Polyploidy mitigates the impact of DNA damage while simultaneously bearing its burden.

Polyploidy is frequently enhanced under pathological conditions, such as tissue injury and cancer in humans. Polyploidization is critically involved in cancer evolution, including cancer initiation and the acquisition of drug resistance. However, the effect of polyploidy on cell fate remains unclear. In this study, we explored the effects of polyploidization on cellular responses to DNA damage and cell cycle progression. Through various comparisons based on ploidy stratifications of cultured cells, we found that polyploidization and the accumulation of genomic DNA damage mutually induce each other, resulting in polyploid cells consistently containing more genomic DNA damage than diploid cells under both physiological and stress conditions. Notably, despite substantial DNA damage, polyploid cells demonstrated a higher tolerance to its impact, exhibiting delayed cell cycle arrest and reduced secretion of inflammatory cytokines associated with DNA damage-induced senescence. Consistently, in mice with ploidy tracing, hepatocytes with high ploidy appeared to potentially persist in the damaged liver, while being susceptible to DNA damage. Polyploidy acts as a reservoir of genomic damage by mitigating the impact of DNA damage, while simultaneously enhancing its accumulation.

[Senescence and Cancer].

Cells experiencing significant stress such as irreparable genomic damage enter a state called cellular senescence, where their cell cycle is irreversibly arrested. Originally, cellular senescence was thought to serve as a crucial mechanism for suppressing carcinogenesis by stopping the growth of abnormal cells that could potentially lead to cancer development. However, it has become evident that senescence induced by treatments such as chemotherapy can contribute to drug resistance in cancer by exhibiting resistance to cell death and allowing cancer cells to survive. Furthermore, senescent cells exhibit a property known as the senescence-associated secretory phenotype(SASP), where they release a variety of inflammatory molecules and growth factors. Through these SASP factors influencing the tumor microenvironment, senescent cells can be deeply involved in various cancer pathologies such as carcinogenesis and the acquisition of drug resistance. This article provides an overview of the multifaceted and complex interplay between cellular senescence and the initiation and progression of cancer.

Impact of intratumoral microbiome on tumor immunity and prognosis in human pancreatic ductal adenocarcinoma.

BACKGROUND: Recent evidence suggests that the presence of microbiome within human pancreatic ductal adenocarcinoma (PDAC) tissue potentially influences cancer progression and prognosis. However, the significance of tumor-resident microbiome remains unclear. We aimed to elucidate the impact of intratumoral bacteria on the pathophysiology and prognosis of human PDAC. METHODS: The presence of intratumoral bacteria was assessed in 162 surgically resected PDACs using quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH) targeting 16S rRNA. The intratumoral microbiome was explored by 16S metagenome sequencing using DNA extracted from formalin-fixed paraffin-embedded tissues. The profile of intratumoral bacteria was compared with clinical information, pathological findings including tumor-infiltrating T cells, tumor-associated macrophage, fibrosis, and alterations in four main driver genes (KRAS, TP53, CDKN2A/p16, SMAD4) in tumor genomes. RESULTS: The presence of intratumoral bacteria was confirmed in 52 tumors (32%) using both qPCR and ISH. The 16S metagenome sequencing revealed characteristic bacterial profiles within these tumors, including phyla such as Proteobacteria and Firmicutes. Comparison of bacterial profiles between cases with good and poor prognosis revealed a significant positive correlation between a shorter survival time and the presence of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus. The abundance of these bacteria was correlated with a decrease in the number of tumor-infiltrating T cells positive for CD4, CD8, and CD45RO. CONCLUSIONS: Intratumoral infection of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus is correlated with the suppressed anti-PDAC immunity and poor prognosis.

Histological diagnosis of polyploidy discriminates an aggressive subset of hepatocellular carcinomas with poor prognosis.

