a ~3-fold decrease in latency-to-onset and a ~2.8-fold increase in growth rates vs. those from rats in dLAN + MLT. Tumor cAMP levels, as well as numerous signaling pathways involved in promoting EMT (Vimentin, β-catenin, and SNAIL) and metastasis (HER2/HER3, pCREB, pERK1/2, pRSK2, and pSTAT3), showed increased expression/phospho-activation at 2400 hrs in response to dLAN but repressed expression in tumors from rats in dLAN + MLT. Follow-up studies with Foxn1nude female mice implanted with non-metastatic luciferase expressing MCF-7 breast cancer cells showed that exposure to dLAN suppressed the nighttime serum levels of MLT by 93% in these mice compared to those in a LD,12:12 photoperiod. Exposure of mice to dLAN induced the rapid growth of MCF-7luc tumor xenografts and, after 5 weeks, induced the metastatic outgrowth of MCF-7 xenografts to form luciferase identifiable metastatic foci in the lungs, livers, and brains of all mice, as measured by IVIS small animal imaging system. Conversely, MCF-7luc tumor xenografts from mice exposed to dLAN and supplemented with nighttime MLT showed a reduced tumor development, 3-fold slower tumor growth, and a small metastatic lesion in one lung of a single mouse. This study is the first to show that circadian/MLT disruption by host exposure to dLAN is able to drive EMT in human epithelial breast cancer xenografts to generate metastatic foci in lung, liver, and brain of mice. Citation Format: Steven M. Hill, Shulin Xiang, Robert T. Dauchy, Melissa Wren-Dail, Murali Anbalagan, Brian Rowan, Tripp Frasch, David E. Blask. Circadian/melatonin disruption by dim light at night drives human epithelial breast cancer to a metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4897. doi:10.1158/1538-7445.AM2017-4897

The suprachiasmatic nucleus is synchronized by the light:dark cycle and is the master biologic clock that serves as a pacemaker to regulate circadian rhythms. We explored the hypothesis that spectral transmittance (tint) of light through caging alters circadian rhythms of endocrine and metabolic plasma constituents in nonpigmented Sprague-Dawley rats. Rats (Crl:SD; n = 12 per group) were housed in a 12:12-h light:dark environment (300 lx; 123.0 μ W/cm(2); lights on, 0600) in either clear-, amber-, blue-, or red-tinted rodent cages. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and measured for melatonin, total fatty acids, pH, glucose, lactic acid, corticosterone, insulin, and leptin. As expected, plasma melatonin levels were low during the light phase but higher during the dark phase in all groups; however, when compared with the clear-cage group, rats in amber-, blue-, and red-tinted cages had 29%, 74%, and 48%, respectively, greater total daily melatonin levels due to an increased duration and, in some cases, amplitude of the nocturnal melatonin signal. No differences were found in dietary and water intake, body growth rates, total fatty acids, pH, or glucose among groups. Disruptions in circadian rhythms, manifesting as alterations in phase timing, amplitude, or duration, occurred in the melatonin, lactic acid, corticosterone, insulin, and leptin levels of rats in tinted compared with clear cages. Therefore, the use of variously tinted animal cages significantly alters circadian rhythms in plasma measures of metabolism and physiology in laboratory rats, thus potentially altering the outcomes of scientific investigations.

Light entrains normal circadian rhythms of physiology and metabolism in all mammals. Previous studies from our laboratory demonstrated that spectral transmittance (color) of light passing through cages affects these responses in rats. Here, we addressed the hypothesis that red tint alters the circadian nocturnal melatonin signal and circadian oscillation of other metabolic and physiologic functions. Female nude rats (Hsd:RH-Foxn1(rnu); n = 12 per group) were maintained on a 12:12-h light (300 lx; 123.0 μW/cm(2); lights on 0600):dark regimen in standard polycarbonate translucent clear or red-tinted cages. After 1 wk, rats underwent 6 low-volume blood draws via cardiocentesis over a 4-wk period. Plasma melatonin levels were low during the light phase (1.0 ± 0.2 pg/mL) in rats in both types of cages but were significantly lower in red-tinted (105.0 ± 2.4 pg/mL) compared with clear (154.8 ± 3.8 pg/mL) cages during the dark. Normal circadian rhythm of plasma total fatty acid was identical between groups. Although phase relationships of circadian rhythms in glucose, lactic acid, pO2, and pCO2 were identical between groups, the levels of these analytes were lower in rats in red-tinted compared with clear cages. Circadian rhythms of plasma corticosterone, insulin, and leptin were altered in terms of phasing, amplitude, and duration in rats in red-tinted compared with clear cages. These findings indicate that spectral transmittance through red-colored cages significantly affects circadian regulation of neuroendocrine, metabolic, and physiologic parameters, potentially influencing both laboratory animal health and wellbeing and scientific outcomes.

