Structral And Biochemical Insights Into The Transition From Transcription Initiation To Elongation

Transcription by RNA polymerase II (Pol II) is a complex process that requires timely and coordinated regulation at multiple steps for proper gene expression. Initiation is the first step in transcription and decades of biochemical and genome-wide studies have identified proteins involved in the process and revealed their functions. Additionally, technological advancements in cryo-EM enabled researchers to visualize initiation complexes and provide mechanistic insights into initiation processes in the last several years. However, the mechanistic understanding of the transition from transcription initiation to elongation has been limited in part due to the lack of an efficient transcription initiation system in vitro. We purified yeast general transcription factors (GTFs: TFIIA, TFIIB, TBP(a component of TFIID), TFIIE, TFIIF, and TFIIH) and Pol II, all of which are necessary and sufficient for basal transcription initiation, and optimized the initiation system. Using this system, we biochemically re-examined effects of two elongation factors (Cet1-Ceg1 and Spt4/5) on promoter escape, a process in which Pol II dissociates from GTFs except TFIIF for elongation. We find that inclusion of these elongation factors has positive effects on promoter escape. Furthermore, we took advantage of our efficient system, and generated and isolated post-initiation complexes in vitro for structural characterization by cryo-EM. Our structure of the initially-transcribing complex (ITC) stalled +26 shows a large conformational change of TFIIH in the way that it is much closer to TFIIE than in the pre-initiation complex (PIC) and it loses contacts with Pol II. These changes most likely prime for Pol II to escape the promoter. In addition, the structural studies of post-initiation complex stalled +49 reveal two elongation complexes (ECs) colliding to each other as well as show the presence of EC+ITC. In the structure the colliding ECs, the trailing EC contained RNA of ~25 nt in length but has backtracked by ~10 nt upon colliding. These studies together provide a model of the process of promoter escape, where TFIIH can get kicked out by the preceding promoter-proximal EC

AKT Plays a Crucial Role in Gastric Cancer.

The AKT protein is involved in the phosphatidylinositol-3 kinase signaling pathway and is a vital regulator of survival. proliferation, and differentiation of various types of cells. Helicobacter pylori induce epithelial cell proliferation and oxidative stress in chronic gastritis. These alterations lead to telomere shortening and resultant telomerase activation. Specifically, AKT is activated by H. pylori-induced inflammation; it subsequently promotes expression of human telomerase reverse transcriptase, which encodes a catalytic subunit of telomerase; and induces telomerase activity, which is an essential process of carcinogenesis. AKT activation is increased in gastric mucosa with carcinogenic properties and is associated with low survival of patients with gastric cancer. These findings suggest that AKT is pivotal in gastric carcinogenesis and progression

Protumoral Effect of Angiotensin System.

Colorectal cancer (CRC) cells possess an angiotensin activation mechanism provided by the expression of renin and chymase. Renin expression is induced by a hyperglycemic condition. Since angiotensinogen is produced in the liver, CRC cells with angiotensin-activating machinery possess an advantage to metastasize to the liver. In human CRC cases, the diabetes-complicated patients show higher concentrations of renin and angiotensin-Ⅱ in the primary tumors, and a more progressed disease stage, especially, liver metastasis in association with HbA1c levels than those in the patients without diabetes. Concurrent treatment with anti-angiotensin and hypoglycemic agents shows a synergic effect of decreasing liver metastasis and improving the survival of diabetic mice in the CRC liver metastasis model. MAS1-angiotensin1-7 is a negative regulator of the AGTR1-angiotensin Ⅱ axis in breast cancer. Notably, MAS1 is overexpressed in triple negative breast cancer, which might be a novel molecular target for the treatment-refractory entity of breast cancer. Nuclear AGTR2 and intracellular angiotensin-Ⅱ play a role in anti-apoptotic and anti-oxidative stress properties. These functions of nuclear AGTR2 might mitigate "anti-tumoral side effects" of AGTR1 and angiotensin-Ⅱ system, which enhance mainly tumor progression. The effect of anti-angiotensin treatment such as ARB and blood sugar control as ab aseline management of many cancer patients needs to be examined in a clinical situation for prevention of RAS-induced tumor progression

Role of CD10 in the Metastasis of Colorectal Cancer to the Liver.

CD10 is a widely expressed endopeptidase that is present in human colorectal cancer (CRC), which shows a high frequency of liver metastasis. CD10 expression in CRC cells is associated with liver metastasis in rodent models, and CD10 expression enhances the phosphorylation of epidermal growth factor (EGF) receptor (EGFR) and extracellular signalregulated kinase (ERK) l/2. Met-enkephalin (MENK), a CD10 substrate, activates its specific receptor δ-opioid receptor (DOR), which is expressed in CRCs. DOR is a partial agonist of ERK1/2, which suppresses EGF-induced phosphorylation of EGFR and ERK1/2. CD10 retains EGF-induced EGFR activation by degrading MENK. Paradoxically, CRCs express MENK at a high frequency. Since MENK suppresses T lymphocytes, CD10-expressing CRCs can escape from T-cell immunity without exhibiting auto-inhibition. CD10 is strongly associated with the metastasis of CRCs to the liver via an immunosuppressive mechanism. Additionally, CD10 may be an excellent serum marker for liver metastasis in patients with CRC and could represent a potential molecular target for antimetastatic treatment in patients with CRC

