An association between clock genes and clock‐controlled cell cycle genes in murine colorectal tumors

Disruption of circadian machinery appears to be associated with the acceleration of tumor development. To evaluate the function of the circadian clock during neoplastic transformation, the daily profiles of the core clock genes Per1, Per2, Rev‐Erbα and Bmal1, the clock‐controlled gene Dbp and the clock‐controlled cell cycle genes Wee1, c‐Myc and p21 were detected by real‐time RT‐PCR in chemically induced primary colorectal tumors, the surrounding normal tissue and in the liver. The circadian rhythmicity of Per1, Per2, Rev‐Erbα and Dbp was significantly reduced in tumor compared with healthy colon and the rhythmicity of Bmal1 was completely abolished. Interestingly, the circadian expression of Per1, Per2, Rev‐Erbα and Dbp persisted in the colonic tissue surrounding the tumor but the rhythmic expression of Bmal1 was also abolished. Daily profiles of Wee1, c‐Myc and p21 did not exhibit any rhythmicity either in tumors or in the colon of healthy animals. The absence of diurnal rhythmicity of cell cycle genes was partially associated with ageing, because young healthy mice showed rhythmicity in the core clock genes as well as in the Wee1 and p21. In the liver of tumor‐bearing mice the clock gene rhythms were temporally shifted. The data suggest that the circadian regulation is distorted in colonic neoplastic tissue and that the gene‐specific disruption may be also observed in the non‐neoplastic tissues. These findings reinforce the role of peripheral circadian clockwork disruption for carcinogenesis and tumor progression.     

Disruption of Circadian Coordination and Malignant Growth

Altered circadian rhythms predicted for poor survival in patients with metastatic colorectal or breast cancer. An increased incidence of cancers has been reported in flying attendants and in women working predominantly at night. To explore the contribution of circadian structure to tumor growth we ablated the 24-h rest-activity cycle and markedly altered the rhythms in body temperature, serum corticosterone and lymphocyte count in mice by complete stereotaxic destruction of the suprachiasmatic nuclei (SCN) or by subjecting the mice to experimental chronic jet-lag. Such disruption of circadian coordination significantly accelerated malignant growth in two transplantable tumor models, Glasgow osteosarcoma and Pancreatic adenocarcinoma. The mRNA expression of clock genes per2 and reverb-α in controls displayed significant circadian rhythms in the liver (Cosinor, p=0.006 and p=0.003, respectively) and in the tumor (p=0.04 and p<0.001, respectively). Both rhythms were suppressed in the liver and in the tumor of jet lagged mice. This functional disturbance of molecular clock resulted in down regulation of p53 and overexpression of c-Myc, two effects which may favor cancer growth. Conclusions:These results indicate that circadian system could play an important role in malignant growth control. This should be taken into consideration in cancer prevention and therapy.     

Expression of the Circadian Clock Genes Per1 and Per2 in Sporadic and Familial Breast Tumors

There is a growing body of evidence implicating aberrant circadian clock expression in the development of cancer. Based on our initial experiments identifying a putative interaction between BRCA1 and the clock proteins Per1 and Per2, as well as the reported involvement of the circadian clock in the development of cancer, we have performed an expression analysis of the circadian clock genes Per1 and Per2 in both sporadic and familial primary breast tumors and normal breast tissues using real-time polymerase chain reaction. Significantly decreased levels of Per1 were observed between sporadic tumors and normal samples (P < .00001), as well as a further significant decrease between familial and sporadic breast tumors for both Per1 (P < .00001) and Per2 (P < .00001). Decreased Per1 was also associated with estrogen receptor negativity (53% vs 15%, P = .04). These results suggest a role for both Per1 and Per2 in normal breast function and show for the first time that deregulation of the circadian clock may be an important factor in the development of familial breast cancer. Aberrant expression of circadian clock genes could have important consequences on the transactivation of downstream targets that control the cell cycle and on the ability of cells to undergo apoptosis, potentially promoting carcinogenesis.     

Tumor‐specific cytotoxic T cell generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti‐tumor immunity

Interleukin (IL‐) 27 is a member of IL‐12 cytokine family with Th1‐promoting and anti‐inflammatory effects. IL‐27 has been shown to facilitate tumor‐specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL‐27 suppresses cytokine production of lymphocytes and antigen‐presenting function of dendritic cells (DCs). To examine the in vivo role of IL‐27 in generation of anti‐tumor immunity, we examined IL‐27‐mediated antitumor‐effects using WSX‐1 (IL‐27 receptor α chain)‐deficient (WSX‐1^?/?) mice. In WSX‐1^?/? mice inoculated with B16 melanoma cells, tumor growth was higher than in wild‐type (WT) mice. Accordingly, tumor‐specific CTL generation was lower in WSX‐1^?/? mice than in WT mice. CTL induction in WSX‐1^?/? mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL‐27 is directly required for generation of tumor‐specific CTLs. However, when transferred into tumor‐bearing mice, WSX‐1^?/? DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL‐27 on DC function. Finally, the combination of WT CD8⁺ T cells and KO DCs is more potent in generation of antigen‐specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor‐specific CD8⁺ T cells were also the highest in the combination of WT CD8+ T cells and WSX‐1^?/? DCs. It was thus revealed that IL‐27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL‐27 signal‐defective DCs may have therapeutic potential against tumors. © 2008 Wiley‐Liss, Inc.     