Melatonin, has been shown repeatedly to inhibit the growth of human breast tumor cells in vitro and in vivo. Its anti-proliferative effects have been well-studied in MCF-7 human breast cancer cells and several other estrogen receptor α (ERα)-positive human breast cancer cell lines. However, the MDA-MB-231 breast cancer cell line, an ERα negative cell line widely used in breast cancer research, has been shown to be unresponsive to melatonin’s growth-suppressive effect in vitro. Here we examined the effect of melatonin on the cell proliferation of several ERα-negative breast cancer cell lines including MDA-MB-231, BT-20 and SK-BR-3 cells. Although the MT1 G-protein-coupled receptor is expressed in all three cell lines, melatonin significantly suppressed the proliferation of SK-BR-3 cells without having any significant effect on the growth of MDA-MB-231 and BT-20 cells. We confirmed that the MT1-associated Gα proteins are expressed in MDA-MB-231 cells. Further studies demonstrated that the melatonin-unresponsiveness in MDA-MB-231 cells may be caused by aberrant signaling downstream of the Gαi proteins, resulting in differential regulation of ERK1/2 activity.

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. Melatonin's 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.

Resistance to chemotherapy is a significant impediment to the treatment of breast cancer. More than 30% of breast cancer patients present with intrinsic resistance to chemotherapy; almost all who initially respond will develop acquired resistance. Resistant tumors frequently exhibit constitutive activation of numerous survival signaling pathways, including ERK, AKT, NF-kB, and STAT3. We have reported that the circadian hormone melatonin inhibits the growth of both ERá+/ERá- breast cancers and, as well as the daytime induced phospho-activation of ERK1/2, AKT and NF-kB in breast tumor xenografts. We also demonstrated that dim light at night (dLAN), by decreasing nocturnal melatonin, resulted in constitutive phospho-activation of ERK1/2, CREB, NF-kB, and STAT3, promoting resistance to tamoxifen and doxorubicin therapy. Here we tested the hypothesis that dLAN, via phospho-activation of ERK1/2 and STAT3, promotes resistance to paclitaxel (Pax). Female nude rats with “tissue-isolated” MCF-7 breast cancer xenografts were housed in photoperiodic conditions of either LD 12:12, 12:12dLAN (0.2 lux), or 12:12dLAN supplemented with nighttime melatonin (0.05 õg/ml) in the drinking water, with lights on at 0600 hrs and off at 1800 hrs. When estimated tumor weights reached 2.5 g, animals were treated daily with either diluent or Pax i.p. (4õã/kg) 2 h prior to onset of dLAN or dLAN with nighttime melatonin supplementation. Blood samples collected during the mid-dark phase (2400 hrs) showed elevated nocturnal melatonin in the LD 12:12 group, but significantly suppressed melatonin in the dLAN group. Tumor xenografts from rats housed in dLAN showed a 3-fold decrease in latency-to-onset and a 2.8-fold increased growth rates vs. those from rats receiving melatonin supplementation. Tumor cAMP levels, linoleic acid, and tumor metabolism (Warburg effect) were significantly elevated in dLAN tumors. Numerous signaling pathways including ERK1/2, RSK2, and STAT3, were phospho-activated and others including AKT and HER2/3 were elevated at 2400 hrs by dLAN but repressed in dLAN melatonin supplemented tumors. Tumors from dLAN rats showed intrinsic resistance to Pax, whereas those in LD 12:12 or dLAN and supplemented with nighttime melatonin rapidly regressed. These findings show that temporally coordinated and integrated metabolic and signal transduction mechanisms, particularly the STAT3 pathway, underlying human breast cancer growth, can be activated by the host's exposure to LAN with profound effects culminating in rapid tumor progression and the development of resistance to chemotherapy. Citation Format: Steven M. Hill, Shulin Xiang, Robert T. Dauchy, Lulu mao, Lin Yuan, Adam Hauch, Victoria P. Belancio, Melissa A. Wren-Dail, David Pointer, Peter W. Lundberg, Whitney M. Summers, David E. Blask. Circadian/melatonin disruption by dim light at night drives paclitaxel resistance in breast cancer via activation of stat3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 874.