BACKGROUND: Although genome duplication, or polyploidization, is believed to drive cancer evolution and affect tumor features, its significance in hepatocellular carcinoma (HCC) is unclear. We aimed to determine the characteristics of polyploid HCCs by evaluating chromosome duplication and to discover surrogate markers to discriminate polyploid HCCs. METHODS: The ploidy in human HCC was assessed by fluorescence in situ hybridization for multiple chromosomes. Clinicopathological and expression features were compared between polyploid and near-diploid HCCs. Markers indicating polyploid HCC were explored by transcriptome analysis of cultured HCC cells. RESULTS: Polyploidy was detected in 36% (20/56) of HCCs and discriminated an aggressive subset of HCC that typically showed high serum alpha-fetoprotein, poor differentiation, and poor prognosis compared to near-diploid HCCs. Molecular subtyping revealed that polyploid HCCs highly expressed alpha-fetoprotein but did not necessarily show progenitor features. Histological examination revealed abundant polyploid giant cancer cells (PGCCs) with a distinct appearance and frequent macrotrabecular-massive architecture in polyploid HCCs. Notably, the abundance of PGCCs and overexpression of ubiquitin-conjugating enzymes 2C indicated polyploidy in HCC and efficiently predicted poor prognosis in combination. CONCLUSIONS: Histological diagnosis of polyploidy using surrogate markers discriminates an aggressive subset of HCC, apart from known HCC subgroups, and predict poor prognosis in HCC.

Implications of Polyploidy and Ploidy Alterations in Hepatocytes in Liver Injuries and Cancers.

Polyploidy, a condition in which more than two sets of chromosomes are present in a cell, is a characteristic feature of hepatocytes. A significant number of hepatocytes physiologically undergo polyploidization at a young age. Polyploidization of hepatocytes is enhanced with age and in a diseased liver. It is worth noting that polyploid hepatocytes can proliferate, in marked contrast to other types of polyploid cells, such as megakaryocytes and cardiac myocytes. Polyploid hepatocytes divide to maintain normal liver homeostasis and play a role in the regeneration of the damaged liver. Furthermore, polyploid hepatocytes have been shown to dynamically reduce ploidy during liver regeneration. Although it is still unclear why hepatocytes undergo polyploidization, accumulating evidence has revealed that alterations in the ploidy in hepatocytes are involved in the pathophysiology of liver cirrhosis and carcinogenesis. This review discusses the significance of hepatocyte ploidy in physiological liver function, liver injury, and liver cancer.

The 6th international cell senescence association conference.

The 6th conference of the international cell senescence association (ICSA) on the theme of "A New Era of Senescence Research: The Challenge of Controlling Aging and Cancer" was held on December 12-15, 2021 in Osaka, Japan as a Hybrid Meeting. The conference brought together basic and translational scientists to discuss recent developments in the field of cellular senescence research. In recent years, the study of cellular senescence has become a very hot field of research. It is clear that the ICSA, founded in 2015, has played an important role in this process. The 6th ICSA conference has provided another opportunity for exchanges and new connections between basic and translational scientists. The scientific program consisted of keynote lectures, invited talks, short talks selected from abstracts, a poster session, and luncheon seminars sponsored by the Japanese Society of Anti-Aging Medicine. In the Meet the Editor session, Dr Christoph Schmitt, Editor-in-Chief of Nature Metabolism, gave a short presentation about the journal and answered questions from the audience. Being a hybrid meeting, there was only so much that could be done, but we hope that the meeting was fruitful.

Development and evaluation of a colorectal cancer screening method using machine learning-based gut microbiota analysis.

Accumulating evidence indicates that alterations of gut microbiota are associated with colorectal cancer (CRC). Therefore, the use of gut microbiota for the diagnosis of CRC has received attention. Recently, several studies have been conducted to detect the differences in the gut microbiota between healthy individuals and CRC patients using machine learning-based gut bacterial DNA meta-sequencing analysis, and to use this information for the development of CRC diagnostic model. However, to date, most studies had small sample sizes and/or only cross-validated using the training dataset that was used to create the diagnostic model, rather than validated using an independent test dataset. Since machine learning-based diagnostic models cause overfitting if the sample size is small and/or an independent test dataset is not used for validation, the reliability of these diagnostic models needs to be interpreted with caution. To circumvent these problems, here we have established a new machine learning-based CRC diagnostic model using the gut microbiota as an indicator. Validation using independent test datasets showed that the true positive rate of our CRC diagnostic model increased substantially as CRC progressed from Stage I to more than 60% for CRC patients more advanced than Stage II when the false positive rate was set around 8%. Moreover, there was no statistically significant difference in the true positive rate between samples collected in different cities or in any part of the colorectum. These results reveal the possibility of the practical application of gut microbiota-based CRC screening tests.

Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion.

Emerging evidence is revealing that alterations in gut microbiota are associated with colorectal cancer (CRC). However, very little is currently known about whether and how gut microbiota alterations are causally associated with CRC development. Here we show that 12 faecal bacterial taxa are enriched in CRC patients in two independent cohort studies. Among them, 2 Porphyromonas species are capable of inducing cellular senescence, an oncogenic stress response, through the secretion of the bacterial metabolite, butyrate. Notably, the invasion of these bacteria is observed in the CRC tissues, coinciding with the elevation of butyrate levels and signs of senescence-associated inflammatory phenotypes. Moreover, although the administration of these bacteria into ApcΔ14/+ mice accelerate the onset of colorectal tumours, this is not the case when bacterial butyrate-synthesis genes are disrupted. These results suggest a causal relationship between Porphyromonas species overgrowth and colorectal tumourigenesis which may be due to butyrate-induced senescence.

Proliferative polyploid cells give rise to tumors via ploidy reduction.

Polyploidy is a hallmark of cancer, and closely related to chromosomal instability involved in cancer progression. Importantly, polyploid cells also exist in some normal tissues. Polyploid hepatocytes proliferate and dynamically reduce their ploidy during liver regeneration. This raises the question whether proliferating polyploids are prone to cancer via chromosome missegregation during mitosis and/or ploidy reduction. Conversely polyploids could be resistant to tumor development due to their redundant genomes. Therefore, the tumor-initiation risk of physiologic polyploidy and ploidy reduction is still unclear. Using in vivo lineage tracing we here show that polyploid hepatocytes readily form liver tumors via frequent ploidy reduction. Polyploid hepatocytes give rise to regenerative nodules with chromosome aberrations, which are enhanced by ploidy reduction. Although polyploidy should theoretically prevent tumor suppressor loss, the high frequency of ploidy reduction negates this protection. Importantly, polyploid hepatocytes that undergo multiple rounds of cell division become predominantly mononucleated and are resistant to ploidy reduction. Our results suggest that ploidy reduction is an early step in the initiation of carcinogenesis from polyploid hepatocytes.

Hes1 Is Essential in Proliferating Ductal Cell-Mediated Development of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is frequently driven by aberrant KRAS activation and develops in the liver with chronic inflammation. Although the Notch signaling pathway is critically involved in ICC development, detailed mechanisms of Notch-driven ICC development are still unknown. Here, we use mice whose Notch signaling is genetically engineered to show that the Notch signaling pathway, specifically the Notch/Hes1 axis, plays an essential role in expanding ductular cells in the liver with chronic inflammation or oncogenic Kras activation. Activation of Notch1 enhanced the development of proliferating ductal cells (PDC) in injured livers, while depletion of Hes1 led to suppression. In correlation with PDC expansion, ICC development was also regulated by the Notch/Hes1 axis and suppressed by Hes1 depletion. Lineage-tracing experiments using EpcamcreERT2 mice further confirmed that Hes1 plays a critical role in the induction of PDC and that ICC could originate from PDC. Analysis of human ICC specimens showed PDC in nonneoplastic background tissues, confirming HES1 expression in both PDC and ICC tumor cells. Our findings provide novel direct experimental evidence that Hes1 plays an essential role in the development of ICC via PDC. SIGNIFICANCE: This study contributes to the identification of the cells of origin that initiate ICC and suggests that HES1 may represent a therapeutic target in ICC.

