Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Trends and Characteristics in Health Care-related Deaths Investigated through Medico-legal Autopsies after System Changes in Japan.

In Japan, a new cause-of-death investigation system and related new laws were enacted in the mid-2010s. These laws provided for an autopsy system for non-criminal unnatural deaths and a medical accident investigation system outside the criminal justice process for health care-related deaths. We retrospectively explored changes in the number and characteristics of medico-legal autopsy cases of health care-related deaths in Chiba Prefecture, Japan, and examined trends over time during these reforms. We found that the percentage of forensic autopsies based on the Code of Criminal Procedure for health care-related deaths had decreased significantly. The number of autopsies of accidental and unintentional deaths in nursing homes, which are not covered by the newly established medical accident investigation system, has been increasing, reflecting the ageing of society. The trend toward decriminalisation of health care-related deaths was expected to contribute more to medical safety if the scope was expanded and a system for disclosure of autopsy information was established.

GPR30-Expressing Gastric Chief Cells Do Not Dedifferentiate But Are Eliminated via PDK-Dependent Cell Competition During Development of Metaplasia.

BACKGROUND & AIMS: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells. METHODS: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-KrasG12D, LSL-KrasG12D, and LSL-HrasG12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition. RESULTS: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30+ chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl+ clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion. CONCLUSIONS: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells.

Tumor microenvironment in gastric cancers.

The tumor microenvironment favors the growth and expansion of cancer cells. Many cell types are involved in the tumor microenvironment such as inflammatory cells, fibroblasts, nerves, and vascular endothelial cells. These stromal cells contribute to tumor growth by releasing various molecules to either directly activate the growth signaling in cancer cells or remodel surrounding areas. This review introduces recent advances in findings on the interactions within the tumor microenvironment such as in cancer-associated fibroblasts (CAFs), immune cells, and endothelial cells, in particular those established in mouse gastric cancer models. In mice, myofibroblasts in the gastric stroma secrete R-spondin and support normal gastric stem cells. Most CAFs promote tumor growth in a paracrine manner, but CAF population appears to be heterogeneous in terms of their function and origin, and include both tumor-promoting and tumor-restraining populations. Among immune cell populations, tumor-associated macrophages, including M1 and M2 macrophages, and myeloid-derived suppressor cells (MDSCs), are reported to directly or indirectly promote gastric tumorigenesis by secreting soluble factors or modulating immune responses. Endothelial cells or blood vessels not only fuel tumors with nutrients, but also interact with cancer stem cells and immune cells by secreting chemokines or cytokines, and act as a cancer niche. Understanding these interactions within the tumor microenvironment would contribute to unraveling new therapeutic targets.

BHLHA15-Positive Secretory Precursor Cells Can Give Rise to Tumors in Intestine and Colon in Mice.

BACKGROUND & AIMS: The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. METHODS: We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. RESULTS: Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors toward those of ISCs. Bhlha15+ enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. CONCLUSIONS: In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs.

A Descriptive Study of the Characteristics of Homicide-Suicide in Forensic Autopsy Cases.

Homicide followed by suicide (HS) is a tragic event with varied characteristics across countries and regions. Compared to Western countries, there are limited studies on HS in Asian countries. Therefore, this study aimed to clarify the characteristics of recent HS cases by examining forensic autopsy records from 2008 to 2020 collected from the Department of Legal Medicine, Chiba University, in Japan. A total of 77 HS cases were identified, involving 77 perpetrators (52 completed suicides, 25 attempted suicides), with 28 perpetrator and 89 victim autopsies. Our findings showed that older adults accounted for nearly half of the victims; victims were mostly females, whereas most perpetrators were male. The most common HS relationship was that between a parent and a child. Autopsy findings showed that the most common cause of death was strangulation, and illegal drugs were detected only in a few cases; however, psychotropic drugs were detected in child victims. No obvious evidence of past child physical abuse by caregivers was found. In contrast, intimate partner violence (IPV) was present, with a history of IPV found in half of HS cases involving adult intimate partner relationships. Notably, gender differences in age and relationship to the victim were identified. Likewise, some perpetrators may have expressed their plans and intentions for HS before the event, which may represent an important sign for HS prevention. However, to accurately reveal the course of HS, nationwide integrated statistics, forensic autopsies, including toxicological analyses of the deceased; and forensic psychiatric perspectives, including psychological autopsy, are required.