Current evidence indicates that rotating night shift workers have an increased risk of developing breast and prostate cancers, which have been associated with light at night (LAN)-induced circadian disruption as the principal risk factor. Previously, we demonstrated that animal room dark phase light contamination with as little as 0.20 lux (0.08 μW/cm2) suppressed the nocturnal production of the circadian oncostatic neurohormone melatonin and stimulated human breast tumor growth and metabolism. The circadian melatonin signal suppresses tumor growth and metabolism via an MT1 melatonin receptor-mediated signaling mechanism involving inhibition of aerobic glycolysis (Warburg effect) and linoleic acid (LA) uptake and conversion to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) culminating in down-regulation of the epidermal growth factor and insulin-like growth factor-1 pathways. We developed a new tissue-isolated androgen-receptor positive (AR+), castration-sensitive VCaP prostate tumor model in adult male athymic nude rats (Crl:NIH-Foxn1rnu), to test the hypothesis that nocturnal melatonin levels inhibit, while dim LAN (dLAN)-induced suppression of nocturnal melatonin production stimulates, tumor signaling, metabolic and growth activity. VCaP xenograft-bearing rats (n = 6/group) maintained on either a control light/dark cycle (LD, 12:12; 300 lux light phase intensity) or an experimental light/dark cycle (LD, 12:12dLAN (0.2 lux; dark phase intensity) for 6 weeks resulted in a 2.5-fold decrease in latency-to-onset (time of implant to first palpable mass) and 2-fold increase in tumor growth rates in experimental animals lacking a nocturnal circadian melatonin signal as compared to control animals with an intact melatonin signal. In control animals, plasma melatonin levels were high in the mid-dark phase (183.4 ± 12.8 pg/mL) and low (2.2 ± 0.4 pg/mL) in mid-light phase, while they were low throughout the 24-hr period in dLAN-exposed animals. Tumors harvested during the mid-dark phase (2400 h) revealed that cAMP levels, Warburg effect (increased glucose uptake and lactate production), LA uptake, 13-HODE production, and DNA [3H]Thymidine incorporation were all significantly elevated (P < 0.001) in dLAN as compared with the controls. Signaling pathways AKT, MEK, ERK ½, STAT3, GSK3ß, and NFκß were all phospho-activated along with increased expression of Aldo-keto reductase family 1 member C3 (AKR1C3) under dLAN conditions. AKR1C3 has been associated with intratumoral androgen synthesis and the development of castration-resistance. These findings are the first to show that the nocturnal melatonin signal inhibits, while dLAN stimulates the Warburg effect, LA metabolism and growth activity, signaling activity and AKR1C3 expression in VCaP human androgen-sensitive prostate cancer. Citation Format: Robert T. Dauchy, Melissa A. Wren, Erin M. Dauchy, Steven M. Hill, Lin Yuan, Shulin Xiang, Yan Dong, Victoria P. Belancio, David M. Blask. Light exposure at night influences host/cancer circadian regulatory dynamics, Warburg effect, and human prostate cancer progression in nude rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 36.

Melatonin, a circadian anti-cancer hormone produced by the pineal gland during darkness at night suppresses the Warburg effect, linoleic acid (LA) uptake/metabolism and tumor cell proliferation in both estrogen receptor (ERα+) and ERα- in tissue-isolated human breast cancer xenografts. The nighttime circadian melatonin signal regulates circadian rhythms in tumor glucose and fatty acid metabolism as well as related signaling pathways that are important in controlling cell proliferative and survival mechanisms. Over-the-counter (OTC) melatonin supplements are used by millions of individuals to treat insomnia and/or jet-lag. The present study addressed the hypothesis that oral ingestion of OTC melatonin supplements by normal adult human female volunteers results in blood levels of melatonin that suppress LA uptake/metabolism, aerobic glycolysis and cell proliferative activity in human breast cancer xenografts, growing in nude female rats, directly perfused in situ with human subject donor whole-blood following melatonin intake. Twelve young, healthy premenopausal women were recruited to ingest an OTC melatonin supplement at a single dose of either 75μg, 150μg, 300μg or 1 mg during midday (low endogenous melatonin levels) resulting in low to high pharmacological blood concentrations of melatonin. A pre-supplement venous blood sample was collected from the antecubital vein of the forearm. Each subject then ingested a randomly selected oral dose of melatonin followed approximately 1 hour later by the withdrawal of a post-supplement venous blood sample. On the following day, the pre- and post-supplement whole-blood samples collected from a given subject were separately placed into a tumor perfusion reservoir. Tissue-isolated ERα- MCF-7 human breast cancer xenografts grown in female nude rats were then directly perfused in situ for 1 hour with either pre- or post-supplement oxygenated blood (37°C). Irrespective of the dose tested, melatonin induced a 50% decrease in both tumor glucose uptake and lactate release, 22% and 44% deceases in O2 uptake and CO2 production, respectively, 50% decrease in cAMP concentrations, and a 100% decrease in linoleic acid (LA) uptake and 13-hydroxyoctadecadienoic acid (13-HODE) formation. Melatonin also caused a marked decrease in the expression of phospho-AKT, GSK3β and ERK1/2, and an 85% decrease in the incorporation of [3H]thymidine into DNA. Similar results were obtained at the lowest dose of melatonin (e.g., 75μg) in ERα+ human breast cancer xenografts. The melatonin-induced suppression tumor proliferative and metabolic activity in both ERα- and ERα+ breast cancer xenografts was completely prevented by the co-perfusion with the non-selective MT1/MT22 melatonin receptor blocker S20928 consistent with the involvement of a melatonin receptor-mediated mechanism. These results indicate that oral administration of an OTC melatonin supplement at a variety of low to high doses were equally effective in suppressing the Warburg effect and key tumor proliferative and survival signaling pathways, cAMP-dependent LA uptake and metabolism to mitogenically active 13-HODE and ultimately cell proliferation in tissue-isolated human breast xenografts irrespective of ERα status via a melatonin receptor-mediated mechanism. These findings suggest that even low doses of OTC melatonin preparations may play a potentially important role as a new chronotherapeutic agent in human breast cancer treatment and/or prevention by targeting aerobic glycolysis and fatty acid signaling and metabolism. Supported by NIH Grants R21CA129875 (DEB) and R01CA54152 (SMH). Citation Format: David E. Blask, Robert T. Dauchy, Erin M. Dauchy, Steven M. Hill, Lulu Mao, Melissa M. Wren, Mary M.C. Meyaski-Schluter, Lin Yuan. Over-the-counter melatonin supplementation in human subjects: A potentially novel chronotherapeutic approach targeting the Warburg effect and fatty acid metabolism in breast cancer therapy/prevention. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A91.