In Vivo Lineage Tracing of Polyploid Hepatocytes Reveals Extensive Proliferation during Liver Regeneration.

The identity of cellular populations that drive liver regeneration after injury is the subject of intense study, and the contributions of polyploid hepatocytes to organ regeneration and homeostasis have not been systematically assessed. Here, we developed a multicolor reporter allele system to genetically label and trace polyploid cells in situ. Multicolored polyploid hepatocytes undergo ploidy reduction and subsequent re-polyploidization after transplantation, providing direct evidence of the hepatocyte ploidy conveyor model. Marker segregation revealed that ploidy reduction rarely involves chromosome missegregation in vivo. We also traced polyploid hepatocytes in several different liver injury models and found robust proliferation in all settings. Importantly, ploidy reduction was seen in all injury models studied. We therefore conclude that polyploid hepatocytes have extensive regenerative capacity in situ and routinely undergo reductive mitoses during regenerative responses.

Comprehensive analysis of genetic aberrations linked to tumorigenesis in regenerative nodules of liver cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) recurrently develops in cirrhotic liver containing a number of regenerative nodules (RNs). However, the biological tumorigenic potential of RNs is still unclear. To uncover the molecular bases of tumorigenesis in liver cirrhosis, we investigated the genetic aberrations in RNs of cirrhotic tissues using next-generation sequencing. METHODS: We isolated 205 RNs and 7 HCC tissues from the whole explanted livers of 10 randomly selected patients who had undergone living-donor liver transplantation. Whole-exome sequencing and additional targeted deep sequencing on 30 selected HCC-related genes were conducted to reveal the mutational landscape of RNs and HCCs. RESULTS: Whole-exome sequencing demonstrated that RNs frequently harbored relatively high-abundance genetic alterations, suggesting a clonal structure of each RN in cirrhotic liver. The mutation signature observed in RNs was similar to those determined in HCC, characterized by a predominance of C>T transitions, followed by T>C and C>A mutations. Targeted deep sequencing analyses of RNs identified nonsynonymous low-abundance mutations in various tumor-related genes, including TP53 and ARID1A. In contrast, TERT promoter mutations were not detected in any of the RNs examined. Consistently, TERT expression levels in RNs were comparable to those in normal livers, whereas every HCC tissue demonstrated an elevated level of TERT expression. CONCLUSION: Analyses of RNs constructing cirrhotic liver indicated that a variety of genetic aberrations accumulate in the cirrhotic liver before the development of clinically and histologically overt HCC. These aberrations in RNs could provide the basis of tumorigenesis in patients with liver cirrhosis.

Expansion of viral variants associated with immune escape and impaired virion secretion in patients with HBV reactivation after resolved infection.

HBV reactivation could be induced under immunosuppressive conditions in patients with resolved infection. This study aimed to clarify the viral factors associated with the pathogenesis of HBV reactivation in association with the immunosuppressive status. Whole HBV genome sequences were determined from the sera of 24 patients with HBV reactivation, including 8 cases under strong immunosuppression mediated by hematopoietic stem cell transplantation (HSCT) and 16 cases without HSCT. Ultra-deep sequencing revealed that the prevalence of genotype B and the ratio of non-synonymous to synonymous evolutionary changes in the surface (S) gene were significantly higher in non-HSCT cases than in patients with HSCT. Those non-synonymous variants included immune escape (6/16 cases) and MHC class II-restricted T-cell epitope variants (6/16 cases). Furthermore, reactivated HBV in 11 of 16 (69%) non-HSCT cases possessed substitutions associated with impaired virion secretion, including E2G, L77R, L98V, T118K, and Q129H in the S region, and M1I/V in the PreS2 region. In conclusion, virologic features of reactivated HBV clones differed depending on the intensity of the immunosuppressive condition. HBV reactivation triggered by immunosuppressive conditions, especially those without HSCT, was characterized by the expansion of variants associated with immune escape, MHC class II-restricted T-cell epitope alterations, and/or impaired virion secretion.

