RNA editing enzyme ADAR2 regulates P-glycoprotein expression in murine breast cancer cells through the circRNA-miRNA pathway.

Among the various RNA modifications, adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) family, ADAR1 and ADAR2, is the most common nucleotide conversion in mammalian cells. The pathological relevance of ADAR expression has been highlighted in recent human genetic studies. Low expression of the ADAR2 gene is correlated with a poor prognosis in breast cancer patients, but the underlying mechanism remains enigmatic. In this study, we constructed Adar2-knockdown (Adar2-KD) murine breast cancer 4T1 cells and observed their reduced susceptibility to chemotherapeutic drug doxorubicin. Downregulation of ADAR2 induced the expression of P-glycoprotein (P-gp), leading to a reduction in the intracellular accumulation of doxorubicin. The upregulation of P-gp occurred at the post-transcriptional level due to the decreased miR-195a-3p function. The search for the underlying cause of the induction of P-gp expression in Adar2-KD 4T1 cells led to the identification of circular RNA (circRNA) circHif1a as a sponge for miR-195a-3p. The enhanced expression of circHif1a inhibited miR-195a-3p function, resulting in the upregulation of P-gp expression. These results suggest that ADAR2 acts as a suppressor of circHif1a biogenesis and then allows miR-195a-3p to interfere with P-gp translation. Our findings may help to improve drug efficacy by clarifying the mechanism of chemoresistance in breast cancer.

Circadian rhythms in CYP2A5 expression underlie the time-dependent effect of tegafur on breast cancer.

The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

Suppression of neuropathic pain in the circadian clock-deficient Per2m/m mice involves up-regulation of endocannabinoid system.

Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.

Time-Dependent Differences in Vancomycin Sensitivity of Macrophages Underlie Vancomycin-Induced Acute Kidney Injury.

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1ß expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1ß and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

Modulation of cell physiology by bispecific nanobodies enabling changes in the intracellular localization of organelle proteins.

Proteins localize to their respective organelles in cells. This localization is changed by activation or repression in response to signal transduction. Therefore, the appropriate intracellular localization of proteins is important for their functions to be exerted. However, difficulties are associated with controlling the localization of endogenous proteins. In the present study, we developed a conceptually new method of controlling the intracellular localization of endogenous proteins using bispecific nanobodies (BiNbs). BiNbs recognize proteins expressed in the inner membrane, cytoskeleton, nucleus, and peroxisomes, but not in mitochondria or endoplasmic reticulum. BiNbs designed to recognize ß-CATENIN and the intrinsic cytosolic protein VIMENTIN (3 × Flag ß-CAT-VIM BiNbs) decreased the ß-CATENIN-mediated transactivation of target genes by preventing its nuclear localization. Furthermore, 3 × Flag ß-CAT-VIM BiNbs suppressed the proliferation and invasion of the VIMENTIN-expressing breast cancer cell line MDA-MB-231, but not MDA-MB-468, in which the expression of VIMENTIN was defective. The present results revealed that changes in the intracellular localization of specific proteins by BiNbs modulated the physiology and functions of cells. The development of BiNbs to recognize proteins specifically expressed in target cells may be a useful approach for eliciting cell-selective effects.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

RNA editing enzyme ADAR1 controls miR-381-3p-mediated expression of multidrug resistance protein MRP4 via regulation of circRNA in human renal cells.

Multidrug resistance-associated protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is highly expressed in the kidneys of mammals and is responsible for renal elimination of numerous drugs. Adenosine deaminase acting on RNA 1 (ADAR1) has been reported to regulate gene expression by catalyzing adenosine-to-inosine RNA editing reactions; however, potential roles of ADAR1 in the regulation of MRP4 expression have not been investigated. In this study, we found that downregulation of ADAR1 increased the expression of MRP4 in human renal cells at the posttranscriptional level. Luciferase reporter assays and microarray analysis revealed that downregulation of ADAR1 reduced the levels of microRNA miR-381-3p, which led to the corresponding upregulation of MPR4 expression. Circular RNAs (circRNAs) are a type of closed-loop endogenous noncoding RNAs that play an essential role in gene expression by acting as miRNA sponges. We demonstrate that ADAR1 repressed the biogenesis of circRNA circHIPK3 through its adenosine-to-inosine RNA editing activity, which altered the secondary structure of the precursor of circHIPK3. Furthermore, in silico analysis suggested that circHIPK3 acts as a sponge of miR-381-3p. Indeed, we found overexpression of circHIPK3 induced the expression of MRP4 through its interference with miR-381-3p. Taken together, our study provides novel insights into regulation of the expression of xenobiotic transporters through circRNA expression by the RNA editing enzyme ADAR1.

