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A 23-year-old man presented with complaints of macrohematuria and hematospermia and was referred to our hospital for further examination. Magnetic resonance imaging revealed a round 30 × 25 mm tumor in the right peripheral zone; hence, a rare prostate tumor was suspected. Grayscale transrectal ultrasonography (TRUS) was performed using the Aplio-i800 PVL-715RST-transducer and revealed a well-defined round tumor. Although regular color Doppler flow imaging could not detect internal blood flow, superb microvascular imaging (SMI) identified the low-velocity blood flow in the tumor. Based on the results of a TRUS-guided targeted biopsy assisted by SMI, the patient was diagnosed with stromal sarcoma. He underwent total pelvic exenteration with construction of ileal conduit and colostomy, the tumor was finally diagnosed as prostate stromal sarcoma (PSS). Since PSS is a rare malignant prostate tumor, reports on the characteristic findings in imaging tests are scarce. To the best of our knowledge, this study reports the first case in which a poor internal blood flow was detected in PSS, but not through regular color Doppler flow imaging. SMI revealed that the blood flow signal to the PSS was relatively poor; however, its definite presence was confirmed, suggesting a malignant disease with relatively poor blood supply and the findings of SMI would assist the adequate targeted biopsy-sampling from the presence site of viable cells with the blood supply.

The circadian clock, which regulates cellular physiology, such as energy metabolism, resides in each cell level throughout the body. Recently, it has been elucidated that the cellular circadian clock is closely linked with cellular differentiation. Moreover, the misregulation of cellular differentiation in mouse embryonic stem cells (ESCs) induced abnormally differentiated cells with impaired circadian clock oscillation, concomitant with the post-transcriptional suppression of CLOCK proteins. Here, we show that the circadian molecular oscillation is disrupted in dysdifferentiation-mediated mouse kidney tumors induced by partial in vivo reprogramming, resembling Wilms tumors. The expression of CLOCK protein was dramatically reduced in the tumor cells despite the Clock mRNA expression. We also showed that a similar loss of CLOCK was observed in human Wilms tumors, suggesting that the circadian molecular clockwork may be disrupted in dysdifferentiation-mediated embryonal tumors such as Wilms tumors, similar to the in vivo reprogramming-induced mouse kidney tumors. These results support our previous reports and may provide a novel viewpoint for understanding the pathophysiological nature of cancers through the correlation between cellular differentiation and circadian clock.

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1+CD44high CD4 T cells as well as CD95+GL7+ germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders.

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of Clock subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10–12 hearts (63, no core circadian genes) than in E17–19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of Clock mRNA, which we showed was due to Dicer/Dgcr8-dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of Clock plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

Circadian clocks in mammals function in most organs and tissues throughout the body. Various renal functions such as the glomerular filtration and excretion of electrolytes exhibit circadian rhythms. Although it has been reported that the expression of the clock genes composing molecular oscillators show apparent daily rhythms in rodent kidneys, functional variations of regional clocks are not yet fully understood. In this study, using macroscopic bioluminescence imaging method of the PER2::Luciferase knock-in mouse kidney, we reveal that strong and robust circadian clock oscillation is observed in the medulla. In addition, the osmotic pressure in the inner medulla shows apparent daily fluctuation, but not in the cortex. Quantitative-PCR analysis of the genes contributing to the generation of high osmotic pressure or the water re-absorption in the inner medulla, such as vasopressin receptors (V1aR, V2R), urea transporter (UT-A2) and water channel (Aqp2) show diurnal variations as well as clock genes. Deficiency of an essential clock gene Bmal1 impairs day-night variations of osmotic pressure gradient in the inner medulla, suggesting that circadian clocks in the medulla part of the kidney may regulate the circadian rhythm of cortico-medullary osmotic pressure gradient, and may contribute physiological day-night rhythm of urination.

Most organisms have evolved intrinsic circadian clock to optimize their behavior and physiology for diurnal environmental changes. The circadian clock exists not only in the organismal level but also in the peripheral organs and cellular levels, and regulates the various physiological aspects. Among these regulation systems, the relationship between circadian clock and cancer has been enthusiastically investigated in this decade. However, the mechanistic link connecting circadian clock and cancer has not been fully understood. Recently, we have clarified that circadian clock is tightly connected with cellular differentiation using the in vitro mouse embryonic stem cells (ESCs) differentiation model, and in addition, misregulation of differentiation leads to the circadian clock disruption via highly expression of KPNA2 and suppression of CLOCK protein. On the other hand, it was revealed that altered dedifferentiation leads to cancer development using in vivo reprogramming mouse model. So we considered that the viewpoint of dysdifferentiation can help to provide significant pathophysiological implications for the relationship of circadian clock and cancer. Firstly, we established Rosa26:M2-rtTA TetO:OSKM ES cells carrying circadian rhythm reporter mPer2:luc, and generated chimeric mice. The chimeric mice were treated with doxycycline (Dox) for transient in vivo reprogramming. The Dox treatment induced Wilms tumor-like kidney tumors and we sectioned the kidney tumor for monitoring real time bioluminescence signals, and examined the RNA and protein expression profiles. Here, we showed that the circadian clock was disrupted in the Wilms tumor-like mouse kidney tumor tissues, while the control mouse kidney exhibited the obvious circadian bioluminescence oscillation. And the gene expression signature of the circadian clock development correlated gene set in the tumor was similar to the dysdifferentiation-mediated circadian clock disrupted cells. Moreover, the highly expressed KPNA2 and suppression of CLOCK protein were also observed in the mouse tumor cells. Next, we examined human Wilms tumor gene and protein expression profiles. Astonishingly, the transcriptional signature of the circadian clock development correlated genes was similar to the mouse kidney tumor induced by dedifferentiation. Furthermore, the protein expression patterns of KPNA2 and CLOCK coincided with them. These findings suggest that the severely dedifferentiated cancers may lose their circadian clocks and the mechanism is common with the suppression mechanism during differentiation coupled circadian clock development. Moreover, circadian clock functionality may reflect the etiology of cancer development including dedifferentiation. In future prospects, a new view point of the circadian clock may help to evaluate the type of cancer cells, and it leads to more improved therapeutic strategy. Citation Format: Munehiro Ohashi, Yasuhiro Umemura, Yoichi Minami, Hitomi Watanabe, Tomoko Tanaka, Tsuneharu Miki, Osamu Ukimura, Tatsuro Tajiri, Gen Kondoh, Yasuhiro Yamada, Kazuhiro Yagita. Disruption of circadian clockwork in in vivo reprogramming induced mouse kidney cancer and human Wilms tumor [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 1039. doi:10.1158/1538-7445.AM2017-1039