胃癌におけるクローディン4標的化によるシスプラチン化学療法感受性の向上

Claudins are major tight-junction proteins that mediate cellular polarity and differentiation. The present study investigated whether the 4D3 antibody to the human CLDN4 extracellular domain (that we previously established) is capable of modulating chemotherapeutic sensitivity in gastric cancer (GC). The results of the present study showed that CLDN4 was overexpressed in 137 of the 192 analyzed GC cases, and that CLDN4 expression was retained in tumors of a lower histological grade (more differentiated), and/or those that were caudal-type homeobox protein 2 (CDX2)-positive, but was reduced in more highly undifferentiated, and CDX2-negative GC cases. The study also compared the synergic effects of combining 4D3 with CDDP treatment and knocking down CLDN4 expression in MKN74 and TMK-1 human GC cells. Co-treatment with 4D3 increased anti-tumor effects of CDDP, whereas CLDN4 knockdown did not. In the TMK-1 cells, non-tight junction CLDN4 associated with integrin β1, increasing stem cell-associated proteins via FAK-c-SRC signals. The anti-tumoral effect of CDDP and 4D3 was examined in a nude mouse subcutaneous tumor model. In the two GC cell lines, concurrent treatment with 4D3 and CDDP synergistically inhibited cell proliferation and increased tumor necrosis and apoptosis to a greater degree than CDDP treatment alone. These findings suggest that 4D3 might increase chemotherapeutic sensitivity by evoking structural disintegration of tight-junction CLDN4 expressed in gastric cancer.博士（医学）・甲第713号・令和元年6月26日Copyright: Nishiguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0 https://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

酸化型HMGB-1は間葉系幹細胞/間葉系細胞を介して大腸癌の転移性を促進する

High mobility group box-1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post-translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three-fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM-MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM-MSCs pretreated with oxidized HMGB1 were co-cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co-culture with reduced HMGB1-pretreated BM-MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1–MSC axis may be an important target for cancer therapy.博士（医学）・甲第874号・令和5年3月15

中鎖脂肪酸と糖質の併用摂取は癌関連骨格筋萎縮から保護する

Skeletal muscle volume is associated with prognosis of cancer patients. Maintenance of skeletal muscle is an essential concern in cancer treatment. In nutritional intervention, it is important to focus on differences in metabolism between tumor and skeletal muscle. We examined the influence of oral intake of glucose (0%, 10%, 50%) and 2% medium-chain fatty acid (lauric acid, LAA, C12:0) on tumor growth and skeletal muscle atrophy in mouse peritoneal metastasis models using CT26 mouse colon cancer cells and HT29 human colon cancer cells. After 2 weeks of experimental breeding, skeletal muscle and tumor were removed and analyzed. Glucose intake contributed to prevention of skeletal muscle atrophy in a sugar concentration-dependent way and also promoted tumor growth. LAA ingestion elevated the level of skeletal muscle protein and suppressed tumor growth by inducing tumor-selective oxidative stress production. When a combination of glucose and LAA was ingested, skeletal muscle mass increased and tumor growth was suppressed. Our results confirmed that although glucose is an important nutrient for the prevention of skeletal muscle atrophy, it may also foster tumor growth. However, the ingestion of LAA inhibited tumor growth, and its combination with glucose promoted skeletal muscle integrity and function, without stimulating tumor growth. These findings suggest novel strategies for the prevention of skeletal muscle atrophy.博士（医学）・甲第733号・令和2年3月16日© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License(https://creativecommons.org/licenses/by-nc/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes

膀胱癌における抗クローディン4細胞外ドメイン中和抗体の化学療法増感効果

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.博士（医学）・甲第649号・平成28年3月15日Copyright © 2015 Elsevier Ireland Ltd. All rights reserved

Cyp27c1 red-shifts the spectral sensitivity of photoreceptors by converting Vitamin A₁ into A₂

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A(1) (the precursor of 11-cis retinal) into vitamin A(2) (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A(2), eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity, and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A(1) and A(2) in the eye

Structral and Biochemical Insights Into the Transition from Transcription Initiation to Elongation

Transcription by RNA polymerase II (Pol II) is a complex process that requires timely and coordinated regulation at multiple steps for proper gene expression. Initiation is the first step in transcription and decades of biochemical and genome-wide studies have identified proteins involved in the process and revealed their functions. Additionally, technological advancements in cryo-EM enabled researchers to visualize initiation complexes and provide mechanistic insights into initiation processes in the last several years. However, the mechanistic understanding of the transition from transcription initiation to elongation has been limited in part due to the lack of an efficient transcription initiation system in vitro. We purified yeast general transcription factors (GTFs: TFIIA, TFIIB, TBP(a component of TFIID), TFIIE, TFIIF, and TFIIH) and Pol II, all of which are necessary and sufficient for basal transcription initiation, and optimized the initiation system. Using this system, we biochemically re-examined effects of two elongation factors (Cet1-Ceg1 and Spt4/5) on promoter escape, a process in which Pol II dissociates from GTFs except TFIIF for elongation. We find that inclusion of these elongation factors has positive effects on promoter escape. Furthermore, we took advantage of our efficient system, and generated and isolated post-initiation complexes in vitro for structural characterization by cryo-EM. Our structure of the initially-transcribing complex (ITC) stalled +26 shows a large conformational change of TFIIH in the way that it is much closer to TFIIE than in the pre-initiation complex (PIC) and it loses contacts with Pol II. These changes most likely prime for Pol II to escape the promoter. In addition, the structural studies of post-initiation complex stalled +49 reveal two elongation complexes (ECs) colliding to each other as well as show the presence of EC+ITC. In the structure the colliding ECs, the trailing EC contained RNA of ~25 nt in length but has backtracked by ~10 nt upon colliding. These studies together provide a model of the process of promoter escape, where TFIIH can get kicked out by the preceding promoter-proximal EC