Sustained Clinical Complete Response after Discontinuation of Trastuzumab-deruxetecan Due to Interstitial Pneumonia for HER2-positive Gastric adenocarcinoma with Enteroblastic Differentiation (GAED): A Case Report.

Trastuzumab deruxtecan (T-DXd) has demonstrated remarkable efficacy as a third- or later-line chemotherapy for human epidermal growth factor receptor 2 (HER2)-positive advanced gastric and gastroesophageal junction adenocarcinomas. However, it may cause pneumonitis, and its efficacy in rare histologies such as gastric adenocarcinoma with enteroblastic differentiation (GAED) remains unclear. A 74-year-old woman with unresectable HER2-positive GAED and lung metastasis received T-DXd as a fifth-line chemotherapy. Treatment was discontinued after 15 cycles owing to drug-induced pneumonitis; however, the patient achieved a sustained complete response for 14 months without subsequent chemotherapy or the exacerbation of pneumonitis. T-DXd was effective in HER2-positive GAED.

Occupational accidental injury deaths in Tokyo and Chiba prefectures, Japan: A 10-year study (2011-2020) of forensic institute evaluations.

Occupational accidental injury deaths (OAIDs) are a major social problem, and the analysis of individual cases is important for developing injury prevention measures. In this study, OAIDs with autopsies performed at forensic facilities in the metropolitan area of Japan (Tokyo and Chiba prefectures) from 2011 to 2020 were reviewed. The epidemiological characteristics of these OAIDs (n = 136), which accounted for 13.5% of OAIDs reported in the region during the study period, were compared with those of non-occupational accidental injury deaths (non-OAID) cases (n = 3926). Among OAID cases, 134 (98.5%) were men and 13 (9.6%) were foreign-born workers, which was significantly more than in non-OAID cases (p < 0.001, respectively). OAIDs were most frequent in construction (39.0%) followed by the manufacturing category (21.3%). The percentage of OAIDs in workers aged 65 and over showed an increasing trend. Most accidents occurred just after the start of work or just before the workday ended, as well as during the peak months of the year. The most common type of accident was fall/crash from a height (25.0%), and the most common injury site was the chest; none of these cases were confirmed to have been wearing a safety belt properly. Among foreign-born workers, the most common type of accident was caught in/between. As the working population is expected to change in the future, and an increase in the number of older adults and foreign workers is expected, it is necessary to take preventive measures such as improving the work environment based on ergonomics and providing safety education.

Dysregulated Immune Responses by ASK1 Deficiency Alter Epithelial Progenitor Cell Fate and Accelerate Metaplasia Development during H. pylori Infection.

The mechanism of H. pylori-induced atrophy and metaplasia has not been fully understood. Here, we demonstrate the novel role of Apoptosis signal-regulating kinase 1 (ASK1) and downstream MAPKs as a regulator of host immune responses and epithelial maintenance against H. pylori infection. ASK1 gene deficiency resulted in enhanced inflammation with numerous inflammatory cells including Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs) recruited into the infected stomach. Increase of IL-1ß release from apoptotic macrophages and enhancement of TH1-polarized immune responses caused STAT1 and NF-κB activation in epithelial cells in ASK1 knockout mice. Dysregulated immune and epithelial activation in ASK1 knockout mice led to dramatic expansion of gastric progenitor cells and massive metaplasia development. Bone marrow transplantation experiments revealed that ASK1 in inflammatory cells is critical for inducing immune disorder and metaplastic changes in epithelium, while ASK1 in epithelial cells regulates cell proliferation in stem/progenitor zone without changes in inflammation and differentiation. These results suggest that H. pylori-induced immune cells may regulate epithelial homeostasis and cell fate as an inflammatory niche via ASK1 signaling.

