Low Exposures to Amphibole or Serpentine Asbestos in Germline Bap1-mutant Mice Induce Mesothelioma Characterized by an Immunosuppressive Tumor Microenvironment.

Asbestos and BAP1 germline mutations are risk factors for malignant mesothelioma (MM). While it is well accepted that amphibole asbestos is carcinogenic, the role of serpentine (chrysotile) asbestos in MM has been debated. To address this controversy, we assessed whether minimal exposure to chrysotile could significantly increase the incidence and rate of MM onset in germline Bap1-mutant mice. With either crocidolite or chrysotile, and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in wild-type littermates. To explore the role of gene-environment interactions in MMs from Bap1-mutant mice, we investigated proinflammatory and protumorigenic factors and the tumor immune microenvironment (TIME). IHC and immunofluorescence staining showed an increased number of macrophages in granulomatous lesions and MMs. The relative number of CD163-positive (CD163+) M2 macrophages in chrysotile-induced MMs was consistently greater than in crocidolite-induced MMs, suggesting that chrysotile induces a more profound immunosuppressive response that creates favorable conditions for evading immune surveillance. MMs from Bap1-mutant mice showed upregulation of CD39/CD73-adenosine and C-C motif chemokine ligand 2 (Ccl2)/C-C motif chemokine receptor 2 (Ccr2) pathways, which together with upregulation of IL6 and IL10, promoted an immunosuppressive TIME, partly by attracting M2 macrophages. Interrogation of published human MM RNA sequencing (RNA-seq) data implicated these same immunosuppressive pathways and connections with CD163+ M2 macrophages. These findings indicate that increased M2 macrophages, along with upregulated CD39/CD73-adenosine and Ccl2/Ccr2 pathways, contribute to an immunosuppressive TIME in chrysotile-induced MMs of Bap1-mutant mice, suggesting that immunotherapeutic strategies targeting protumorigenic immune pathways could be beneficial in human BAP1 mutation carriers who develop MM. SIGNIFICANCE: We show that germline Bap1-mutant mice have enhanced susceptibility to MM upon minimal exposure to chrysotile asbestos, not only amphibole fibers. Chrysotile induced a more profound immune tumor response than crocidolite in Bap1-mutant mice by upregulating CD39/CD73-adenosine and Ccl2/Ccr2 pathways and recruiting more M2 macrophages, which together contributed to an immunosuppressive tumor microenvironment. Interrogation of human MM RNA-seq data revealed interconnected immunosuppressive pathways consistent with our mouse findings.

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Epithelial overexpression of IL-33 induces eosinophilic esophagitis dependent on IL-13.

BACKGROUND: Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. OBJECTIVE: We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. METHODS: We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2-/-, eosinophil-deficient, and IL-13-/- mice. Finally, EoE33 mice were treated with dexamethasone. RESULTS: EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and TH2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. CONCLUSIONS: IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery.

ALDH2 dysfunction and alcohol cooperate in cancer stem cell enrichment.

The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three-dimensional organoids and xenograft models. We find that alcohol exposure and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents.

Autophagy contributes to homeostasis in esophageal epithelium where high autophagic vesicle content marks basal cells with limited proliferation and enhanced self-renewal potential.

Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .

p53 Gain-of-Function Mutation Induces Metastasis via BRD4-Dependent CSF-1 Expression.

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease. SIGNIFICANCE: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489.

Systemic exposure to bacterial amyloid curli alters the gut mucosal immune response and the microbiome, exacerbating Salmonella-induced arthritis.