缺氧诱导因子-3α（HIF-3α）对肝癌细胞生物钟基因表达的影响

目的:研究缺氧诱导因子-3α(HIF-3α)对肝癌细胞生物钟基因表达的影响。方法:将HIF-3α过表达的慢病毒载体感染HepG2细胞，构建稳定过表达HIF-3α肝癌细胞系，同时通过siRNA干扰HepG2细胞中HIF-3α的表达。然后通过Real-time PCR和Western Blot检测HIF-3α感染前后肝癌细胞中生物钟基因Clock、Bmal1、NPAS2、Per1、Per2、Per3、Timeless、Cry1、Cry2、REV-ERBA、Rora、CKIε的表达水平。结果:Real-time PCR结果显示HIF-3α使Per3、CKIε mRNA表达升高(P均<0.05)，Bmal1、Per1、Per2、Cry1、REV-ERBA、Rora mRNA表达降低(P均<0.05)，HIF-3α对Clock、NPAS2、Timeless、Cry2 mRNA的表达无明显影响(P均>0.05)。Western Blot结果与PCR结果相一致。结论:HIF-3α可引起肝癌细胞生物钟基因的紊乱，可能是肝癌生物钟基因表达异常的原因之一。     

Time‐dependent changes in proliferation,DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time‐of‐day dependent changes of proliferation and DNA damage in HCC. Using transgenic c‐myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ‐H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev‐erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ‐H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time‐of‐day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.     

Daily coordination of cancer growth and circadian clock gene expression

Background.Circadian coordination in mammals is accomplished, in part, by coordinate, rhythmic expression of a series of circadian clock genes in the central clock within the suprachiasmatic nuclei (SCN) of the hypothalamus. These same genes are also rhythmically expressed each day within each peripheral tissue.Methods.We measured tumor size, tumor cell cyclin E protein, tumor cell mitotic index, and circadian clock gene expression in liver and tumor cells at six equispaced times of day in individual mice of a 12-h light, 12-h dark schedule.Results.We demonstrate that C3HFeJ/HeB mice with transplanted syngeneic mammary tumor maintain largely normal circadian sleep/activity patterns, and that the rate of tumor growth is highly rhythmic during each day. Two daily 2.5-fold peaks in cancer cell cyclin E protein, a marker of DNA synthesis, are followed by two daily up-to-3-fold peaks in cancer cell mitosis (one minor, and one major peak). These peaks are, in turn, followed by two prominent daily peaks in tumor growth rate occurring during mid-sleep and the second, during mid-activity. These data indicate that all therapeutic targets relevant to tumor growth and tumor cell proliferation are ordered in tumor cells within each day. The daily expression patterns of the circadian clock genes Bmal1, mPer1, and mPer2, remain normally circadian coordinated in the livers of these tumor bearing mice. Bmal1 gene expression remains circadian rhythmic in cancer cells, although damped in amplitude, with a similar circadian pattern to that in normal hepatocytes. However, tumor cell mPer1 and mPer2 gene expression patterns fail to maintain statistically significant daily rhythms.Conclusion. We conclude that, if core circadian clock gene expression is essential to gate tumor cell proliferation within each day, then there may be substantial redundancy in this timing system. Alternatively, the daily ordering of tumor cell clock gene expression may not be essential to the daily gating of cancer cell DNA synthesis, mitosis and growth. This would indicate that host central SCN-mediated neuro–humoro-behavioral controls and/or daily light-induced changes in melatonin or peripherally-induced rhythms such as those resulting from feeding, may be adequate for the daily coordination of cancer cell expression of proliferation related therapeutic targets.     

Down regulation of circadian clock gene Period 2 accelerates breast cancer growth by altering its daily growth rhythm

Purpose Per2, a core circadian clock gene, has tumor suppressor properties and is mutated or down regulated in human breast cancers. We have manipulated the expression of this gene in vitro and in vivo to more fully understand how the Per2 clock gene product affects cancer growth. Methods We used siRNA and shRNA to down regulate Per2 expression in vitro and in vivo and measured cancer cell proliferation, tumor growth rate and several molecular pathways relevant to cancer growth and their circadian organizations. All statistical tests were two-sided. Results Down regulation of functional Per2 gene expression increases Cyclin D and Cyclin E levels and doubles in vitro breast cancer cell proliferation (P < 0.05). Down regulation of Per2 also accelerates in vivo tumor growth and doubles the daily amplitude of the tumor growth rhythm (P < 0.05). Conclusions The clock gene Per2 exerts its tumor suppressor function in a circadian time dependent manner. Therefore, Per2 and perhaps other clock genes represent a new class of potential therapeutic targets whose manipulation will modulate cancer growth and cancer cell proliferation.     