Age estimation from ossification of sternum and true ribs using 3D post-mortem CT images in a Japanese population.

Age estimation of cadavers from post-mortem "chest plate" using conventional radiography, which involves radiographic assessment of ossification around the sternum and rib ends, has been evaluated without fruitful results. This study examined the value of images of the chest plate obtained by three-dimensional post-mortem CT for estimation of age at time of death in a Japanese population. Five chest plate ossification scores were evaluated in 320 subjects, including ossification of the first costal cartilage (OF), ossification of the second to seventh costal cartilages at the rib (OR) and sternal (OS) ends, fusion of the manubriosternal joint (FM), and fusion of the xiphisternal joint (FX). OS was found to have the highest correlation with age while FM had no significant correlation. The best composite score for age estimation was the summative score for both sides of the OS and the right side of the OF and FX, for which the coefficient of determination (R2) and the standard error of estimation (SEE) were 0.608 and 12.44 years, respectively, for men and 0.590 and 14.65 years for women. The accuracy of the model was tested in a further 26 male and 24 female subjects, and the accuracy rate within the first SEE was 57.69% and 70.83%, respectively. This rapid and non-invasive method of age estimation in the chest plate area is superior to conventional methods and could be useful for estimation of age at time of death in the Japanese population.

[Fall risk assessment and knee extensor muscle activity in elderly people].

PURPOSE: The purpose of this study was to analyze relationships between the history of falls, tripping, sway, and knee extensor muscle strengths as a tool for fall risk assessment in elderly people. We examined effective fall prevention measures. METHODS: We investigated 102 elderly volunteers in the community. The subjects were classified according to history of falls, tripping, sway and 5 performance tests conducted to assess fall risk including Timed up-and-go test (TUG), Functional Reach test (FR), Hand grip and Reaction time (RT). In addition, the time serial data of the knee extensor muscle strength were acquired using a hand-held dynamometer. RESULTS: In comparison to the non-faller group, the faller group showed a significantly higher incident rate of tripping and sway. A frequency analysis using the Maximum Entropy Method revealed that the fallers group showed lower peak frequency (p=0.025). Also, the slope of the logarithmical spectrum was less steep in the fallers group (p=0.035). Also results from analysis of the peak force latency from the beginning of measurement to 50%, 80%, and 100% muscle strength, also showed that the faller group took more time for maximal voluntary contraction. CONCLUSIONS: The frequency analysis of the time series date of peak force latency of knee extensor muscle strength revealed that the muscle activity differs in faller compared to non-fallers. This study suggested that knee extensor muscle isometric performance could possibly be used as a new tool for fall risk assessment. We concluded that exercises to raise maximal muscle strength and muscle response speed are useful for the prevention of falls.

Inhibition of TNFα-interacting protein α (Tipα)-associated gastric carcinogenesis by BTG2/TIS21 via downregulating cytoplasmic nucleolin expression.

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Down-regulation of histone deacetylase 4, -5 and -6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin.

(-)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, -5 and -6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and -5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, -5 and -6 and stimulates apoptotic induction.

Expression patterns of class I KNOX and YABBY genes in Ruscus aculeatus (Asparagaceae) with implications for phylloclade homology.

STM (RaSTM) and YAB2 (RaYAB2) homologues were isolated from Ruscus aculeatus (Asparagaceae, monocots), and their expressions were analyzed by real-time polymerase chain reaction (PCR) to assess hypotheses on the evolutionary origin of the phylloclade in the Asparagaceae. In young shoot buds, RaSTM is expressed in the shoot apex, while RaYAB2 is expressed in the scale leaf subtending the shoot bud. This expression pattern is shared by other angiosperms, suggesting that the expression patterns of RaSTM and RaYAB2 are useful as molecular markers to identify the shoot and leaf, respectively. RaSTM and RaYAB2 are expressed concomitantly in phylloclade primordia. These results suggest that the phylloclade is not homologous to either the shoot or leaf, but that it has a double organ identity.