The Salmonella biofilm-associated amyloid protein, curli, is a dominant instigator of systemic inflammation and autoimmune responses following Salmonella infection. Systemic curli injections or infection of mice with Salmonella Typhimurium induce the major features of reactive arthritis, an autoimmune disorder associated with Salmonella infection in humans. In this study, we investigated the link between inflammation and microbiota in exacerbating autoimmunity. We studied C57BL/6 mice from two sources, Taconic Farms and Jackson Labs. Mice from Taconic Farms have been reported to have higher basal levels of the inflammatory cytokine IL - 17 than do mice from Jackson Labs due to the differences in their microbiota. When we systemically injected mice with purified curli, we observed a significant increase in diversity in the microbiota of Jackson Labs mice but not in that of the Taconic mice. In Jackson Labs, mice, the most striking effect was the expansion of Prevotellaceae. Furthermore, there were increases in the relative abundance of the family Akkermansiaceae and decreases in families Clostridiaceae and Muribaculaceae in Jackson Labs mice. Curli treatment led to significantly aggravated immune responses in the Taconic mice compared to Jackson Labs counterparts. Expression and production of IL - 1ß, a cytokine known to promote IL - 17 production, as well as expression of Tnfa increased in the gut mucosa of Taconic mice in the first 24 hours after curli injections, which correlated with significant increases in the number of neutrophils and macrophages in the mesenteric lymph nodes. A significant increase in the expression of Ccl3 in colon and cecum of Taconic mice injected with curli was detected. Taconic mice injected with curli also had elevated levels of inflammation in their knees. Overall, our data suggest that autoimmune responses to bacterial ligands, such as curli, are amplified in individuals with a microbiome that promote inflammation.

Tumor-Derived CCL5 Recruits Cancer-Associated Fibroblasts and Promotes Tumor Cell Proliferation in Esophageal Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas. IMPLICATIONS: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC.

Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

Eosinophilic esophagitis-associated epithelial remodeling may limit esophageal carcinogenesis.

Introduction: Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. Methods: In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. Results: In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Discussion: Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

A role for age-associated alterations in esophageal epithelium in eosinophilic esophagitis-associated fibrosis.

Subepithelial fibrosis occurs in a subset of eosinophilic esophagitis (EoE) patients and is associated with esophageal stricture. While mechanisms driving EoE fibrosis remain incompletely understood, findings from experimental systems support roles for epithelial-fibroblast crosstalk in this type of tissue remodeling. The current paradigm presents EoE as a progressive fibrostenotic disease in which aged patients develop fibrosis as a function of disease chronicity. In the current study we provide evidence that altered epithelial biology in the aging esophagus may also contribute to EoE-associated fibrosis. We find that induction of EoE inflammation in young and aged mice using the MC903/Ovalbumin protocol for the same time period results in increased lamina propria thickness uniquely in aged animals. Additionally, epithelial cells from aged mice less efficiently limit fibroblast contractility in collagen plug contraction assays compared to those from their young counterparts. Finally, to identify potential mechanisms through which aged esophageal epithelial cells may stimulate fibrotic remodeling, we perform cytokine array experiments in young and aged mice. These studies are significant as identification of age-associated factors that contribute to fibrotic remodeling may aid in the design of strategies toward early detection, prevention, and therapy of fibrostenotic EoE.

Alcohol Metabolism Enriches Squamous Cell Carcinoma Cancer Stem Cells That Survive Oxidative Stress via Autophagy.

BACKGROUND: Alcohol (ethanol) consumption is a major risk factor for head and neck and esophageal squamous cell carcinomas (SCCs). However, how ethanol (EtOH) affects SCC homeostasis is incompletely understood. METHODS: We utilized three-dimensional (3D) organoids and xenograft tumor transplantation models to investigate how EtOH exposure influences intratumoral SCC cell populations including putative cancer stem cells defined by high CD44 expression (CD44H cells). RESULTS: Using 3D organoids generated from SCC cell lines, patient-derived xenograft tumors, and patient biopsies, we found that EtOH is metabolized via alcohol dehydrogenases to induce oxidative stress associated with mitochondrial superoxide generation and mitochondrial depolarization, resulting in apoptosis of the majority of SCC cells within organoids. However, CD44H cells underwent autophagy to negate EtOH-induced mitochondrial dysfunction and apoptosis and were subsequently enriched in organoids and xenograft tumors when exposed to EtOH. Importantly, inhibition of autophagy increased EtOH-mediated apoptosis and reduced CD44H cell enrichment, xenograft tumor growth, and organoid formation rate. CONCLUSIONS: This study provides mechanistic insights into how EtOH may influence SCC cells and establishes autophagy as a potential therapeutic target for the treatment of EtOH-associated SCC.