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.

Dark-phase light contamination can significantly disrupt chronobiologic rhythms, thereby potentially altering the endocrine physiology and metabolism of experimental animals and influencing the outcome of scientific investigations. We sought to determine whether exposure to low-level light contamination during the dark phase influenced the normally entrained circadian rhythms of various substances in plasma. Male Sprague-Dawley rats (n = 6 per group) were housed in photobiologic light-exposure chambers configured to create 1) a 12:12-h light:dark cycle without dark-phase light contamination (control condition; 123 μW/cm(2), lights on at 0600), 2) experimental exposure to a low level of light during the 12-h dark phase (with 0.02, 0.05, 0.06, or 0.08 μW/cm(2) light at night), or 3) constant bright light (123 μW/cm(2)). Dietary and water intakes were recorded daily. After 2 wk, rats underwent 6 low-volume blood draws at 4-h intervals (beginning at 0400) during both the light and dark phases. Circadian rhythms in dietary and water intake and levels of plasma total fatty acids and lipid fractions remained entrained during exposure to either control conditions or low-intensity light during the dark phase. However, these patterns were disrupted in rats exposed to constant bright light. Circadian patterns of plasma melatonin, glucose, lactic acid, and corticosterone were maintained in all rats except those exposed to constant bright light or the highest level of light during the dark phase. Therefore even minimal light contamination during the dark phase can disrupt normal circadian rhythms of endocrine metabolism and physiology and may alter the outcome of scientific investigations.

Melatonin and eicosapentaenoic and 10t,12c-conjugated linoleic acids suppress the growth-stimulating effects of linoleic acid (LA) and its metabolism to the mitogenic agent 13-(S)-hydroxyoctadecadienoic acid (13-(S)-HODE) in established rodent tumors and human cancer xenografts. Here we compared the effects of these 3 inhibitory agents on growth and LA uptake and metabolism in human FaDu squamous cell carcinoma xenografts perfused in situ in male nude rats. Results demonstrated that these agents caused rapid inhibition of LA uptake, tumor cAMP content, 13-(S)-HODE formation, extracellular signal-regulated kinase p44/ p42 (ERK 1/2) activity, mitogen-activated protein kinase kinase (MEK) activity, and [3H]thymidine incorporation into tumor DNA. Melatonin's inhibitory effects were reversible with either the melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-bromoadenosine-cAMP, suggesting that its growth-inhibitory effect occurs in vivo via a receptor-mediated, pertussis-toxin-sensitive pathway.

