Paper Review – Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Hello there,

This paper was a bit shocking when it first came out, and it is a bit complicated, but I think it is worth reviewing because it is immensely interesting and raises profound questions about the link between various environmental factors and behaviours with tumour progression.

The paper in question is “Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice
Susie Lee et al” You may duly retrieve the full paper here, which you will actually need to do since I will be leaving the reading of some parts to you to do from the original paper.

Before moving on to pay attention to all the details, it is worth reviewing the abstract, in my opinion. I quote verbatim and then highlight the key implications.

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.
Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.

[1] They note that circadian rhythms are disrupted in advanced stage cancers.
[2] They use genes implicated in circadian rhythm maintenance as reference standards in this paper, namely the genes Per1 and Per2 , and also Cry1 and Cry2 , they note that mice carrying mutant versions of these genes are prone to cancer, this already indicates a correlation of statistical significance.

If you go through the introduction of the full paper, you will come to a brief understanding of how the Circadian rhythm is maintained, they note that there is a peripheral clock that alone cannot regulate the circadian rhythm of cell proliferation due to it being based on external mitogenic signals, they then postulate that a central circadian clock is involved to account for this extraneous factor, and this takes the form of a molecular clock in which some of the genes they have mentioned above play an active role. The presence of such a system, the authors note, entails some of the following…

The molecular clock regulates clock-controlled genes (CCGs) to control tissue/organ function. Most CCGs follow tissue-specific expression patterns. Only a small group of CCGs, which include key cell cycle regulators and tumor suppressors, are expressed in all tissues studied. Such circadian control leads to the coupling of cell proliferation with key tissue functions in vivo [19], [20], [21], [22], [23], [24]. Disruption of circadian rhythm in cell proliferation is frequently associated with tumor development and progression in mammals [4], [5], [12], [25], [26], [27], [28].

Both positive and negative loops of the molecular clock are involved in cell cycle control. For example, BMAL1 suppresses proto-oncogene c-myc but stimulates the tumor suppressor Wee1 [19], [22], [29], CRY2 indirectly regulates the intra S-check point [30], [31], and PER1 directly interacts with ATM in response to γ-radiation in vitro [32]. In mice, mutation in Per2 leads to deregulation of DNA-damage response and increased neoplastic growth [19], [24], [29]. In humans, deregulation or polymorphism of Per1, Per2, Cry2, Npas2 and Clock is associated with acute myelogenous leukemia, hepatocellular carcinoma, breast, lung, endometrial and pancreatic cancers, and non-Hodgkin’s lymphoma [12], [33], [34], [35], [36], [37], [38], [39], [40], [41].

As already mentioned above, they develop a hypothesis concerning such a synchronization while also stating that such a thing cannot be reduced to peripheral mechanisms alone. They note that this is because at least some known mitogenic cues (signals that tell cells to divide) are external,and in the G1 phase of the cell cycle, it is completely externally controlled (I’ve written a review on the cell cycle on this blog before, you may find it useful to refer to it at this juncture) which means that the internal, peripheral clocks of individual cells and tissues cannot account for these.

Along with references, they present knowledge of the fact that mitogen mediated cellular replication is synchronized with the activity of tumour suppressors, this led them to investigate one possible mechanism of circadian rhythm control through such synchronization by means of the sympathetic nervous system.

At this juncture, I will leave the discerning reader an opportunity to read the rest of that particular section elaborating why such a desynchronization (which upsets what we call an axis) should lead to problems regulating the cell cycle (which is one of the hallmarks of cancer)

The way the authors tested their hypothesis is the next item up for discussion.

