Metabolic regulator ERRγ governs gastric stem cell differentiation into acid-secreting parietal cells.

Parietal cells (PCs) produce gastric acid to kill pathogens and aid digestion. Dysregulated PC census is common in disease, yet how PCs differentiate is unclear. Here, we identify the PC progenitors arising from isthmal stem cells, using mouse models and human gastric cells, and show that they preferentially express cell-metabolism regulator and orphan nuclear receptor Estrogen-related receptor gamma (Esrrg, encoding ERRγ). Esrrg expression facilitated the tracking of stepwise molecular, cellular, and ultrastructural stages of PC differentiation. EsrrgP2ACreERT2 lineage tracing revealed that Esrrg expression commits progenitors to differentiate into mature PCs. scRNA-seq indicated the earliest Esrrg+ PC progenitors preferentially express SMAD4 and SP1 transcriptional targets and the GTPases regulating acid-secretion signal transduction. As progenitors matured, ERRγ-dependent metabolic transcripts predominated. Organoid and mouse studies validated the requirement of ERRγ for PC differentiation. Our work chronicles stem cell differentiation along a single lineage in vivo and suggests ERRγ as a therapeutic target for PC-related disorders.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Loss of Dok-3 in Non-tumor Cells Induces Malignant Transformation of Benign Epithelial Tumor Cells of the Intestine.

The fundamental difference between benign and malignant tumors lies in their invasive ability. It is believed that malignant conversion of benign tumor cells is induced by a tumor cell-intrinsic accumulation of driver gene mutations. Here, we found that disruption of the Dok-3 tumor suppressor gene led to malignant progression in the intestinal benign tumor model ApcMin/+ mice. However, Dok-3 gene expression was undetectable in epithelial tumor cells and the transplantation of bone marrow cells lacking the Dok-3 gene-induced malignant conversion of epithelial tumor cells in ApcMin/+ mice, indicating a previously unrecognized tumor cell-extrinsic mechanism. Moreover, the Dok-3 loss-induced tumor invasion in ApcMin/+ mice required CD4+ and CD8+ T lymphocytes, but not B lymphocytes. Finally, whole-genome sequencing showed an indistinguishable pattern and level of somatic mutations in tumors irrespective of the Dok-3 gene mutation in ApcMin/+ mice. Together, these data indicate that Dok-3 deficiency is a tumor-extrinsic driving force of malignant progression in ApcMin/+ mice, providing a novel insight into microenvironments in tumor invasion. Significance: This study uncovers tumor cell-extrinsic cues that can induce malignant conversion of benign tumors without intensifying mutagenesis in tumors, a novel concept potentially providing a new therapeutic target in malignancy.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

Loss of C/EBPδ enhances apoptosis of intestinal epithelial cells and exacerbates experimental colitis in mice.

Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation involving intestinal tissue damage, which include ulcerative colitis and Crohn's disease as major entities. Accumulating evidence suggests that excessive apoptosis of intestinal epithelial cells (IECs) contributes to the development of IBD. It was recently reported that the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is involved in inflammation; however, its role in colitis remains unclear. Here, we found that C/EBPδ knockout mice showed enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis, a mouse model of IBD, which was associated with severe colonic inflammation and mucosal damage with increased IEC apoptosis. Additionally, DSS stimulation induced increased expression of pro-apoptotic BH3-only protein Bim in the colon of C/EBPδ knockout mice. Collectively, our findings demonstrate that C/EBPδ plays an essential role in suppressing DSS-induced colitis, likely by attenuating IEC apoptosis.

Dok-3 and Dok-1/-2 adaptors play distinctive roles in cell fusion and proliferation during osteoclastogenesis and cooperatively protect mice from osteopenia.