Introduction: The purpose of this study was to test the hypothesis that dim light exposure at night (dLEN)-induced melatonin suppression promotes chemotherapeutic resistance to doxorubicin (Dox) by inducing a circadian-disrupted, hyper-metabolic state relative to linoleic acid (LA) metabolism and the Warburg effect together with constitutive activation of key proliferative and survival signaling pathways. Breast cancer patients often present with de novo (intrinsic) resistance to chemotherapy and many that initially respond will eventually develop acquired resistance. Tumors with chemotherapeutic resistance often exhibit elevated expression/activation of key proliferative and survival signaling pathways and frequently show alterations in tumor metabolism, particularly the biochemical pathways involved in stimulating aerobic glycolysis (Warburg effect). The pineal hormone melatonin inhibits breast cancer proliferation in vitro and the endogenous nocturnal circadian melatonin signal modulates circadian rhythms in tissue-isolated human breast cancer xenografts including LA uptake and metabolism to 13-hydroxyoctadecadeinoic acid (13-HODE), 13-HODE-stimulated Warburg effect, and proliferative and survival signaling. As we have previously reported, these circadian rhythms are induced during the light phase in breast tumor xenografts but inhibited in the dark phase by melatonin. dLEN-induced melatonin suppression drives the constitutive activation of these pathways resulting in tumor hyper-metabolism, increased proliferative and survival signaling activity, and resistance to endocrine therapy with tamoxifen; these effects are repressed by melatonin supplementation. Experimental Procedures: Female nude rats with tissue-isolated ERα+ MCF-7 breast cancer xenografts were housed in environmentally controlled light boxes under photoperiodic conditions of LD, 12L:12D, 12:12dLEN (0.2 lux), or 12:12dLEN with melatonin supplementation during dLEN in the drinking water (lights on at 0600 hrs and off or dLEN on at 1800 hrs). When estimated tumor weights reached ~2.5 g, animals in both dLEN groups were treated daily with either diluent or Dox (6 mg/kg BW) administered by intraperitoneal injection 2 h prior to onset of dLEN. Data Summary: Blood samples collected during the mid-dark phase (2400 hrs) showed elevated nocturnal melatonin levels (118.4 pg/ml) in the LD,12L:12D group, but significantly suppressed melatonin (10.0 pg/ml) in the dLEN group. Rats housed in 12L:12dLEN showed a 3-fold decrease in latency-to-onset of tumor development and a 2.8-fold increase in tumor growth rates vs. those on the 12L:12dLEN photoperiod receiving nighttime melatonin supplementation. Under dLEN circadian-disrupted conditions, tumor metabolism was characterized by a hyper-metabolic state with tumor cAMP-dependent LA-uptake/metabolism to 13-HODE, glucose uptake and lactate release (Warburg effect), O2 uptake and CO2 production, and [3H] thymidine incorporation into DNA all markedly elevated. Furthermore, numerous proliferative and survival signaling pathways including many down stream of the human epidermal growth factor receptors (HER2, HER3) including extracellular signal-related kinase (ERK1/2), protein kinase B (AKT), protein kinase C (PKCα and γ), signal transducer and activator of transcription 3 (STAT3) etc. were highly elevated at 2400 hrs in response to dLEN, but repressed in dLEN melatonin supplemented tumors. Tumor Xenografts from dLEN rats showed complete intrinsic resistance to Dox, whereas tumors from rats on dLEN and supplemented with melatonin exhibited marked sensitivity to Dox and regressed. Conclusions: When circadian-regulated and integrated metabolic and signal transduction mechanisms underlying human breast cancer growth are disrupted by dLEN-induced melatonin suppression, this results in rapid tumor progression and the development of resistance to chemotherapy. Thus, melatonin acts as both a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor (CRKI) to reestablish the sensitivity of breast tumors to Dox and drive tumor regression. Citation Format: Steven M. Hill, Shulin Xiang, Robert T. Dauchy, Lulu Mao, Adam Hauch, Samantha Brimmer, Victoria P. Belancio, Melissa Wren, Debasis Mondal, David E. Blask. Circadian/melatonin disruption by dim light at night drive chemotherapy resistance in breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR13.

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 μW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 μW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.

The type and content of dietary PUFAs have profound influences on the growth rate of transplantable human breast cancers in immunodeficient rodents. Diets enriched in linoleic acid (LA), an (n-6) fatty acid, stimulate tumor growth, whereas dietary fats containing (n-3) fatty acids slow such growth. Interactions between LA and (n-3) fatty acids capable of regulating cell proliferation in solid tumors in vivo are not yet well defined. Here we tested the hypothesis that plasma eicosapentaenoic acid (EPA), an (n-3) fatty acid, suppresses cell proliferation in MCF-7 human breast cancer xenografts via a pertussis toxin-sensitive reduction of intratumor cAMP, LA uptake, and formation of the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) from LA. Plasma fatty acid uptake and 13-HODE release were determined in control and EPA-treated xenografts from arteriovenous differences measured during perfusion in situ. Intratumor cAMP, extracellular signal-regulated kinase p44/p42 (ERK1/2) phosphorylation, and [3H]thymidine incorporation (TTI) were measured in tumors freeze-clamped at the end of the perfusions. Arterial blood containing EPA caused significant decreases (P < 0.05) in cAMP, uptake of SFA, monounsaturated fatty acids, and (n-6) PUFA, 13-HODE formation, ERK1/2 phosphorylation, and TTI in MCF-7 xenografts. These effects of EPA were reversed by the addition of either pertussis toxin or 8-bromoadenosine-cAMP to the EPA-containing arterial blood. Addition of 13-HODE to the EPA-containing arterial blood restored phosphorylated ERK1/2 and TTI but not FA uptake. The results suggest that EPA regulates cell proliferation in MCF-7 xenografts via a novel inhibitory G protein-coupled, (n-3) FFA receptor-mediated signal transduction pathway.

Both physiological and pharmacological levels of the pineal hormone melatonin exhibit substantial anticancer activity in tissue-isolated rat hepatoma 7288CTC via melatonin receptor-mediated blockade of tumor uptake of linoleic acid (LA) and its metabolism to the mitogenic signaling molecule 13-hydroxyoctadecadienoic acid (13-HODE). Melatonin is also present in significant amounts in edible plants and is supplied in nutritional supplements. We confirmed the presence of significant quantities of melatonin in 20 varieties of edible plants. In pinealectomized tumor-free rats, 3 weeks of ingestion of either 5 or 50 microg/day of melatonin contained in a semi-purified diet resulted in a dose-dependent elevation in steady-state plasma melatonin levels within the nocturnal physiological range. In pineal-intact tumor-bearing rats, the daily intake of 5 microg/day of melatonin for 3 weeks resulted in an enhanced amplitude and duration of the nocturnal melatonin levels within physiological circulating limits. The nocturnal melatonin amplitude in rats ingesting 500 ng of melatonin/day remained within the physiological range. A dose-related increase in tumor concentrations of melatonin occurred in animals ingesting melatonin from the diet. Perfusion of tumors in situ with physiological, nocturnal blood levels of melatonin resulted in a mean 31% uptake and retention of the melatonin. Chronic ingestion of 50 ng, 500 ng or 5 microg of melatonin/day supplied in a semi-purified 5% corn oil diet led to a significant dose-dependent reduction in the rates of tumor total fatty acid uptake, LA uptake, 13-HODE production and tumor growth. The co-ingestion of melatonin receptor antagonist S20928 completely blocked the effects and prevented the intra-tumoral accumulation of melatonin. Melatonin receptor-mediated suppression of tumor growth, LA uptake and metabolism, and stimulation of tumor melatonin uptake and retention in response to the dietary intake of phytomelatonin from edible plants or melatonin from nutritional supplements, could play an important role in cancer growth prevention.

