Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression

Abstract

Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(⌬19) tumors expressing the clock-mutant protein (CLOCK ⌬19 ) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK ⌬19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.Circadian rhythms affect blood pressure, locomotor activity, core body temperature, and the sleep-wake cycle. These circadian controls of physiology and behavior are driven by a master pacemaker located in the suprachiasmatic nucleus of the hypothalamus, which relies on the interplay of interconnected transcriptional and translational feedback loops (1). The brain and muscle Arnt-like 1 (BMAL1) 3 and circadian locomotor output cycles kaput (CLOCK) heterodimer drive the transcription of cryptochrome (CRY) and period (PER) genes by binding to the E-box in their promoter regions. CRY and PER homodimers and heterodimers inhibit the function of BMAL1/CLOCK, thus decreasing their own expression (2-5). This core loop creates a 24-h rhythmic oscillation of clock-controlled gene expression in normal and tumor cells.Iron is an important metal for cell proliferation, metabolism, respiration, and DNA synthesis (6 -8). Iron homeostasis is dependent on the expression of transferrin receptor 1 (TfR1) (9, 10). TfR1 is a membrane receptor responsible for iron uptake and ferritin is an intracellular protein that stores iron. When present in excess, cellular iron is toxic (11-13). Therefore, iron concentration must be tightly regulated. In general, the expression of TfR1 is regulated by iron-regulatory proteins (IRPs), which are sensors of intracellular iron levels. IRPs control TfR1 and divalent metal transporter 1 (Dmt1) at the post-transcriptional level by binding to RNA stem-loop structures known as iron-responsive elements (IREs) in the 3Ј-untranslated region (UTR) of mRNA. IRP binding stabilizes transcripts in these genes (14 -16).Iron metabolism is critical for the rapid growth of tumors and iron is also required for DNA synthesis in tumor cells. Therefor...

Administering xCT Inhibitors Based on Circadian Clock Improves Antitumor Effects

“…Furthermore, we demonstrated that Bmal1 knockdown did not suppress the induction of xCT expression 36 hours after the serum treatment, whereas the knockdown of Clock did. xCT mRNA and membrane protein expression were shown to be higher between 17:00 and 21:00 and lower between 5:00 and 9:00 in colon 26 tumors, whereas it was demonstrated in our previous report that Clock protein expression is increased between 17:00 and 21:00 as well (22). Therefore, taken together, these results indicate that Clock may represent the main regulator of the circadian rhythm of xCT expression.…”

“…On the other hand, the inhibition of iron metabolism reduces cystine and GSH levels (32). We previously demonstrated that iron metabolism exhibited circadian rhythms, whereas iron levels were shown to be higher at 21:00 in the colon 26 tumors (22,33). Here, we showed that the expression of xCT increases between 17:00 and 21:00, suggesting that the circadian rhythm of xCT expression is involved in the protection from the oxidative stress and in the cell cycle through iron metabolism.…”

“…PCR products were purified and ligated into a pcDNA3.1 expression vector (Thermo Fisher Scientific; ref. 22).…”

Molecular regulation of cellular iron balance

“…Moreover, IRPs do not respond only to iron levels in the LIP, but are specifically controlled by a number of agents and conditions that modify iron metabolism in distinct pathophysiological settings , in particular under hypoxia, given the interplay of iron metabolism and oxygen homeostasis . A remarkable example is represented by the recent observation that circadian clock genes promote the transcription of IRP2, which in turn regulates the 24‐H rhythm of TfR1 expression post‐transcriptionally .…”

Copper in the suprachiasmatic circadian clock: A possible link between multiple circadian oscillators

“…The relationship between trace metals, circadian rhythms, and metabolism is still largely uninvestigated, but several studies in plants demonstrate rhythms in copper and iron homeostasis and how they pertain to circadian activity and metabolism (Andres‐Colas et al., ; Penarrubia et al., ; Perea‐Garcia, Sanz, et al., ; Tissot et al., ). As Cu plays a role in iron homeostasis, it is noteworthy that a circadian rhythm in iron levels in the midbrain of mice has been reported (Unger et al., , ), and there is growing evidence of evolutionarily conserved, circadian control of iron homeostasis and vice‐versa (Ben‐Shlomo et al., ; Botebol et al., ; Cao, Li, Jin, & Hu, ; Dean, Allen, O'Donnell, & Earley, ; Mandilaras & Missirlis, ; Marelja, Leimkuhler, & Missirlis, ; Okazaki et al., ; Robles et al., ; Saha et al., ; Simcox et al., ; Tissot et al., ; Zhang et al., ). In addition, iron deficiency has been shown to affect circadian wheel‐running activity and is strongly associated with restless leg syndrome, which has a circadian component and manifests as a sleep disorder (Dowling et al., ; Earley et al., ; Furudate, Komada, Kobayashi, Nakajima, & Inoue, ).…”