To quote

We found that when kept in 24 hour alternating light-dark conditions (24hr LD cycles), mice deficient in Bmal1 (Bmal1+/−), Cry1 and Cry2 (Cry1−/−;Cry2−/−), Per1 and Per2 (Per1−/−;Per2m/m) or Per2 alone (Per2−/−) were all cancer-prone. About 10–15% of Bmal1−/− mice also showed hyperplasia of salivary glands in spite of a significant reduction in the size of other major organ systems due to aggressive aging [49]. Bmal1+/−, Cry- and Per-mutant mice all showed increased risk of ulcerative dermatitis and hyperplasia in the salivary gland, preputial gland, liver and uterus as well as spontaneous lymphoma, liver and ovarian tumor development, although spontaneous tumors in Cry-mutants were mostly identified after 50 weeks of age, later than that of Per1−/−;Per2m/m and Per2−/− mice

You may ignore the allelic notation at this point, for the formatting is screwed due to the act of having to copy it in, but they noticed that mice with mutations in critical circadian rhythm genes were prone to cancer, thus establishing a correlation. This was followed up by an insight they derived through another cool experimental trick.

A sublethal dose of γ-Radiation induced premature aging on the external appearance of all circadian gene-mutant mouse models studied and further increased incidence of tumor and hyperplasia as well as ulcerative dermatitis in Bmal1+/−, Per- and Cry-mutant mice (Fig. 1b, Table 1 and Fig. S1b). About 8% of irradiated Bmal1−/− mice also developed lymphoma despite an average lifespan of 27 weeks due to a further accelerated rate of aging (Fig. 1c and Table 1). Irradiated Bmal1+/− mice showed a similar rate of tumor development as did irradiated Per2−/− mice (Fig. 1d–e and Table 1). Since all circadian gene-mutant mouse models show increased sensitivity to γ-radiation, we conclude that the molecular clock functions in tumor suppression in vivo.

They used increased sensitivity of said mice to gamma radiation to arrive at the conclusion that tumour suppression was associated with the circadian rhythm.

They then tested what would happen if one were to alter the external visual cues that drive the circadian rhythm (there is a new paper out on PLoS Computational Biology on a mathematical model that explains how such a system works in a plant, but that is beside the point at the moment, so let us carry on), they did this by simulating jet lag.

I quote the relevant section below.

We then examined the role of the central clock in tumor suppression by studying the effects of jet-lag (an 8hr phase-advance followed by an 8hr phase-delay in the onset of the light period every 3 days) on tumor development in mice. We found that jet-lag further increased and hastened tumor development in wt, Cry- and Per-mutant mice and induced pancreatic, kidney and intestinal tumors in mutant mice. However, jet-lag did not significantly change the overall survival and tumor development of irradiated Bmal1−/− mice that were deficient in responding to circadian light cues in the central clock (Table 1, Fig. 2a, Fig. S1c and data not shown) [50]. Jet-lag also significantly changed tumor spectrum and induced osteosarcoma, liver and ovarian tumors, hyperplasia of the salivary gland, liver and uterus as well as severe seminal vesicle enlargement in wt mice (Table 1. Fig. 2b–g, Fig. S1d–l and data not shown). In addition, although when kept 24hr LD cycles, female circadian gene-mutant mice showed an earlier onset and a higher tumor incidence compared to male mutant mice, we did not find a significant gender difference in total tumor incidence and the time of tumor onset among irradiated and jet-lagged male and female mutant mice (data not shown).

They found screwing up the rhythm further by upsetting the cues the genes were linked to hastened tumour development, and this also altered the spectrum of the resulting tumours. They noticed that changing these cues in mice which had genes that could respond to them had an effect but those mutants which did not respond to cues experienced no difference as compared to the first dataset.