Melatonin (MLT), the circadian neurohormone secreted by the pineal gland in mammals during darkness, eicosapentanoic acid (EPA), and conjugated linoleic acid (CLA) have established regulatory roles in cancer growth. Investigations in our laboratory have indicated that these agents inhibit fatty acid (FA) transport by tumors and several sub-types of white adipose tissue via inhibitory G protein-coupled receptor mechanisms. Skeletal muscle constitutes over 45% of human body mass and plays an important role in cancer cachexia and obesity-related diseases. Since fatty acid oxidation is a major source of energy for this tissue, we tested the hypothesis that physiologic MLT levels, EPA, or CLA injected intravenously, inhibit FA uptake in rat skeletal muscle in vivo. We used a surgical technique for catheterizing the femoral vein in rats that allows rapid blood collection from the entire hind limb, while ensuring continuous blood flow to the tissue. Blood acid/gas tensions and hematocrit were monitored and remained constant during the course of each experiment. The MLT, EPA, and CLA inhibited FA uptake by the tissue and lowered cAMP values. Glucose uptake and glycerol production in the hind limb were not affected. These investigations suggest a novel role for MLT, omega-3 FAs, and CLA in the regulation of FA transport and fat metabolism in skeletal muscle.

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.

In this study, we tested the hypothesis that dietary linoleic acid intake controls the arterial blood plasma linoleic acid concentration and the rates of tumor growth and linoleic acid metabolism in vivo. Seven groups of young male Buffalo rats (11-21 rats/group) were given free access to semipurified diets containing different amounts of corn and/or olive oils. Four other groups (7-11 rats/group) were 30% energy-restricted. Each experiment included periods for rat growth and plasma lipid stabilization (6 wk), measurement of mean daily arterial blood plasma fatty acid concentrations (3 wk), surgical implantation of a subcutaneous tissue-isolated hepatoma 7288CTC, tumor growth and harvest (2-4 wk). Linoleic + arachidonic acid (P = 0.007) and oleic acid (P = 0.002) concentrations in arterial blood plasma were increased as dietary intake of linoleic and oleic acids was increased, respectively. In rats given free access to food, tumor growth was directly dependent on the plasma concentrations of linoleic (P < 0.001) and arachidonic acids (P = 0.04). Tumor growth in energy-restricted rats was dependent only on the linoleic acid concentration (P = 0.008). Energy restriction itself caused a growth inhibition independent of plasma linoleic acid. The linoleic acid and total fatty acid concentrations of tumor triacylglycerols were directly dependent on the plasma linoleic acid concentration in rats given free access to food (P = 0.009). Hepatoma 7288CTC (both in vivo and during perfusion in situ) supported a dose-dependent conversion (P < 0.001) of plasma linoleic acid to the mitogen, 13-hydroxy-9, 11-octadecadienoic acid. We conclude that increased arterial blood plasma linoleic acid concentrations, caused by increased dietary intakes, specifically stimulate growth, lipid storage and linoleic acid metabolism in hepatoma 7288CTC in vivo.

