PAF remodels the DREAM complex to bypass cell quiescence and promote lung tumorigenesis.

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion.

BACKGROUND & AIMS: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown. METHODS: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids. Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing. Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC single-cell RNA sequencing data sets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion. RESULTS: We established 32 genetically engineered esophageal organoids and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN knockout also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via nuclear factor-κB. Moreover, comparative single-cell transcriptomic analyses stratified patients with ESCC and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. CONCLUSIONS: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.

TMEM9-v-ATPase Activates Wnt/ß-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.

A Pilot Study of Machine Learning-Based Algorithms to Assist Integrated Care for Older Community-Dwelling Adults.

As life expectancy increases, there is a growing consensus on the development of integrated care encompassing the health and daily activities of older adults. In recent years, although the demand for machine learning applications in healthcare has increased, only a few studies have implemented machine learning-based systems in integrated care for older adults owing to the complex needs of older adults and the coarseness of the available data. Our study aims to explore the possibility of implementing machine learning decision-support algorithms in the integrated care of older community-dwelling adults. Our experiment uses secondary data based on the community-based integrated service model. Such data were collected from 511 older adults through 162 assessment items in which tailored services were selected from 18 available services. We implemented four machine learning models: decision tree, random forest, K-nearest neighbors, and multilayer perceptron. The area under the receiver operating characteristic curve results of the four models were decision tree = 0.89, K-nearest neighbors = 0.88, random forest = 0.93, and multilayer perceptron = 0.88. The results suggest that machine learning-based decision-assisting algorithms can improve the quality of tailored services for integrated care with intensive involvement of face-to-face tasks by reducing the simple, repetitive tasks of care managers.

PAF and EZH2 induce Wnt/ß-catenin signaling hyperactivation.

Fine control of Wnt signaling is essential for various cellular and developmental decision-making processes. However, deregulation of Wnt signaling leads to pathological consequences, one of which is cancer. Here, we identify a function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells and is required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing a secondary axis with ß-catenin target gene upregulation. Upon Wnt signaling activation, PAF dissociates from PCNA and binds directly to ß-catenin. Then, PAF recruits EZH2 to the ß-catenin transcriptional complex and specifically enhances Wnt target gene transactivation, independently of EZH2's methyltransferase activity. In mice, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling and propose a regulatory mechanism of Wnt signaling during tumorigenesis.

Telomerase modulates Wnt signalling by association with target gene chromatin.

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.

Dyrk2-associated EDD-DDB1-VprBP E3 ligase inhibits telomerase by TERT degradation.

Telomerase maintains the telomere, a specialized chromosomal end structure that is essential for genomic stability and cell immortalization. Telomerase is not active in most somatic cells, but its reactivation is one of the hallmarks of cancer. In this study, we found that dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (Dyrk2) negatively regulates telomerase activity. Dyrk2 phosphorylates TERT protein, a catalytic subunit of telomerase. Phosphorylated TERT is then associated with the EDD-DDB1-VprBP E3 ligase complex for subsequent ubiquitin-mediated TERT protein degradation. During the cell cycle, Dyrk2 interacts with TERT at the G2/M phase and induces degradation. In contrast, depletion of endogenous Dyrk2 disrupts the cell cycle-dependent regulation of TERT and elicits the constitutive activation of telomerase. Similarly, a Dyrk2 nonsense mutation identified in breast cancer compromises ubiquitination-mediated TERT protein degradation. Our findings suggest the novel molecular mechanism of kinase-associated telomerase regulation.

HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.

Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

Tumor niche network-defined subtypes predict immunotherapy response of esophageal squamous cell cancer.

Despite the promising outcomes of immune checkpoint inhibitors (ICIs), resistance to ICI presents a new challenge. Therefore, selecting patients for specific ICI applications is crucial for maximizing therapeutic efficacy. Herein, we curated 69 human esophageal squamous cell cancer (ESCC) patients' tumor microenvironment (TME) single-cell transcriptomic datasets to subtype ESCC. Integrative analyses of the cellular network and transcriptional signatures of T cells and myeloid cells define distinct ESCC subtypes characterized by T cell exhaustion, and interleukin (IL) and interferon (IFN) signaling. Furthermore, this approach classifies ESCC patients into ICI responders and non-responders, as validated by whole tumor transcriptomes and liquid biopsy-based single-cell transcriptomes of anti-PD-1 ICI responders and non-responders. Our study stratifies ESCC patients based on TME transcriptional network, providing novel insights into tumor niche remodeling and potentially predicting ICI responses in ESCC patients.

CRACD loss induces neuroendocrine cell plasticity of lung adenocarcinoma.

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, some lung adenocarcinoma (LUAD) cells transform into neuroendocrine (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD (capping protein inhibiting regulator of actin dynamics), a capping protein inhibitor, is frequently inactivated in cancers. CRACD knockout (KO) is sufficient to de-repress NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE cell plasticity is associated with cell de-differentiation and stemness-related pathway activation. The single-cell transcriptomic analysis of LUAD patient tumors recapitulates that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with impaired actin remodeling. This study reveals the crucial role of CRACD in restricting NE cell plasticity that induces cell de-differentiation of LUAD.

E-cadherin loss drives diffuse-type gastric tumorigenesis via EZH2-mediated reprogramming.

Diffuse-type gastric adenocarcinoma (DGAC) is a deadly cancer often diagnosed late and resistant to treatment. While hereditary DGAC is linked to CDH1 mutations, the role of CDH1/E-cadherin inactivation in sporadic DGAC tumorigenesis remains elusive. We discovered CDH1 inactivation in a subset of DGAC patient tumors. Analyzing single-cell transcriptomes in malignant ascites, we identified two DGAC subtypes: DGAC1 (CDH1 loss) and DGAC2 (lacking immune response). DGAC1 displayed distinct molecular signatures, activated DGAC-related pathways, and an abundance of exhausted T cells in ascites. Genetically engineered murine gastric organoids showed that Cdh1 knock-out (KO), KrasG12D, Trp53 KO (EKP) accelerates tumorigenesis with immune evasion compared with KrasG12D, Trp53 KO (KP). We also identified EZH2 as a key mediator promoting CDH1 loss-associated DGAC tumorigenesis. These findings highlight DGAC's molecular diversity and potential for personalized treatment in CDH1-inactivated patients.

Tumor Niche Network-Defined Subtypes Predict Immunotherapy Response of Esophageal Squamous Cell Cancer.

Despite the promising outcomes of immune checkpoint blockade (ICB), resistance to ICB presents a new challenge. Therefore, selecting patients for specific ICB applications is crucial for maximizing therapeutic efficacy. Herein we curated 69 human esophageal squamous cell cancer (ESCC) patients' tumor microenvironment (TME) single-cell transcriptomic datasets to subtype ESCC. Integrative analyses of the cellular network transcriptional signatures of T cells, myeloid cells, and fibroblasts define distinct ESCC subtypes characterized by T cell exhaustion, Interferon (IFN) a/b signaling, TIGIT enrichment, and specific marker genes. Furthermore, this approach classifies ESCC patients into ICB responders and non-responders, as validated by liquid biopsy single-cell transcriptomics. Our study stratifies ESCC patients based on TME transcriptional network, providing novel insights into tumor niche remodeling and predicting ICB responses in ESCC patients.

CDH1 loss promotes diffuse-type gastric cancer tumorigenesis via epigenetic reprogramming and immune evasion.

Diffuse-type gastric adenocarcinoma (DGAC) is a deadly cancer often diagnosed late and resistant to treatment. While hereditary DGAC is linked to CDH1 gene mutations, causing E-Cadherin loss, its role in sporadic DGAC is unclear. We discovered CDH1 inactivation in a subset of DGAC patient tumors. Analyzing single-cell transcriptomes in malignant ascites, we identified two DGAC subtypes: DGAC1 (CDH1 loss) and DGAC2 (lacking immune response). DGAC1 displayed distinct molecular signatures, activated DGAC-related pathways, and an abundance of exhausted T cells in ascites. Genetically engineered murine gastric organoids showed that Cdh1 knock-out (KO), KrasG12D, Trp53 KO (EKP) accelerates tumorigenesis with immune evasion compared to KrasG12D, Trp53 KO (KP). We also identified EZH2 as a key mediator promoting CDH1 loss-associated DGAC tumorigenesis. These findings highlight DGAC's molecular diversity and potential for personalized treatment in CDH1-inactivated patients.

CRACD suppresses neuroendocrinal plasticity of lung adenocarcinoma.

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, lung adenocarcinoma (LUAD) cells transform into neuroendocrinal (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD, a capping protein inhibitor, is frequently inactivated in cancers. CRACD knock-out (KO) de-represses NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE plasticity is associated with cell de-differentiation and activated stemness-related pathways. The single-cell transcriptomes of LUAD patient tumors recapitulate that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with SOX2, OCT4, and NANOG pathway activation, and impaired actin remodeling. This study reveals an unexpected role of CRACD in restricting NE cell plasticity that induces cell de-differentiation, providing new insights into cell plasticity of LUAD.

CRACD loss promotes small cell lung cancer tumorigenesis via EZH2-mediated immune evasion.