Diurnal Expression of PD-1 on Tumor-Associated Macrophages Underlies the Dosing Time-Dependent Antitumor Effects of the PD-1/PD-L1 Inhibitor BMS-1 in B16/BL6 Melanoma-Bearing Mice.

Cancer cells have acquired several pathways to escape from host immunity in the tumor microenvironment. Programmed death 1 (PD-1) receptor and its ligand PD-L1 are involved in the key pathway of tumor immune escape, and immune checkpoint therapy targeting PD-1 and PD-L1 has been approved for the treatment of patients with certain types of malignancies. Although PD-1 is a well-characterized receptor on T cells, the immune checkpoint receptor is also expressed on tumor-associated macrophages (TAM), a major immune component of the tumor microenvironment. In this study, we found significant diurnal oscillation in the number of PD-1-expressing TAMs collected from B16/BL6 melanoma-bearing mice. The levels of Pdcd1 mRNA, encoding PD-1, in TAMs also fluctuated in a diurnal manner. Luciferase reporter and bioluminescence imaging analyses revealed that a NF-κB response element in the upstream region of the Pdcd1 gene is responsible for its diurnal expression. A circadian regulatory component, DEC2, whose expression in TAMs exhibited diurnal oscillation, periodically suppressed NF-κB-induced transactivation of the Pdcd1 gene, resulting in diurnal expression of PD-1 in TAMs. Furthermore, the antitumor efficacy of BMS-1, a small molecule inhibitor of PD-1/PD-L1, was enhanced by administering it at the time of day when PD-1 expression increased on TAMs. These findings suggest that identification of the diurnal expression of PD-1 on TAMs is useful for selecting the most appropriate time of day to administer PD-1/PD-L1 inhibitors. IMPLICATIONS: Selecting the most appropriate dosing time of PD-1/PD-L1 inhibitors may aid in developing cancer immunotherapy with higher efficacy.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene.

The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates.

Fluorescence detection of metabolic activity of the fatty acid beta oxidation pathway in living cells.

Detection of metabolic activity in living cells facilitates the understanding of the cell mechanism. Here, we report a fluorescent probe that can detect fatty acid beta oxidation (FAO) in living cells. This probe is metabolically degraded by the sequential enzyme reactions of FAO and can visualize the FAO activity with turn-on fluorescence.

Epithelial cell adhesion molecule expression in hepatic stem/progenitor cells is controlled by the molecular clock system.

The circadian rhythm, which regulates various body functions, is transcriptionally controlled by a series of clock gene clusters. The clock genes are related to the pathology of various kinds of diseases. Although there is evidence of serious sleep disorders in patients with chronic hepatitis, the liver regeneration mechanism under chronic hepatitis conditions and its association with the clock genes are not clear. In this study, the influence of the circadian locomotor output cycles kaput (CLOCK), which is one of the clock genes, on a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced hepatitis animal model was investigated. The appearance of potential hepatic stem-like cells (epithelial cell adhesion molecule [EpCAM]-positive cells) is an initial critical step in liver regeneration during chronic inflammation. The results showed a considerable number of hepatic EpCAM-positive cells in the wild-type (WT) mice 1 week after the DDC feeding. However, the number of EpCAM-positive cells in the Clock-mutant (Clk/Clk) mice decreased, and their hepatitis was worse compared with the WT mice. In addition, the expression of Epcam mRNA, which is a functional marker of potential hepatic stem-like cells, was controlled by LEF1, which was regulated by CLOCK. The results of this study will facilitate the elucidation of the liver regeneration mechanisms, including those at the molecular level, and may assist in the development of new treatment modalities in the future.

Inhibition of G0/G1 Switch 2 Ameliorates Renal Inflammation in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G0/G1 switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.

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

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 thatIrp2mRNA 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(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) 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(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.