Over 630,000 people in the U.S. alone this year will be diagnosed with either breast, prostate, colorectal, or cervical cancers. Epidemiological studies have indicated that the risk of breast, prostate and colorectal cancer is increased in night-shift workers. These individuals experience circadian disruption in response to ocular exposure to light at night, which suppresses the nocturnal circadian production of melatonin by the pineal gland. In previous studies, we determined that nocturnal blood levels of melatonin inhibit tissue-isolated human breast cancer xenograft growth via suppression of 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), culminating in down-regulation of the epidermal growth factor and insulin-like growth factor-1 signaling pathways. Here we showed the effects in vivo of a physiological, nocturnal concentration of melatonin (500 pM) on tumor proliferative activity mediated via aerobic glycolysis (Warburg effect) and LA metabolic signaling in tissue-isolated human HT-29 colorectal and HeLa cervical cancer xenografts perfused in situ in nude rats. Following tumor implantation, HT29 colorectal and HeLa cervical cancer xenografts exhibited latency-to-onset and growth rates that were 12 and 8 days, and 0.12 ± 0.01 and 0.09 ± 0.01 g/day, respectively; mean tumor weights were 5.6 ± 0.2 g (n = 20/group). These tissue-isolated human cancer xenografts in nude rats perfused in situ for 60 min with rat donor blood containing melatonin resulted in a significant reduction in tumor aerobic glycolysis that included a 30% reduction in glucose uptake, lactate release, O2 uptake and CO2 production. Additionally, melatonin induced a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, MEK, Akt, and GSK3β expression, as well as significant reductions in tumor cAMP levels, DNA content and [3H]thymidine incorporation into tumor DNA. Addition of the non-selective MT1/MT2 melatonin antagonist S20928, forskolin, 8-Bromo-cyclic-AMP, or pertussis toxin to the tumor perfusate completely reversed the inhibitory effects of melatonin on aerobic glycolysis, LA metabolic signaling and proliferative activity in both types of xenografts. These results demonstrate in HT29 colorectal and HeLa cervical adenocarcinomas that nocturnal melatonin levels directly and rapidly inhibit tumor growth activity via a melatonin receptor-mediated suppression of the Warburg effect and LA metabolic and other related signaling mechanisms. An understanding of this novel signaling pathway for the control of aerobic glycolysis and LA metabolism in cancer could lead to new circadian-based approaches for cancer therapy and/or prevention. Citation Format: Robert T. Dauchy, Erin M. Dauchy, Lulu Mao, Melissa A. Wren, Victoria P. Belancio, Steven M. Hill, David E. Blask. The circadian neurohormone melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human colorectal and cervical cancer. [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 4001. doi:10.1158/1538-7445.AM2013-4001

In the United States alone this year approximately 3,500 people will be diagnosed with leiomyosarcoma (LMS), a rare, malignant soft tissue cancer derived from smooth muscle cells, and about 36% will perish from this disease, despite the current medical interventions of surgery, radio- and chemotherapy. Epidemiological studies have indicated that the risk of many types of epithelial cancer including breast, prostate, colorectal, and endometrial cancer is increased in night-shift workers. These individuals experience circadian disruption in response to ocular exposure to light at night, which suppresses the nocturnal circadian production of melatonin by the pineal gland. In previous studies, we demonstrated that human LMS perfused with rodent donor blood containing physiological nocturnal levels of melatonin (1nM) showed a complete inhibition of LA uptake, 13-HODE release, and marked reduction in tumor cAMP levels and [3H]thymidine incorporation into tumor DNA. Here we showed the in vivo effects of a physiological, nocturnal concentration of melatonin on tumor proliferative activity mediated via aerobic glycolysis (Warburg effect) and LA metabolic signaling in tissue-isolated LMS xenografts perfused in situ in nude rats with human donor blood. Following tumor implantation, LMS xenografts exhibited latency-to-onset and growth rates that were 22 days and 0.21 ± 0.04 g/day, respectively; mean tumor weights were 5.1 ± 0.2 g (n = 72). The tissue-isolated human LMS xenografts in nude rats perfused in situ for 60 min with human donor blood containing melatonin resulted in a significant reduction in tumor aerobic glycolysis that included a 30-50% reduction in glucose uptake, lactate release, O2 uptake and CO2 production. Additionally, melatonin induced a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, Akt, GSK3β (Ser9), and NF-kB (p65) phosphorylation, as well as significant reductions in tumor cAMP levels, DNA content and [3H]thymidine incorporation into tumor DNA. Addition of the non-selective MT1/MT2 melatonin antagonist S20928 (1 μM) completely reversed the inhibitory effects of melatonin on aerobic glycolysis, LA metabolic signaling and proliferative activity in LMS xenografts. Moreover, melatonin at physiological concentrations (1 nM) induced a 30-50% inhibition in cell proliferation in culture, and suppressed cell invasion by 30% in the transwell assay. These results demonstrate that in human LMS nocturnal melatonin levels directly and rapidly inhibit tumor growth activity and invasion via suppression of the Warburg effect and LA metabolic and other related signaling mechanisms. An understanding of this novel signaling pathway for the control of aerobic glycolysis and LA metabolism in human leiomyosarcoma could lead to new circadian-based approaches for cancer therapy and/or prevention of rare but highly aggressive mesenchymally-derived solid tumors. Citation Format: Lulu Mao, Robert T. Dauchy, Erin M. Dauchy, Melissa A. Wren, Samantha D. Zeringue, Victoria P. Belancio, David E. Blask, Steven M. Hill. Melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human leiomyosarcoma. [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 5423. doi:10.1158/1538-7445.AM2013-5423 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Over 620,000 people in the U.S. alone this year will be diagnosed with either breast, prostate, or colorectal cancers, which represent nearly 50% of all reported malignancies. Patients with advanced stage breast, prostate, or colorectal cancer have only a 35% survival rate at 5 years. Epidemiological studies have indicated that the risk of these cancers is increased in persons exposed to excessive sunlight or, interestingly, ocular light at night, as experienced by night-shift workers. This latter finding was shown to be associated with a suppression of the nighttime production of the circadian neurohormone melatonin. Previous work from this laboratory using perfused, tissue-isolated human breast and prostate tumors showed that tumor growth and metabolism in vivo is dependent upon uptake of linoleic acid (LA), the most prominent fatty acid in our western diet, and its conversion to 13-hydroxyoctadecadienoic acid (13-HODE). In tissue-isolated human MCF-7 (steroid responsive and non-responsive) and PC3 prostate tumors perfused in situ with arterial blood, melatonin inhibits LA-uptake and conversion to 13-HODE, a lipoxygenase product that stimulates EGF mitogenesis. The present study explores the effects in vivo of melatonin (500 pM) on perfused, tissue isolated HT29 colorectal carcinomas in nude rats. HT29 colorectal carcinoma latency-to-onset and growth rates, respectively, of 12 days and 0.12 ± 0.01 g/day; mean tumor weights were 5.6 ± 0.2 g (n = 20). Colorectal carcinomas perfused in situ with donor rodent blood for 2 hours (Controls) revealed a tumor LA uptake and 13-HODE release of 0.86 ± 0.08 μg/min/g (20.0 ± 1.6% of arterial supply) and 3.23 ± 0.81 ng/min/g, respectively. Incorporation of 3H-thymidine into tumor DNA and DNA content, respectively were 25.9 ± 1.6 dpms/μg DNA and 2.7 ± 0.1 μg/g tumor. Carcinomas perfused with arterial donor blood augmented with physiological nocturnal levels of 500 pM melatonin showed a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, MEK, Akt, and GSK3β activities, and over a 90% reduction in tumor cAMP levels and [3H]thymidine incorporation into tumor DNA. Addition of the non-selective MT1/MT2 melatonin antagonist S20928, forskolin, 8-Bromo-cyclic-AMP, or pertussis toxin completely reversed the tumor growth inhibitory response. Also, addition of 13-HODE reversed the inhibition of 3H-thymidine incorporation into tumor DNA, while having no effect on LA uptake. These results are the first to demonstrate in HT29 colorectal carcinoma that nocturnal melatonin levels suppress tumor growth via a melatonin receptor-mediated signal transduction mechanism. An understanding of this signaling pathway for the control of LA uptake in cancer could lead to new approaches for therapeutic intervention and/or chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 165. doi:1538-7445.AM2012-165