Disruption of Circadian Rhythm Promotes Tumor Development in Wild-type Mice. (a) The Kaplan-Meier survival curves of wt, Cry1−/−;Cry2−/−, Per1−/−;Per2m/m and Bmal1−/− mice (−IR: untreated, +IR: irradiated, +IR/Shift: irradiated and jet-lagged, p1: untreated wt vs. untreated mutant mice, p2: untreated wt mice vs. irradiated mice, p3: untreated wt mice vs. irradiated/jet-lagged mice, and p4: untreated vs. irradiated Bmal1−/− mice). Median survival times in weeks (95% CI) are 79.7 weeks (79.2–80.1) for untreated, 78.6 weeks (77.5–79.7) for irradiated and 67.9 weeks (63.3–72.5) for irradiated/jet-lagged wt mice, 76.9 weeks (73.9–80.0) for untreated, 69.3 weeks (64.4–74.2) for irradiated and 54.9 weeks (47.8–62.0) for irradiated/jet-lagged Cry1−/−;Cry2−/− mice, 74.8 weeks (71.8–77.8) for untreated, 67.1 weeks (62.5–71.7) for irradiated and 56.1 weeks (51.0–61.2) for irradiated/jet-lagged Per1−/−;Per2m/m mice, and 35.5 weeks (30.73–40.1) for untreated, 27.3 weeks (23.3–31.4) for irradiated and 28.5 weeks (24.4–32.5) for irradiated/jet-lagged Bmal1−/− mice. Representative pictures of (b) lymphomas in the salivary glands of irradiated/jet-lagged Bmal1−/−, Bmal1+/−, Cry1−/−;Cry2−/−, Per2−/−, Per1−/−;Per2m/m and wt (Per1+/+;Per2+/+) mice, (c) hepatocellular carcinomas in irradiated/jet-lagged Bmal1+/−, Cry1−/−;Cry2−/−, Per1−/−;Per2m/m, Per2−/− and wt (Bmal1+/+ and Per1+/+;Per2+/+) mice, (d) ovarian granulosa cell tumors in irradiated/jet-lagged Cry1−/−;Cry2−/−, Per1−/−;Per2m/m, Per2−/− and wt (Cry1+/+;Cry2+/+) mice, (e) osteosarcoma growing out from the spine into the chest cavity of an irradiated/jet-lagged wt (Per1+/+;Per2+/+) mouse and on the back of an irradiated/jet-lagged Per2−/− mouse, (f) severe cystic hyperplasia of the uterus in irradiated/jet-lagged Cry1−/−;Cry2−/−, Per1−/−;Per2m/m and wt (Per1+/+;Per2+/+) mice, and (g) seminal vesicles from an untreated 60-week old wt (Per1+/+;Per2+/+) mouse and age-matched irradiated/jet-lagged Cry1−/−;Cry2−/− and Per1−/−;Per2m/m mice.

So now we have

[1] Mutant circadian genes —-> increased incidence of cancer relative to fully functional circadian genes.
[2] Mutant circadian genes that respond to external cues + radically altered cues —-> higher incidence than [1]
[3] Mutant genes that don’t respond to external cues + radically altered cues —–> incidence similar to case [1]

Then, in a section I will leave to readers again, they established through the occurrence of renal failure when such deviations occured, using the fact that renal failure is strongly indicative of sympathetic nervous system (SNS) dysfunction, that said time cues must involve the SNS.

They then looked for evidence that the SNS could rhythmically be associated with regulating gene expression. They used Ucp1 mRNA levels as a marker for gene expression, they noted that said mRNA is produced in two peaks and one of them doesn’t vary regardless of whether circadian genes are mutated or not, and the other does, this led them to recognize the fact that the ZT10 peak, as they name it, is controlled by collaboration between both clocks (central and peripheral) while ZT22 is not.

(a) Northern blot of Ucp1 mRNA expression in wt and Per1−/−;Per2m/m BAT in 24hr LD cycles. (b) A summary of three independent Northern blot analyses described in (a) (±SEM). (c) Ucp1 mRNA expression in wt and Per1−/−;Per2m/m BAT in 24hr DD cycles. (d) A summary of three independent Northern blot analyses described in (c) (±SEM). (e) Per2 and Bmal1 expression in wt and Per1−/−;Per2m/m BAT in 24hr LD cycles. Summaries of (f) Per2 and (g) Bmal1 mRNA expression in 24hr LD cycles from three independent experiments (±SEM). (h) Per2, Bmal1 and Ucp1 mRNA expression in BAT from untreated (Control) wt mice and wt mice treated with one cycle of jet-lag (Shift). (i) A summary of three independent Northern blot analyses described in (h) (±SEM). (j) Ucp1 mRNA expression in BAT from age-matched control wt mice and wt mice treated with 10 months of chronic jet-lag. (k) A summary of Ucp1 mRNA expression from three independent Northern blot analyses described in (j). doi:10.1371/journal.pone.0010995.g004

They then repeated this marker associated study with jet lag simulation thrown into the mix,they noted that either the collaboration between the central and peripheral clocks is disrupted or the latter is screwed permanently. We now have evidence that Circadian rhythm disruption, through the SNS, can affect gene expression, but the hypothesis necessitates that tumour suppression be of special importance, how did they solve this problem?

