Melissa A. Wren

Circadian and Melatonin Disruption by Exposure to Light at Night Drives Intrinsic Resistance to Tamoxifen Therapy in Breast Cancer

Resistance to endocrine therapy is a major impediment to successful treatment of breast cancer. Preclinical and clinical evidence links resistance to antiestrogen drugs in breast cancer cells with the overexpression and/or activation of various pro-oncogenic tyrosine kinases. Disruption of circadian rhythms by night shift work or disturbed sleep-wake cycles may lead to an increased risk of breast cancer and other diseases. Moreover, light exposure at night (LEN) suppresses the nocturnal production of melatonin that inhibits breast cancer growth. In this study, we used a rat model of estrogen receptor (ERα(+)) MCF-7 tumor xenografts to demonstrate how altering light/dark cycles with dim LEN (dLEN) speed the development of breast tumors, increasing their metabolism and growth and conferring an intrinsic resistance to tamoxifen therapy. These characteristics were not observed in animals in which the circadian melatonin rhythm was not disrupted, or in animals subjected to dLEN if they received nocturnal melatonin replacement. Strikingly, our results also showed that melatonin acted both as a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to tamoxifen and tumor regression. Together, our findings show how dLEN-mediated disturbances in nocturnal melatonin production can render tumors insensitive to tamoxifen.

Doxorubicin Resistance in Breast Cancer is Driven by Light at Night Induced Disruption of the Circadian Melatonin Signal

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.

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

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

Abstract 36: Light exposure at night influences host/cancer circadian regulatory dynamics, Warburg effect, and human prostate cancer progression in nude rats

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 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.

Abstract PR13: Circadian/melatonin disruption by dim light at night drive chemotherapy resistance in breast cancer

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.

Abstract 4001: The circadian neurohormone melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human colorectal and cervical cancer.

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 MT 1 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, O 2 uptake and CO 2 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 [ 3 H]thymidine incorporation into tumor DNA. Addition of the non-selective MT 1 /MT 2 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

Abstract 5423: Melatonin inhibits aerobic glycolysis (Warburg effect) and fatty acid metabolic signaling in human leiomyosarcoma.

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 [ 3 H]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, O 2 uptake and CO 2 production. Additionally, melatonin induced a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, Akt, GSK3β (Ser 9 ), and NF-kB (p65) phosphorylation, as well as significant reductions in tumor cAMP levels, DNA content and [ 3 H]thymidine incorporation into tumor DNA. Addition of the non-selective MT 1 /MT 2 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.