Over 12,000 women in the U.S. alone this year will be diagnosed with cervical cancer, ranking third behind endometrial and ovarian cancers. Patients with an advanced stage cervical cancer have only a 15-20% survival rate at 5 years. Observational studies of the night-shift worker population have shown an increased incidence of risk in persons exposed to ocular light at night in a number of cancers, including cervical cancer. This finding was associated with an inhibition of nighttime pineal gland production of melatonin, the circadian neurohormone shown to suppress tumor metabolic and proliferative activity. This occurs via melatonin inhibition of tumor linoleic acid (LA) uptake and conversion to 13-hydroxyoctadecadienoic acid (13-HODE), a molecule that upregulates epidermal growth factor and insulin-like growth factor-I-induced mitogenesis. Here, we investigate the effects in vivo of melatonin (500 pM) on perfused, tissue-isolated HeLa human adenocarcinoma xenografts in nude rats. HeLa adenocarcinoma latency-to-onset and tumor growth rates were, respectively, 8 days and 0.09 ± 0.01 g/day; mean tumor weights were 5.9 ± 0.3 g (n = 32). Cervical adenocarcinomas perfused in situ with donor rodent blood for 150 minutes (Controls) revealed a tumor glucose uptake and lactate release of 11.4 ± 0.9 ng/min/g and −20.4 ± 2.2 nmol/min/g, respectively; control tumor LA uptake and 13-HODE release was 0.71 ± 0.08 μg/min/g and 0.35 ± 0.07 ng/min/g, respectively. Incorporation of 3H-thymidine into tumor DNA and DNA content, respectively, were 21.2 ± 0.6 dpms/μg DNA and 2.5 ± 0.1 mg/g tumor. Adenocarcinomas perfused with arterial donor blood augmented with physiological nocturnal levels of 500 pM melatonin showed over a 20% abrogation of tumor glucose uptake and lactate production, O2 consumption and CO2 production, and a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, MEK, Akt, GSK3β proliferative activities, and over an 85% reduction in tumor cAMP levels and 3H-thymidine incorporation into tumor DNA. Supplementation with the non-selective MT1/MT2 melatonin antagonist S20928, forskolin, 8-Bromo-cyclic-AMP, or pertusiss toxin completely reversed the tumor growth inhibitory response. Also, addition of 13-HODE reversed 3H-thymidine into tumor DNA, but had no effect on LA uptake. The preliminary results presented here demonstrate, for the first time, that nocturnal melatonin levels suppress HeLa cervical adenocarcinoma metabolism and proliferation in vivo via a melatonin receptor mediated signal transduction mechanism. A better understanding of this signaling pathway and the regulation of LA in cancer metabolism and proliferation may lead to new therapeutic and/or chemopreventative interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5167. doi:1538-7445.AM2012-5167

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 melatonin's 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