Firstly, they demonstrated that tampering with receptors involved in extracellular mitogenic signalling (i.e growth signals from outside the cell) resulted in varied proliferation, thus establishing that cell cycle progression could be linked to external mitogenic cues. This section I will again leave to the discerning reader. The nature of the agonist used, being linked to the signalling pathways mentioned before, illustrated that the SNS is a source of, through those pathways , mitogenic signals.

We now have evidence that the SNS is associated with cell cycle control.

Next, using the same agonist and the mechanism to study the effect of activation of said pathways on cellular function, they investigated the functioning of p53 which can be regarded as the master guardian of the cell, and this is a tumour suppressor. They note…

Since p53 plays a key role in preventing Myc oncogenic activation [48], we studied whether iso also induces p53 expression in preosteoblasts. We found that p53 was induced by iso with kinetics similar to AP1 transcription factors in wt osteoblasts. However, iso-treated Per1−/−;Per2m/m osteoblasts showed AP1 overexpression in the absence of p53 induction (Fig. 6a). The expression of p53 is mainly controlled by its interaction with the E3 ubiquitin ligase MDM2 in vivo [56], [57]. Thus, we examined MDM2 expression in iso-treated preosteoblasts. We found that MDM2 was suppressed by iso with the same kinetics as p53 induction in wt osteoblasts, but remained high in Per1−/−;Per2m/m osteoblasts (Fig. 6a). The MDM2-p53 interaction is best studied in γ-radiation response, which activates protein kinase ATM that phosphorylates p53 at Ser18 (S18) and MDM2 at Ser395 (S395) in mice. The phosphorylation at these two sites blocks MDM2-p53 interaction, leading to MDM2 autoubiquitination and p53 induction [58]. Since we used an anti-MDM2 2A10 antibody raised against a MDM2 C-terminal region containing the S395 residue, the interaction of 2A10 with MDM2 could be blocked by MDM2 S395 phosphorylation [58]. Thus, we studied MDM2 and p53 expression in iso-treated preosteoblasts using an anti-p53 S18 antibody and an anti-MDM2 AB4 antibody that interacts with MDM2 at the N-terminal region. We found that these two antibodies detected a similar rate of p53 induction and MDM2 degradation in wt osteoblasts as shown by the p53 PAb421 and MDM2 2A10 antibodies but failed to detect p53 S18 induction and MDM2 decrease in Per1−/−;Per2m/m osteoblasts (Fig. 6b). We then studied p53 induction in iso-treated wt and Atm−/− preosteoblasts. We found that in wt osteoblasts, ATM was activated by iso, as shown by ATM S1981 phosphorylation [59], in an iso dose-dependent manner and that the duration and the levels of ATM activation correlated with the level of p53 S18 phosphorylation and total p53 accumulation. In contrast, Atm−/− osteoblasts that lacked p53 S18 phosphorylation failed to show p53 induction by iso (Fig. 6c–d). Together, our findings indicate that the SNS activates p53 via ATM in a peripheral clock-dependent manner.

They established that the SNS can activate p53 through a mechanism that requires the peripheral clock too.

So now, circadian rhythm disruption, which can also involve the SNS, especially when jet lag et cetera is present, through the SNS can screw up cell cycle control, firstly and also affect p53 function, which is critical to tumour suppression, we have seen evidence of how this happens and through what components it may occur as well.
It therefore enables aberrant cells to proceed to full blown cancer with much more ease than normal.

There endeth a very brief exposition of what is a content heavy paper, which of course can always be found using the link at the top. It is a brilliant paper, absolutely cutting edge and insightful and I think you will be immensely satisfied if you excercise the due diligence required to assimilate the original paper. My review was just an effort at facilitating something like that.

Moral of the story – sleep regularly, and too much jet-lag is bad for you if you want to keep your tumour suppression systems running smoothly.

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One response to “Paper Review – Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

  1. Pingback: Cues Signals Reading

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