Bone mass is determined by coordinated acts of osteoblasts and osteoclasts, which control bone formation and resorption, respectively. Osteoclasts are multinucleated, macrophage/monocyte lineage cells from bone marrow. The Dok-family adaptors Dok-1, Dok-2 and Dok-3 are expressed in the macrophage/monocyte lineage and negatively regulate many signaling pathways, implying roles in osteoclastogenesis. Indeed, mice lacking Dok-1 and Dok-2, the closest homologues with redundant functions, develop osteopenia with increased osteoclast counts compared to the wild-type controls. Here, we demonstrate that Dok-3 knockout (KO) mice also develop osteopenia. However, Dok-3 KO, but not Dok-1/-2 double-KO (DKO), mice develop larger osteoclasts within the normal cell-count range, suggesting a distinctive role for Dok-3. Indeed, Dok-3 KO, but not Dok-1/-2 DKO, bone marrow-derived cells (BMDCs) generated larger osteoclasts with more nuclei due to augmented cell-to-cell fusion in vitro. In addition, while Dok-1/-2 DKO BMDCs generated more osteoclasts, Dok-1/-2/-3 triple-KO (TKO) BMDCs generated osteoclasts increased in both number and size. Furthermore, Dok-1/-2/-3 TKO mice showed the combined effects of Dok-3 and Dok-1/-2 deficiency: severe osteopenia with more and larger osteoclasts. Together, our findings demonstrate that Dok-3 and Dok-1/-2 play distinctive but cooperative roles in osteoclastogenesis and protect mice from osteopenia, providing physiological and pathophysiological insight into bone homeostasis.

DOK7 gene therapy enhances motor activity and life span in ALS model mice.

Amyotrophic lateral sclerosis (ALS) is a progressive, multifactorial motor neurodegenerative disease with severe muscle atrophy. The glutamate release inhibitor riluzole is the only medication approved by the FDA, and prolongs patient life span by a few months, testifying to a strong need for new treatment strategies. In ALS, motor neuron degeneration first becomes evident at the motor nerve terminals in neuromuscular junctions (NMJs), the cholinergic synapse between motor neuron and skeletal muscle; degeneration then progresses proximally, implicating the NMJ as a therapeutic target. We previously demonstrated that activation of muscle-specific kinase MuSK by the cytoplasmic protein Dok-7 is essential for NMJ formation, and forced expression of Dok-7 in muscle activates MuSK and enlarges NMJs. Here, we show that therapeutic administration of an adeno-associated virus vector encoding the human DOK7 gene suppressed motor nerve terminal degeneration at NMJs together with muscle atrophy in the SOD1-G93A ALS mouse model. Ultimately, we show that DOK7 gene therapy enhanced motor activity and life span in ALS model mice.

Loss of Dok-1 and Dok-2 in mice causes severe experimental colitis accompanied by reduced expression of IL-17A and IL-22.

Appropriate immune responses and mucosal barrier functions are required for the maintenance of intestinal homeostasis. Defects in this defense system may lead to inflammatory disorders such as inflammatory bowel disease. Downstream of tyrosine kinases 1 (Dok-1) and its closest homolog, Dok-2, are preferentially expressed in immune cells, and play essential roles in the negative regulation of multiple signaling pathways in both innate and adaptive immunity. However, the function of these proteins in intestinal homeostasis remained unclear. Here we show that Dok-1/-2 double knockout (DKO) mice were highly susceptible to dextran sodium sulfate (DSS)-induced colitis compared with Dok-1 or Dok-2 single KO and wild type (WT) mice. Furthermore, DSS-treated Dok-1/-2 DKO mice exhibited increased colonic tissue damage accompanied by reduced proliferation of the epithelial cells relative to WT controls, suggesting that Dok-1/-2 DKO mice have defects in the repair of intestinal epithelial lesions. In addition, the levels of the Th17 cytokines IL-17A and IL-22, which have protective roles in DSS-induced colitis, were reduced in DSS-treated Dok-1/-2 DKO mice compared with WT mice. Taken together, our results demonstrate that Dok-1 and Dok-2 negatively regulate intestinal inflammation, apparently through the induction of IL-17A and IL-22 expression.

Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction.

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.

Dok adaptors play anti-inflammatory roles in pulmonary homeostasis.