Mutant p53 regulates Survivin to foster lung metastasis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53R175H (homologous to Trp53R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53R175H in ESCC remains to be investigated. To investigate p53R175H-mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53R172H/- mice to model ESCC. In the primary Trp53R172H/- tumor cell lines, we depleted Trp53R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP-BIRC5 axis as a potential mediator of Trp53R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5, increases in the presence of Trp53R172H Furthermore, depletion of Survivin specifically decreases Trp53R172H-driven lung metastasis. Mechanistically, Trp53R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis.

LIN28B induces a differentiation program through CDX2 in colon cancer.

Most colorectal cancers (CRCs) are moderately differentiated or well differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a potentially novel posttranscriptional regulatory mechanism via a LIN28B/CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration, and tumorigenesis. Our RNA-binding protein IP (RIP) assay revealed that LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression resulted in enhanced CDX2 expression to promote differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. A ChIP sequence for CDX2 identified α-methylacyl-CoA racemase (AMACR) as a potentially novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found that AMACR enhanced intestinal alkaline phosphatase activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrated that LIN28B promotes CRC differentiation through the CDX2/AMACR axis.

Dll1+ quiescent tumor stem cells drive chemoresistance in breast cancer through NF-κB survival pathway.

Development of chemoresistance in breast cancer patients greatly increases mortality. Thus, understanding mechanisms underlying breast cancer resistance to chemotherapy is of paramount importance to overcome this clinical challenge. Although activated Notch receptors have been associated with chemoresistance in cancer, the specific Notch ligands and their molecular mechanisms leading to chemoresistance in breast cancer remain elusive. Using conditional knockout and reporter mouse models, we demonstrate that tumor cells expressing the Notch ligand Dll1 is important for tumor growth and metastasis and bear similarities to tumor-initiating cancer cells (TICs) in breast cancer. RNA-seq and ATAC-seq using reporter models and patient data demonstrated that NF-κB activation is downstream of Dll1 and is associated with a chemoresistant phenotype. Finally, pharmacological blocking of Dll1 or NF-κB pathway completely sensitizes Dll1+ tumors to chemotherapy, highlighting therapeutic avenues for chemotherapy resistant breast cancer patients in the near future.

Rab11-FIP1 mediates epithelial-mesenchymal transition and invasion in esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer worldwide. The most commonly mutated gene in ESCC is TP53. Using a combinatorial genetic and carcinogenic approach, we generate a novel mouse model of ESCC expressing either mutant or null p53 and show that mutant p53 exhibits enhanced tumorigenic properties and displays a distinct genomic profile. Through RNA-seq analysis, we identify several endocytic recycling genes, including Rab Coupling Protein (Rab11-FIP1), which are significantly downregulated in mutant p53 tumor cells. In 3-dimensional (3D) organoid models, genetic knockdown of Rab11-FIP1 results in increased organoid size. Loss of Rab11-FIP1 increases tumor cell invasion in part through mutant p53 but also in an independent manner. Furthermore, loss of Rab11-FIP1 in human ESCC cell lines decreases E-cadherin expression and increases mesenchymal lineage-specific markers, suggesting induction of epithelial-mesenchymal transition (EMT). Rab11-FIP1 regulates EMT through direct inhibition of Zeb1, a key EMT transcriptional factor. Our novel findings reveal that Rab11-FIP1 regulates organoid formation, tumor cell invasion, and EMT.