Effects of chronic jet lag on tumor progression in mice

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F(1) mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbalpha in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbalpha mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.     

Tumor Suppression by the Mammalian Period Genes

The Period (Per) genes are key circadian rhythm regulators in mammals. Expression of the mouse Per (mPer) genes have diurnal pattern in the suprachiamstic nuclei and in peripheral tissues. Genetic ablation mPER1 and mPER2 function results in a complete loss of circadian rhythm control based on wheel running activity in mice. In addition, these animals also display apparent premature aging and significant increase in neoplastic and hyperplastic phenotypes. When challenged by γ-radiation, mPer2 deficient mice response by rapid hair graying, are deficient in p53-mediated apoptosis in thymocytes and have robust tumor occurrences. Our studies have demonstrated that the circadian clock function is very important for cell cycle, DNA damage response and tumor suppression in vivo. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as c-Myc, Cyclin D1, Cyclin A, Mdm-2 and Gadd45α is deregulated in mPer2 mutant mice. In addition, genetic studies have demonstrated that many key regulators of cell cycle and growth control are also important circadian clock regulators confirming the critical role of circadian function in organismal homeostasis. Recently studies of human breast and endometrial cancers revealed that the loss and deregulation of PERIOD proteins is common in the tumor cells.     

Ileal FGF15 contributes to fibrosis‐associated hepatocellular carcinoma development

Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut‐derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15^+/+ and Fgf15^?/? mice were subjected to a clinically relevant model of liver inflammation and fibrosis‐associated carcinogenesis. Fgf15^?/? mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15^+/+ animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15^?/? mice, which also expressed lower levels of the HCC marker alpha‐fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro‐fibrogenic and pro‐tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15‐triggered CTGF‐mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis.     

Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer

It has been reported that disruption of the circadian clock may lead to increased risk of breast cancer in humans and to a high rate or ionizing radiation-induced tumors and mortality in mice. Cryptochrome 1 and cryptochrome 2 proteins are core components of the mammalian circadian clock and mice mutated in both genes are arrhythmic. We tested Cry1-/- Cry2-/- mice and fibroblasts derived from these mice for radiation-induced cancer and killing and DNA damage checkpoints and killing, respectively. We find that the mutant mice are indistinguishable from the wild-type controls with respect to radiation-induced morbidity and mortality. Similarly, the Cry1-/- Cry2-/-mutant fibroblasts are indistinguishable from the wild-type controls with respect to their sensitivity to ionizing radiation and UV radiation and ionizing radiation-induced DNA damage checkpoint response. Our data suggest that disruption of the circadian clock in itself does not compromise mammalian DNA repair and DNA damage checkpoints and does not predispose mice to spontaneous and ionizing radiation-induced cancers. We conclude that the effect of circadian clock disruption on cellular response to DNA damage and cancer predisposition in mice may depend on the mechanism by which the clock is disrupted.     

Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor‐derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non‐bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel‐running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.     

Modulatory effects of adiponectin on the polarization of tumor‐associated macrophages

The plasticity of macrophages with selective functional phenotypes partially arises in respective to their microenvironment. Tumor‐associated macrophages (TAMs) may promote disease progression with tumor specific manner. Here we report that in pediatric malignant soft‐tissue tumors, the presence of TAMs and expression of adiponectin (APN) are heterogeneous. Both APN and TAMs had high expression in rhabdomyosarcoma, especially in the malignant subtype, alveolar rhabdomyosarcoma. To investigate the mode of action of APN on TAM activation, a murine MN/MCA1 sarcoma model was used. The Results revealed that exogenous APN had no effect on MN/MCA1 proliferation but tumor size was markedly reduced in apn^?/? mice versus WT controls. The accumulation of TAMs in apn^?/? mice was also reduced which correlated to downregulated serum levels of MCP‐1. Likewise, TAMs in apn^?/? mice exhibited a M1‐like phenotype, characterized by increase in MHC II^high population and M1 phenotypic markers, such as iNOS gene and serum TNF‐α accompanied by a decrease in M2 markers, namely YM1 gene and serum IL‐10. In addition, APN deficiency increased the number of CD4⁺ T cells, CD8⁺ T cells and NK cells in tumors and reduced tumor metastasis. The altered phenotype of TAMs in apn^?/? mice was associated with a marked decrease in phospho‐p38 and treatment with a p38 MAPK inhibitor significantly reduced tumor size and increased MHC II expression on TAMs in WT mice, implying p38 MAPK signaling pathway may contribute to APN‐mediated TAM polarization. Collectively, our findings suggest that APN may have a potential role in regulating soft tissue sarcoma growth.     

Liver-specific expression of the <Emphasis Type="Italic">agouti</Emphasis> gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes

Background  

The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (A ^y ) and viable yellow (A ^vy ) are dominant regulatory mutations in the mouse agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the α-melanocyte stimulating hormone (αMSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded.