Met receptor is essential for MAVS-mediated antiviral innate immunity in epithelial cells independent of its kinase activity.

Epithelial tissue is at the forefront of innate immunity, playing a crucial role in the recognition and elimination of pathogens. Met is a receptor tyrosine kinase that is necessary for epithelial cell survival, proliferation, and regeneration. Here, we showed that Met is essential for the induction of cytokine production by cytosolic nonself double-stranded RNA through retinoic acid-inducible gene-I-like receptors (RLRs) in epithelial cells. Surprisingly, the tyrosine kinase activity of Met was dispensable for promoting cytokine production. Rather, the intracellular carboxy terminus of Met interacted with mitochondrial antiviral-signaling protein (MAVS) in RLR-mediated signaling to directly promote MAVS signalosome formation. These studies revealed a kinase activity-independent function of Met in the promotion of antiviral innate immune responses, defining dual roles of Met in both regeneration and immune responses in the epithelium.

RB1 loss induces quiescent state through downregulation of RAS signaling in mammary epithelial cells.

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

RECK/GPR124-driven WNT signaling in pancreatic and gastric cancer cells.

RECK has been described to modulate extracellular matrix components through negative regulation of MMP activities. Recently, RECK was demonstrated to bind to an orphan G protein-coupled receptor GPR124 to mediate WNT7 signaling in nontumor contexts. Here, we attempted to clarify the role of RECK in driving WNT signaling in cancer cells. RECK and GPR124 formed a complex in 293T cells, and when both were expressed, WNT signaling was significantly enhanced in a WNT7-dependent manner. This cooperation was abolished when RECK mutants unable to bind to GPR124 were transduced. RECK stimulated the growth of KRAS-mutated pancreatic ductal adenocarcinoma (PDAC) cells with increased sensitivity to WNT inhibitor in a GPR124-dependent manner. A gastric cancer cell line SH10TC endogenously expresses both RECK and GPR124 under regular culture conditions. In this cell line, inhibited cell growth and WNT signaling as well as increased apoptosis in the GPR124 depletion was dominantly found over those in the RECK deletion. These findings suggest that RECK promotes tumor cell growth by positively modulating WNT signaling through GPR124. This study proposes that the RECK/GPR124 complex might be a good therapeutic target in PDAC and gastric cancer.

The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

Combined inhibition of Bcl-2 family members and YAP induces synthetic lethality in metastatic gastric cancer with RASA1 and NF2 deficiency.

BACKGROUND: Targetable molecular drivers of gastric cancer (GC) metastasis remain largely unidentified, leading to limited targeted therapy options for advanced GC. We aimed to identify molecular drivers for metastasis and devise corresponding therapeutic strategies. METHODS: We performed an unbiased in vivo genome-wide CRISPR/Cas9 knockout (KO) screening in peritoneal dissemination using genetically engineered GC mouse models. Candidate genes were validated through in vivo transplantation assays using KO cells. We analyzed target expression patterns in GC clinical samples using immunohistochemistry. The functional contributions of target genes were studied through knockdown, KO, and overexpression approaches in tumorsphere and organoid assays. Small chemical inhibitors against Bcl-2 members and YAP were tested in vitro and in vivo. RESULTS: We identified Nf2 and Rasa1 as metastasis-suppressing genes through the screening. Clinically, RASA1 mutations along with low NF2 expression define a distinct molecular subtype of metastatic GC exhibiting aggressive traits. NF2 and RASA1 deficiency increased in vivo metastasis and in vitro tumorsphere formation by synergistically amplifying Wnt and YAP signaling in cancer stem cells (CSCs). NF2 deficiency enhanced Bcl-2-mediated Wnt signaling, conferring resistance to YAP inhibition in CSCs. This resistance was counteracted via synthetic lethality achieved by simultaneous inhibition of YAP and Bcl-2. RASA1 deficiency amplified the Wnt pathway via Bcl-xL, contributing to cancer stemness. RASA1 mutation created vulnerability to Bcl-xL inhibition, but the additional NF2 deletion conferred resistance to Bcl-xL inhibition due to YAP activation. The combined inhibition of Bcl-xL and YAP synergistically suppressed cancer stemness and in vivo metastasis in RASA1 and NF2 co-deficiency. CONCLUSION: Our research unveils the intricate interplay between YAP and Bcl-2 family members, which can lead to synthetic lethality, offering a potential strategy to overcome drug resistance. Importantly, our findings support a personalized medicine approach where combined therapy targeting YAP and Bcl-2, tailored to NF2 and RASA1 status, could effectively manage metastatic GC.

Clinical significance and immune infiltration analyses of a novel coagulation-related signature in ovarian cancer.

Ovarian cancer (OV) is the most lethal gynecological malignancies worldwide. The coagulation cascade could induce tumor cell infiltration and contribute to OV progression. However, coagulation-related gene (CRG) signature for OV prognosis hasn't been determined yet. In this study, we evaluated the prognostic value of coagulation scores through receiver operating characteristics (ROC) analysis and K-M curves, among OV patients at our institution. Based on the transcriptome data of TCGA-OV cohort, we stratified two coagulation-related subtypes with distinct differences in prognosis and tumor immune microenvironment (p < 0.05). Moreover, from the 6406 differentially-expressed genes (DEGs) between the GTEx (n = 180) and TCGA-OV cohorts (n = 376), we identified 138 potential CRGs. Through LASSO-Cox algorithm, we finally distinguished a 3-gene signature (SERPINA10, CD38, and ZBTB16), with promising prognostic ability in both TCGA (p < 0.001) and ICGC cohorts (p = 0.040). Stepwise, we constructed a nomogram based on the clinical features and coagulation-related signature for overall survival prediction, with the C-index of 0.6761, which was evaluated by calibration curves. Especially, based on tissue microarrays analysis, Quantitative real-time fluorescence PCR (qRT-PCR), and Western Blot, we found that aberrant upregulation of CRGs was related to poor prognosis in OV at both mRNA and protein level (p < 0.05). Collectively, the coagulation-related signature was a robust prognostic biomarker, which could provide therapeutic benefits for chemotherapy/immunotherapy and assist clinical decision in OV patients.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Interleukin 1 Up-regulates MicroRNA 135b to Promote Inflammation-Associated Gastric Carcinogenesis in Mice.

BACKGROUND & AIMS: Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice. METHODS: We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan. RESULTS: We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130F/F mice and patients compared with nontumor tissues. In gastric organoids and immortalized cell lines, expression of miR-135B was induced by interleukin 1 signaling. K19-C2mE mice with disruption of Mir-135b developed hyperplastic lesions that were 50% smaller than mice without Mir-135b disruption and had significant reductions in cell proliferation. Expression of miR-135B in gastric cancer cell lines increased their colony formation, migration, and sphere formation. We identified FOXN3 and RECK messenger RNAs (mRNAs) as targets of miR-135B; their knockdown reduced migration of gastric cancer cell lines. Levels of FOXN3 and RECK mRNAs correlated inversely with levels of miR-135B in human gastric tumors and in inflamed mucosa from K19-C2mE mice. CONCLUSIONS: We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.

Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3∆IEC mouse-derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli ( Apc) Δ716 and Apc∆716 Tgfbr2∆IEC mice, thus indicating that Stat3 is not required for Wnt activation-driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells.-Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Identification of Stem Cells in the Epithelium of the Stomach Corpus and Antrum of Mice.

BACKGROUND & AIMS: Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice. METHODS: We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence. RESULTS: Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in human gastric atrophy. CONCLUSIONS: Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.

Multi-omics analysis identifies pathways and genes involved in diffuse-type gastric carcinogenesis induced by E-cadherin, p53, and Smad4 loss in mice.

The molecular mechanisms underlying the pathogenesis of diffuse-type gastric cancer (DGC) have not been adequately explored due to a scarcity of appropriate animal models. A recently developed tool well suited for this line of investigation is the Pdx-1-Cre;Cdh1F/+ ;Trp53F/F ;Smad4F/F (pChe PS) mouse model that spontaneously develops metastatic DGC showing nearly complete E-cadherin loss. Here, we performed a proteogenomic analysis to uncover the molecular changes induced by the concurrent targeting of E-cadherin, p53, and Smad4 loss. The gene expression profiles of mouse DGCs and in vivo gastric phenotypes from various combinations of gene knockout demonstrated that these mutations collaborate to activate cancer-associated pathways to generate aggressive DGC. Of note, WNT-mediated epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM)-cytokine receptor interactions were prominently featured. In particular, the WNT target gene osteopontin (OPN) that functions as an ECM cytokine is highly upregulated. In validation experiments, OPN contributed to DGC stemness by promoting cancer stem cell (CSC) survival and chemoresistance. It was further found that Bcl-xL acts as a targetable downstream effector of OPN in DGC CSC survival. In addition, Zeb2 and thymosin-ß4 (Tß4) were identified as prime candidates as suppressors of E-cadherin expression from the remaining Cdh1 allele during DGC development. Specifically, Tß4 suppressed E-cadherin expression and anoikis while promoting cancer cell growth and migration. Collectively, these proteogenomic analyses broaden and deepen our understanding of the contribution of key driver mutations in the stepwise carcinogenesis of DGC through novel effectors, namely OPN and Tß4.

The Emerging Roles of RUNX Transcription Factors in Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is an evolutionary conserved morphogenetic program necessary for the shaping of the body plan during development. It is guided precisely by growth factor signaling and a dedicated network of specialised transcription factors. These are supported by other transcription factor families serving auxiliary functions during EMT, beyond their general roles as effectors of major signaling pathways. EMT transiently induces in epithelial cells mesenchymal properties, such as the loss of cell-cell adhesion and a gain in cell motility. Together, these newly acquired properties enable their migration to distant sites where they eventually give rise to adult epithelia. However, it is now recognized that EMT contributes to the pathogenesis of several human diseases, notably in tissue fibrosis and cancer metastasis. The RUNX family of transcription factors are important players in cell fate determination during development, where their spatio-temporal expression often overlaps with the occurrence of EMT. Furthermore, the dysregulation of RUNX expression and functions are increasingly linked to the aberrant induction of EMT in cancer. The present chapter reviews the current knowledge of this emerging field and the common themes of RUNX involvement during EMT, with the intention of fostering future research.

The RUNX complex: reaching beyond haematopoiesis into immunity.

Among their diverse roles as transcriptional regulators during development and cell fate specification, the RUNX transcription factors are best known for the parts they play in haematopoiesis. RUNX proteins are expressed throughout all haematopoietic lineages, being necessary for the emergence of the first haematopoietic stem cells to their terminal differentiation. Although much progress has been made since their discoveries almost two decades ago, current appreciation of RUNX in haematopoiesis is largely grounded in their lineage-specifying roles. In contrast, the importance of RUNX to immunity has been mostly obscured for historic, technical and conceptual reasons. However, this paradigm is likely to shift over time, as a primary purpose of haematopoiesis is to resource the immune system. Furthermore, recent evidence suggests a role for RUNX in the innate immunity of non-haematopoietic cells. This review takes a haematopoiesis-centric approach to collate what is known of RUNX's contribution to the overall mammalian immune system and discuss their growing prominence in areas such as autoimmunity, inflammatory diseases and mucosal immunity.

Phase-separated super-enhancers confer an innate radioresistance on genomic DNA.

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.

Differential requirement for IL-2 and IL-23 in the differentiation and effector functions of Th17/ILC3-like cells in a human T cell line.

A well-documented Achilles heel of current cancer immunotherapy approaches is T cell exhaustion within solid tumor tissues. The proinflammatory cytokine interleukin (IL)-23 has been utilized to augment chimeric antigen receptor (CAR) T cell survival and tumor immunity. However, in-depth interrogation of molecular events downstream of IL-23/IL-23 receptor signaling is hampered by a paucity of suitable cell models. The current study investigates the differential contribution of IL-2 and IL-23 to the maintenance and differentiation of the IL-23 responsive Kit225 T-cell line. We observed that IL-23 enhanced cellular fitness and survival but was insufficient to drive proliferation. IL-23 rapidly induced phosphorylation of STAT1, STAT3, and STAT4, and messenger RNA expression of IL17A, the archetypal effector cytokine of T helper 17 (Th17) cells, but not their lineage markers RORC and NCR1. These observations suggest that IL-23 endowed Th17/ILC3-like effector function but did not promote their differentiation. In contrast, spontaneous differentiation of Kit225 cells toward a Th17/ILC3-like phenotype was induced by prolonged IL-2 withdrawal. This was marked by strongly elevated basal IL17A and IL17F expression and the secretion of IL-17. Together, our data present Kit225 cells as a valuable model for studying the interplay between cytokines and their contribution to T cell survival, proliferation, and differentiation.

