p53-dependent Nestin regulation links tumor suppression to cellular plasticity in liver cancer.

The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.

Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.

Author Correction: Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of SUV420H2-Dependent Chromatin Compaction Drives Right-Sided Colon Cancer Progression.

BACKGROUND & AIMS: Epigenetic processes regulating gene expression contribute markedly to epithelial cell plasticity in colorectal carcinogenesis. The lysine methyltransferase SUV420H2 comprises an important regulator of epithelial plasticity and is primarily responsible for trimethylation of H4K20 (H4K20me3). Loss of H4K20me3 has been suggested as a hallmark of human cancer due to its interaction with DNMT1. However, the role of Suv4-20h2 in colorectal cancer is unknown. METHODS: We examined the alterations in histone modifications in patient-derived colorectal cancer organoids. Patient-derived colorectal cancer organoids and mouse intestinal organoids were genetically manipulated for functional studies in patient-derived xenograft and orthotopic transplantation. Gene expression profiling, micrococcal nuclease assay, and chromatin immunoprecipitation were performed to understand epigenetic regulation of chromatin states and gene expression in patient-derived and mouse intestinal organoids. RESULTS: We found that reduced H4K20me3 levels occurred predominantly in right-sided patient-derived colorectal cancer organoids, which were associated with increased chromatin accessibility. Re-compaction of chromatin by methylstat, a histone demethylase inhibitor, resulted in reduced growth selectively in subcutaneously grown tumors derived from right-sided cancers. Using mouse intestinal organoids, we confirmed that Suv4-20h2-mediated H4K20me3 is required for maintaining heterochromatin compaction and to prevent R-loop formation. Cross-species comparison of Suv4-20h2-depleted murine organoids with right-sided colorectal cancer organoids revealed a large overlap of gene signatures involved in chromatin silencing, DNA methylation, and stemness/Wnt signaling. CONCLUSIONS: Loss of Suv4-20h2-mediated H4K20me3 drives right-sided colorectal tumorigenesis through an epigenetically controlled mechanism of chromatin compaction. Our findings unravel a conceptually novel approach for subtype-specific therapy of this aggressive form of colorectal cancer.

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFß, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

Synergistic targeting and resistance to PARP inhibition in DNA damage repair-deficient pancreatic cancer.

OBJECTIVE: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC). DESIGN: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy. RESULTS: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance. CONCLUSION: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice.

BACKGROUND & AIMS: Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. METHODS: We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. RESULTS: Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6ß4 into sEVs-a process that required CD82. CONCLUSIONS: Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Iron at the Interface of Hepatocellular Carcinoma.

Cancer incidence and mortality are rapidly growing, with liver cancer being the sixth most diagnosed cancer worldwide and the third leading cause of cancer death in 2020. A number of risk factors have been identified that trigger the progression to hepatocellular carcinoma. In this review, we focus on iron as a potential risk factor for liver carcinogenesis. Molecules involved in the regulation of iron metabolism are often upregulated in cancer cells, in order to provide a supply of this essential trace element for all stages of tumor development, survival, proliferation, and metastasis. Thus, cellular and systemic iron levels must be tightly regulated to prevent or delay liver cancer progression. Disorders associated with dysregulated iron metabolism are characterized with increased susceptibility to hepatocellular carcinoma. This review discusses the association of iron with metabolic disorders such as hereditary hemochromatosis, non-alcoholic fatty liver disease, obesity, and type 2 diabetes, in the background of hepatocellular carcinoma.

Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage.

Aging and carcinogenesis coincide with the accumulation of DNA damage and mutations in stem and progenitor cells. Molecular mechanisms that influence responses of stem and progenitor cells to DNA damage remain to be delineated. Here, we show that niche positioning and Wnt signaling activity modulate the sensitivity of intestinal stem and progenitor cells (ISPCs) to DNA damage. ISPCs at the crypt bottom with high Wnt/ß-catenin activity are more sensitive to DNA damage compared to ISPCs in position 4 with low Wnt activity. These differences are not induced by differences in cell cycle activity but relate to DNA damage-dependent activation of Wnt signaling, which in turn amplifies DNA damage checkpoint activation. The study shows that instructed enhancement of Wnt signaling increases radio-sensitivity of ISPCs, while inhibition of Wnt signaling decreases it. These results provide a proof of concept that cell intrinsic levels of Wnt signaling modulate the sensitivity of ISPCs to DNA damage and heterogeneity in Wnt activation in the stem cell niche contributes to the selection of ISPCs in the context of DNA damage.