The mechanisms underlying immune evasion and immunotherapy resistance in small cell lung cancer (SCLC) remain unclear. Herein, we investigate the role of CRACD tumor suppressor in SCLC. We found that CRACD is frequently inactivated in SCLC, and Cracd knockout (KO) significantly accelerates SCLC development driven by loss of Rb1, Trp53, and Rbl2. Notably, the Cracd-deficient SCLC tumors display CD8+ T cell depletion and suppression of antigen presentation pathway. Mechanistically, CRACD loss silences the MHC-I pathway through EZH2. EZH2 blockade is sufficient to restore the MHC-I pathway and inhibit CRACD loss-associated SCLC tumorigenesis. Unsupervised single-cell transcriptomic analysis identifies SCLC patient tumors with concomitant inactivation of CRACD, impairment of tumor antigen presentation, and downregulation of EZH2 target genes. Our findings define CRACD loss as a new molecular signature associated with immune evasion of SCLC cells and proposed EZH2 blockade as a viable option for CRACD-negative SCLC treatment.

Frodo links Dishevelled to the p120-catenin/Kaiso pathway: distinct catenin subfamilies promote Wnt signals.

p120-catenin is an Arm repeat protein that interacts with varied components such as cadherin, small G proteins, kinases, and the Kaiso transcriptional repressor. Despite recent advances in understanding the roles that p120-catenin and Kaiso play in downstream modulation of Wnt/beta-catenin signaling, the identity of the upstream regulators of the p120-catenin/Kaiso pathway have remained unclear. Here, we find that p120-catenin binds Frodo, which itself interacts with the Wnt pathway protein Dishevelled (Dsh). In Xenopus laevis, we demonstrate that Wnt signals result in Frodo-mediated stabilization of p120-catenin, which, in turn, promotes Kaiso sequestration or removal from the nucleus. Our results point to Dsh and Frodo as upstream regulators of the p120-catenin/Kaiso signaling pathway. Importantly, this suggests that Wnt signals acting through Dsh regulate the stability of p120-catenin in addition to that of beta-catenin, and that each catenin promotes its respective signal in parallel to regulate distinct, as well as shared, direct downstream gene targets.

TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program.

Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia.

Organoids mimic the physiologic and pathologic events of organs. However, no consensus on esophageal organoid (EO) culture methods has been reached. Moreover, organoid models reproducing esophageal squamous cell carcinoma (ESCC) initiation have been unavailable. Herein, we sought to develop an esophageal minimum essential organoid culture medium (E-MEOM) for culturing murine EOs and establishing an early ESCC model. We formulated E-MEOM to grow EOs from a single cell with clonal expansion, maintenance, and passage. We found that EOs cultured in E-MEOM were equivalent to the esophageal epithelium by histological analysis and transcriptomic study. Trp53 knockout and Kras G12D expression in EOs induced the development of esophageal squamous neoplasia, an early lesion of ESCC. Here we propose the new formula for EO culture with minimum components and the organoid model recapitulating ESCC initiation, laying the foundation for ESCC research and drug discovery.

Blockers of Wnt3a, Wnt10a, or ß-Catenin Prevent Chemotherapy-Induced Neuropathic Pain In Vivo.

Although chemotherapy is a key cancer treatment, many chemotherapeutic drugs produce chronic neuropathic pain, called chemotherapy-induced neuropathic pain (CINP), which is a dose-limiting adverse effect. To date, there is no medicine that prevents CINP in cancer patients and survivors. We determined whether blockers of the canonical Wnt signaling pathway prevent CINP. Neuropathic pain was induced by intraperitoneal injection of paclitaxel (PAC) on four alternate days in male Sprague-Dawley rats or male Axin2-LacZ knock-in mice. XAV-939, LGK-974, and iCRT14, Wnt/ß-catenin blockers, were administered intraperitoneally as a single or multiple doses before or after injury. Mechanical allodynia, phosphoproteome profiling, Wnt ligands, and inflammatory mediators were measured by von Frey filament, phosphoproteomics, reverse transcription-polymerase chain reaction, and Western blot analysis. Localization of ß-catenin was determined by immunohistochemical analysis in the dorsal root ganglia (DRGs) in rats and human. Our phosphoproteome profiling of CINP rats revealed significant phosphorylation changes in Wnt signaling components. Importantly, repeated systemic injections of XAV-939 or LGK-974 prevented the development of CINP in rats. In addition, XAV-939, LGK-974, and iCRT14 ameliorated CINP. PAC increased Wnt3a and Wnt10a, activated ß-catenin in DRG, and increased monocyte chemoattractant protein-1 and interleukin-1ß in DRG. PAC also upregulated rAxin2 in mice. Furthermore, ß-catenin was expressed in neurons, including calcitonin gene-related protein-expressing neurons and satellite cells in rat and human DRG. In conclusion, chemotherapy increases Wnt3a, Wnt10a, and ß-catenin in DRG and their pharmacological blockers prevent and ameliorate CINP, suggesting a target for the prevention and treatment of CINP.