Asthma is a chronic inflammatory disease of the lung with airflow obstruction and bronchospasm, characterized by pulmonary eosinophilia, airway remodeling, increased airway hyperresponsiveness to environmental stimuli, and excessive Th2-type cytokine production. Recent studies indicate that crosstalk between the innate and adaptive immune systems is crucial for this disease. We and others have showed that the Dok (downstream of tyrosine kinases) family adaptors, Dok-1, Dok-2, and Dok-3, play essential roles in negative regulation of a wide variety of signaling pathways in both innate and adaptive immunities. Here, histopathology and bronchoalveolar lavage fluid (BALF) cellularity showed spontaneous pulmonary inflammation in Dok-1-/- Dok-2-/- Dok-3-/- (TKO) mice, but not in Dok-1-/- Dok-2-/- or Dok-3-/- mice, with hallmarks of asthma, including eosinophilia, goblet cell hyperplasia, and subepithelial fibrosis. Consistently, TKO mice, but not the other mutants, showed increased airway hyperresponsiveness to methacholine inhalation. In addition, Th2-type cytokine concentrations in BALF were increased in TKO mice. These findings provide strong evidence that Dok-1, Dok-2, and Dok-3 cooperatively play critical anti-inflammatory roles in lung homeostasis.

Mice lacking Dok-1, Dok-2, and Dok-3 succumb to aggressive histiocytic sarcoma.

Histiocytic sarcoma (HS), a rare hematological malignancy, is an aggressive neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy, and there remains a need for more, and more realistic, animal models. HS cells typically show a histiocytic (ie, tissue macrophage-like) morphology and express histiocyte/macrophage markers in the absence of lymphocyte markers. In this study, we report that Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice develop HS, but do not exhibit elevated incidence of other types of tumors. These mutant mice showed earlier mortality than wild-type (WT) or the other mutant mice, and this mortality was associated with HS. In total, 17 of 21 tumor-bearing Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice necropsied at 25-66 weeks of age showed multiple organ spread, with osteolytic lesions and orthotopic invasion from the bone marrow to skeletal muscle. Tumors from the mice were transplantable. In addition, all Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice, but only a small proportion of Dok-3(-/-) mice and no Dok-1(-/-)Dok-2(-/-) mice, exhibited abnormal accumulation of macrophages in the lung on necropsy at 8-12 weeks of age. Macrophages derived from Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice displayed an exaggerated proliferative response to macrophage colony-stimulating factor (M-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF) compared with WT and mutant controls. Together, these findings indicate that Dok-1, Dok-2, and Dok-3 cooperatively suppress aggressive HS, and commend Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice as a useful model for the study of this neoplasia.

Reduced level of smoothened suppresses intestinal tumorigenesis by down-regulation of Wnt signaling.

BACKGROUND & AIMS: Although the Hedgehog (Hh) pathway regulates development and progression of several types of cancer, its involvement in colon cancer remains unclear. We aimed to clarify the roles of Hh signaling in intestinal tumorigenesis. METHODS: We studied expression of the Hh signaling components in the intestinal tumors of Apc(+/Delta716) mouse, a model for familial adenomatous polyposis. We used small interfering RNAs against Smoothened (SMO), which encodes the major signal transducer of the Hh pathway, to knockdown SMO expression and explore its function in human colon cancer cell lines. We also compared the intestinal tumor phenotypes of Apc(+/Delta716)Smo(+/-) mice with those of Apc(+/Delta716) mice. RESULTS: Expression of Smo was markedly increased in the intestinal adenoma epithelium of Apc(+/Delta716) mice. Importantly, SMO knockdown in human colon cancer cell lines suppressed proliferation in culture; cells arrested at the G1/S phase. Furthermore, Apc(+/Delta716)Smo(+/-) mice had decreased numbers of polyps in the large size class (Phi >or= 1-2 mm) and recessed polyp morphology, accompanied by reduced proliferation of the tumor epithelial cells. Unexpectedly, reduced expression of Smo suppressed beta-catenin-dependent transcription, rather than Hh-responsive Gli-dependent transcription. Interestingly, SMO knockdown reduced protein levels of active beta-catenin and induced its nuclear exclusion. CONCLUSIONS: Smo contributes to intestinal tumorigenesis by increasing Wnt signaling. SMO might be a good therapeutic target for patients with colorectal polyps and carcinomas, even in the absence of Hh signal activation.