YAP Activation Drives Liver Regeneration after Cholestatic Damage Induced by Rbpj Deletion.

Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.

Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling.

OBJECTIVE: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment. DESIGN: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny. RESULTS: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids. CONCLUSIONS: Taken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures.

Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation.

Telomere shortening limits the proliferative lifespan of human cells by activation of DNA damage pathways, including upregulation of the cell cycle inhibitor p21 (encoded by Cdkn1a, also known as Cip1 and Waf1)) (refs. 1-5). Telomere shortening in response to mutation of the gene encoding telomerase is associated with impaired organ maintenance and shortened lifespan in humans and in mice. The in vivo function of p21 in the context of telomere dysfunction is unknown. Here we show that deletion of p21 prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres. p21 deletion improved hematolymphopoiesis and the maintenance of intestinal epithelia without rescuing telomere function. Moreover, deletion of p21 rescued proliferation of intestinal progenitor cells and improved the repopulation capacity and self-renewal of hematopoietic stem cells from mice with dysfunctional telomeres. In these mice, apoptotic responses remained intact, and p21 deletion did not accelerate chromosomal instability or cancer formation. This study provides experimental evidence that telomere dysfunction induces p21-dependent checkpoints in vivo that can limit longevity at the organismal level.

Telomere length in mantle cell lymphoma.

Telomere shortening is of pathogenic and prognostic importance in cancers. In the present study, we analyzed telomere length in 73 mantle cell lymphoma (MCL), 55 chronic lymphocytic leukemia (CLL), and 20 normal B-cell samples using quantitative PCR (Q-PCR) to study its association with disease characteristics and outcome. Telomere length was found to be highly variable in MCL (range, 2.2-13.8 kb; median, 4.3 kb). Telomere dysfunction in MCL was evident from comparison with normal B cells (median, 7.5 kb), but had no significant association with any biologic or clinical feature. This was in contrast to CLL, in which a significant correlation of short telomeres with poor prognostic subgroups was confirmed. There was a trend toward an increased number of genomic aberrations with shortening of telomeres in MCL. No difference in survival was observed between the groups with short and long telomeres, indicating that, as opposed to CLL, telomere length is not of prognostic relevance in MCL.

p21 promotes sustained liver regeneration and hepatocarcinogenesis in chronic cholestatic liver injury.

BACKGROUND AND AIMS: The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. METHODS: The degree of liver injury, regeneration and tumour formation was assessed in Mdr2(-/-) mice and compared with Mdr2/ p21(-/-) mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). RESULTS: Mdr2(-/-) mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21(-/-) mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21(-/-) mice. Moreover, the regenerative capacity of the Mdr2/ p21(-/-) livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21(-/-) microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. CONCLUSIONS: We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.

Disruption of Trp53 in livers of mice induces formation of carcinomas with bilineal differentiation.

BACKGROUND & AIMS: p53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. METHODS: We used AlfpCre mice to create mice with liver-specific disruption of Trp53 (AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice). We analyzed colony formation and genomic features and gene expression patterns in liver cells during hepatocarcinogenesis in mice with homozygous, heterozygous, and no disruption of Trp53. RESULTS: Liver-specific disruption of Trp53 consistently induced formation of liver carcinomas that had bilineal differentiation. In nontransformed liver cells and cultured primary liver cells, loss of p53 (but not p21) resulted in chromosomal imbalances and increased clonogenic capacity of liver progenitor cells (LPCs) and hepatocytes. Primary cultures of hepatocytes and LPCs from AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice, but not Cdkn1a(-/-) mice, formed tumors with bilineal differentiation when transplanted into immunocompromised mice. Spontaneous liver tumors that developed in AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice had significant but complex alterations in expression of Rb checkpoint genes compared with chemically induced liver tumors that developed mice with wild-type Trp53. CONCLUSIONS: Deletion of p53 from livers of mice is sufficient to induce tumor formation. The tumors have bilineal differentiation and dysregulation of Rb checkpoint genes.