Genetic features of multicentric/multifocal intramucosal gastric carcinoma.

Chronic gastritis caused by Helicobacter pylori (H. pylori) infection could lead to the development of gastric cancer. The finding that multiple gastric cancers can develop synchronously and/or metachronously suggests the development of field cancerization in chronically inflamed, H. pylori-infected gastric mucosa. The genetic basis of multiple tumorigenesis in the inflamed stomach, however, is not well understood. In this study, we analyzed the microsatellite instability (MSI) status and copy number aberrations (CNAs) of 41 multiple intramucosal early gastric cancers that synchronously or metachronously developed in 19 patients with H. pylori infection. Among the 41 intramucosal gastric carcinomas, 9 (22%) exhibited MSI, and the remaining 32 (78%) exhibited the microsatellite stable (MSS) phenotype. Metachronous multiple intramucosal gastric carcinoma exhibit inter-tumor heterogeneity by individually acquiring genetic aberrations. All synchronous multiple intramucosal gastric carcinoma pairs shared a common MSI/MSS profile, and CNA analysis revealed that synchronous multiple intramucosal gastric carcinoma pairs with the MSS phenotype shared common aberrations of representative tumor-suppressor genes, including focal deletion of APC, TP53, CDKN2A, and CDKN2B. Multiregional CNA analysis revealed that heterogeneous gene amplifications/deletions, including PDL1 amplification, evolved under the presence of shared "trunk" genetic alterations in a subpopulation of individual intramucosal gastric carcinomas. These data suggest that multiple gastric carcinomas develop in a multicentric/multifocal manner exhibiting features of inter- and intra-tumor heterogeneity in H. pylori-infected gastric mucosa, whereas synchronous multiple intramucosal gastric carcinomas could share partially common genetic alterations, possibly via common oncogenic pathways.

Proliferating EpCAM-Positive Ductal Cells in the Inflamed Liver Give Rise to Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) originates from regenerating liver cells with genetic alterations in chronically inflamed liver. Ductal cells and hepatocytes proliferate for liver regeneration, and proliferating ductal cells (PDC) derived from bile ductules have long been considered putative liver stem/progenitor cells and candidate cellular origins of HCC. The potential of PDC as tumor-originating cells, however, remains controversial in contrast to accumulating evidence that HCC originates from hepatocytes. Here, we demonstrate that PDCs expressing the established surface and cancer stem cell marker EpCAM give rise to HCC in inflamed liver. EpCAM-expressing PDCs were specifically labeled in newly developed EpcamCreERT2 mice and traced in a chemically induced liver injury model. Stepwise accumulation of genetic alterations in EpCAM-positive cells was induced by the mutagenesis activity of activation-induced cytidine deaminase using conditional transgenic mice. Lineage-tracing experiments revealed that labeled PDC differentiated into cholangiocytes, but not into hepatocytes, in the chemically damaged liver. Nevertheless, EpCAM-positive PDC with genetic alterations gave rise to HCC after 8 months of chemical administration. PDC-derived HCC showed histologic characteristics of concomitant ductule-like structures resembling human cholangiolocellular carcinoma (CLC) and exhibited serial transitions from PDC-like CLC cells to hepatocyte-like HCC cells. The Wnt signaling pathway was specifically upregulated in the CLC components of PDC-derived HCC. Our findings provide direct experimental evidence that EpCAM-expressing PDC could be a cellular origin of HCC, suggesting the existence of stem/progenitor-derived hepatocarcinogenesis. Cancer Res; 77(22); 6131-43. ©2017 AACR.

Activation of TNF-α-AID axis and co-inhibitory signals in coordination with Th1-type immunity in a mouse model recapitulating hepatitis B.