Aberrant Upregulation of RUNX3 Activates Developmental Genes to Drive Metastasis in Gastric Cancer.

Gastric cancer metastasis is a major cause of mortality worldwide. Inhibition of RUNX3 in gastric cancer cell lines reduced migration, invasion, and anchorage-independent growth in vitro. Following splenic inoculation, CRISPR-mediated RUNX3-knockout HGC-27 cells show suppression of xenograft growth and liver metastasis. We interrogated the potential of RUNX3 as a metastasis driver in gastric cancer by profiling its target genes. Transcriptomic analysis revealed strong involvement of RUNX3 in the regulation of multiple developmental pathways, consistent with the notion that Runt domain transcription factor (RUNX) family genes are master regulators of development. RUNX3 promoted "cell migration" and "extracellular matrix" programs, which are necessary for metastasis. Of note, we found pro-metastatic genes WNT5A, CD44, and VIM among the top differentially expressed genes in RUNX3 knockout versus control cells. Chromatin immunoprecipitation sequencing and HiChIP analyses revealed that RUNX3 bound to the enhancers and promoters of these genes, suggesting that they are under direct transcriptional control by RUNX3. We show that RUNX3 promoted metastasis in part through its upregulation of WNT5A to promote migration, invasion, and anchorage-independent growth in various malignancies. Our study therefore reveals the RUNX3-WNT5A axis as a key targetable mechanism for gastric cancer metastasis. SIGNIFICANCE: Subversion of RUNX3 developmental gene targets to metastasis program indicates the oncogenic nature of inappropriate RUNX3 regulation in gastric cancer.

Runx3 protects gastric epithelial cells against epithelial-mesenchymal transition-induced cellular plasticity and tumorigenicity.

The transcription factor RUNX3 functions as a tumor suppressor in the gastrointestinal epithelium, where its loss is an early event in carcinogenesis. While RUNX3 acts concurrently as a mediator of TGF-ß signaling and an antagonist of Wnt, the cellular changes that follow its loss and their contribution to tumorigenicity are not fully understood. Here, we report that the loss of Runx3 in gastric epithelial cells results in spontaneous epithelial-mesenchymal transition (EMT). This produces a tumorigenic stem cell-like subpopulation, which remarkably expresses the gastric stem cell marker Lgr5. This phenomenon is due to the compounding effects of the dysregulation of the TGF-ß and Wnt pathways. Specifically, Runx3(-/-) p53(-/-) gastric epithelial cells were unexpectedly sensitized for TGF-ß-induced EMT, during which the resultant induction of Lgr5 was enhanced by an aberrantly activated Wnt pathway. These data demonstrate a protective role for RUNX3 in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness.

A synergistic partnership between IL-33/ST2 and Wnt pathway through Bcl-xL drives gastric cancer stemness and metastasis.

ST2 functions as a receptor for the cytokine IL-33. It has been implicated in carcinogenesis. In this study, we sought to mechanistically determine how ST2 and IL-33 function to support cancer stem cell (CSC) activity and drive gastric cancer (GC) pathogenesis. ST2+ subpopulation spontaneously arose during gastric tumorigenesis. A thorough evaluation of ST2 and IL-33 expression in gastric tumors revealed that they show an overlapping expression pattern, notably in poor differentiated GC and metastasis foci. Moreover, their expression levels are clinically correlated to cancer progression. Using a genetic model of CSC-driven gastric carcinogenesis, ST2+ subpopulation displays increased tumorigenicity, chemoresistance and metastatic potentials through increased survival fitness endowed by an elevated MAPK-regulated Bcl-xL. The IL-33/ST2 axis enhances the self-renewal and survival of GC stem cells and organoids. Importantly, we observed a synergistic cooperation between IL-33/ST2 and the canonical Wnt pathway in transactivating Wnt-dependent transcription and supporting CSC activity, a partnership that was abrogated by inhibiting Bcl-xL. Concordant with this, ST2+ subpopulation was targeted by MEK1/2 and Bcl-xL-specific inhibitors. These findings establish ST2 as a functional CSC marker that fortifies the Wnt signal while availing a novel therapeutic strategy to suppress GC progression by targeting the IL-33/ST2/Bcl-xL signaling axis.