Hepatitis B virus (HBV) infection evokes host immune responses that primarily determine the outcome of HBV infection and the clinical features of HBV-associated liver disease. The precise mechanisms by which host factors restrict HBV replication, however, are poorly understood due to the lack of useful animal models that recapitulate immune responses to HBV. Here, we performed comprehensive immunologic gene expression profiling of the liver of a mouse model recapitulating anti-HBV immune response using a high sensitivity direct digital counting system. Anti-HBV cellular immunity with liver inflammation was elicited in mice hydrodynamically injected with a CpG-depleted plasmid encoding hepatitis B surface antigen (HBsAg) gene after preimmunization with HBsAg vaccine. Comprehensive expression analyses revealed the upregulation of Th1-associated genes including tumor necrosis factor (Tnf) and negative regulators of T cell function in the inflamed liver. Interestingly, activation-induced cytidine deaminase (Aicda, termed AID in humans), which reportedly suppresses HBV infection in vitro, was upregulated in hepatocytes in the course of anti-HBV immunity. Hepatocytic expression of Aicda in a Tnf-dependent manner was confirmed by the administration of Tnf antagonist into Aicda-tdTomato mice with anti-HBV immunity. Our findings suggest that activation of Tnf-Aicda axis and co-inhibitory signals to T cells in coordination with Th1-type immunity has critical roles in the immune response against HBV infection.

Sonazoid-enhanced ultrasonography guidance improves the quality of pathological diagnosis in the biopsy of focal hepatic lesions.

BACKGROUND/AIM: Contrast-enhanced ultrasonography (US) has improved the detection and characterization of focal hepatic lesions. Recently, the importance of obtaining high-quality samples in the biopsy of hepatic lesions has been increasing not only in the field of pathological diagnosis but also in molecular analysis for predicting the effectiveness of anticancer agents and molecular targeted drugs. We evaluated the utility of Sonazoid-enhanced ultrasonography (SEUS) in guiding percutaneous biopsy of focal hepatic lesions by comparing the results of histopathological diagnosis between B-mode US and SEUS guidance. METHODS AND MATERIALS: This retrospective study examined 121 focal hepatic lesions in 108 patients (mean age: 63.8 years) referred for US-guided percutaneous biopsy. The technical success rate was defined as the percentage of the lesions diagnosed clearly at the initial biopsy. RESULTS: Among 121 lesions, 56 lesions were subjected to biopsy with B-mode US guidance whereas 65 were subjected to SEUS guidance. The technical success rate was significantly higher under SEUS guidance than under B-mode US guidance (92.3 vs. 76.8%, respectively, P<0.05). When biopsies were performed to diagnose or rule out malignancy in indeterminate lesions, the technical success rate was also significantly higher under SEUS guidance than under B-mode US guidance (100 vs. 73.9%, respectively, P<0.05). SEUS guidance resulted in a significantly higher rate of successful single-puncture attempts compared with B-mode US guidance (55.4 vs. 35.7%, respectively, P<0.05). CONCLUSION: SEUS guidance is recommended for more accurate localization of suitable hepatic lesion biopsy areas as it increases conspicuity and differentiates viable areas from denaturalization or necrosis.

MSH2 Dysregulation Is Triggered by Proinflammatory Cytokine Stimulation and Is Associated with Liver Cancer Development.

Inflammation predisposes to tumorigenesis in various organs by potentiating a susceptibility to genetic aberrations. The mechanism underlying the enhanced genetic instability through chronic inflammation, however, is not clear. Here, we demonstrated that TNFα stimulation induced transcriptional downregulation of MSH2, a member of the mismatch repair family, via NF-κB-dependent miR-21 expression in hepatocytes. Liver cancers developed in ALB-MSH2(-) (/) (-)AID(+), ALB-MSH2(-) (/) (-), and ALB-AID(+) mice, in which MSH2 is deficient and/or activation-induced cytidine deaminase (AICDA) is expressed in cells with albumin-producing hepatocytes. The mutation signatures in the tumors developed in these models, especially ALB-MSH2(-) (/) (-)AID(+) mice, closely resembled those of human hepatocellular carcinoma. Our findings demonstrated that inflammation-mediated dysregulation of MSH2 may be a mechanism of genetic alterations during hepatocarcinogenesis. Cancer Res; 76(15); 4383-93. ©2016 AACR.