Direct cellular reprogramming enables development of viral T antigen-driven Merkel cell carcinoma in mice.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that frequently carries an integrated Merkel cell polyomavirus (MCPyV) genome and expresses viral transforming antigens (TAgs). MCC tumor cells also express signature genes detected in skin-resident, postmitotic Merkel cells, including atonal bHLH transcription factor 1 (ATOH1), which is required for Merkel cell development from epidermal progenitors. We now report the use of in vivo cellular reprogramming, using ATOH1, to drive MCC development from murine epidermis. We generated mice that conditionally expressed MCPyV TAgs and ATOH1 in epidermal cells, yielding microscopic collections of proliferating MCC-like cells arising from hair follicles. Immunostaining of these nascent tumors revealed p53 accumulation and apoptosis, and targeted deletion of transformation related protein 53 (Trp53) led to development of gross skin tumors with classic MCC histology and marker expression. Global transcriptome analysis confirmed the close similarity of mouse and human MCCs, and hierarchical clustering showed conserved upregulation of signature genes. Our data establish that expression of MCPyV TAgs in ATOH1-reprogrammed epidermal cells and their neuroendocrine progeny initiates hair follicle-derived MCC tumorigenesis in adult mice. Moreover, progression to full-blown MCC in this model requires loss of p53, mimicking the functional inhibition of p53 reported in human MCPyV-positive MCCs.

Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1-Mediated Secretion of Extracellular Vesicles.

Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment. SIGNIFICANCE: This study shows how HSF1 regulates a stromal transcriptional program associated with aggressive gastric cancer and identifies multiple proteins within this program as candidates for therapeutic intervention. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1639/F1.large.jpg.

Aim2-mediated/IFN-ß-independent regulation of gastric metaplastic lesions via CD8+ T cells.

Development of gastric cancer is often preceded by chronic inflammation, but the immune cellular mechanisms underlying this process are unclear. Here we demonstrated that an inflammasome molecule, absent in melanoma 2 (Aim2), was upregulated in patients with gastric cancer and in spasmolytic polypeptide-expressing metaplasia of chronically Helicobacter felis-infected stomachs in mice. However, we found that Aim2 was not necessary for inflammasome function during gastritis. In contrast, Aim2 deficiency led to an increase in gastric CD8+ T cell frequency, which exacerbated metaplasia. These gastric CD8+ T cells from Aim2-/- mice were found to have lost their homing receptor expression (sphingosine-1-phosphate receptor 1 [S1PR1] and CD62L), a feature of tissue-resident memory T cells. The process was not mediated by Aim2-dependent regulation of IFN-ß or by dendritic cell-intrinsic Aim2. Rather, Aim2 deficiency contributed to an increased production of CXCL16 by B cells, which could suppress S1PR1 and CD62L in CD8+ T cells. This study describes a potentially novel function of Aim2 that regulates CD8+ T cell infiltration and retention within chronically inflamed solid organ tissue. This function operates independent of the inflammasome, IFN-ß, or dendritic cells. We provide evidence that B cells can contribute to this mechanism via CXCL16.

Indoleamine 2,3-Dioxygenase 1, Increased in Human Gastric Pre-Neoplasia, Promotes Inflammation and Metaplasia in Mice and Is Associated With Type II Hypersensitivity/Autoimmunity.

BACKGROUND & AIMS: Chronic gastrointestinal inflammation increases the risk of cancer by mechanisms that are not well understood. Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-binding enzyme that regulates the immune response via catabolization and regulation of tryptophan availability for immune cell uptake. IDO1 expression is increased during the transition from chronic inflammation to gastric metaplasia. We investigated whether IDO1 contributes to the inflammatory response that mediates loss of parietal cells leading to metaplasia. METHODS: Chronic gastric inflammation was induced in Ido1-/- and CB57BL/6 (control) mice by gavage with Helicobacter felis or overexpression of interferon gamma in gastric parietal cells. We also performed studies in Jh-/- mice, which are devoid of B cells. Gastric tissues were collected and analyzed by flow cytometry, immunostaining, and real-time quantitative polymerase chain reaction. Plasma samples were analyzed by enzyme-linked immunosorbent assay. Gastric tissues were obtained from 20 patients with gastric metaplasia and 20 patients without gastric metaplasia (controls) and analyzed by real-time quantitative polymerase chain reaction; gastric tissue arrays were analyzed by immunohistochemistry. We collected genetic information on gastric cancers from The Cancer Genome Atlas database. RESULTS: H felis gavage induced significantly lower levels of pseudopyloric metaplasia in Ido1-/- mice, which had lower frequencies of gastric B cells, than in control mice. Blood plasma from H felis-infected control mice had increased levels of autoantibodies against parietal cells, compared to uninfected control mice, but this increase was lower in Ido1-/- mice. Chronically inflamed stomachs of Ido1-/- mice had significantly lower frequencies of natural killer cells in contact with parietal cells, compared with stomachs of control mice. Jh-/- mice had lower levels of pseudopyloric metaplasia than control mice in response to H felis infection. Human gastric pre-neoplasia and carcinoma specimens had increased levels of IDO1 messenger RNA compared with control gastric tissues, and IDO1 protein colocalized with B cells. Co-clustering of IDO1 messenger RNA with B-cell markers was corroborated by The Cancer Genome Atlas database. CONCLUSIONS: IDO1 mediates gastric metaplasia by regulating the B-cell compartment. This process appears to be associated with type II hypersensitivity/autoimmunity. The role of autoimmunity in the progression of pseudopyloric metaplasia warrants further investigation.

Invasive mouse gastric adenocarcinomas arising from Lgr5+ stem cells are dependent on crosstalk between the Hedgehog/GLI2 and mTOR pathways.

Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.

Transgenic expression of interferon-γ in mouse stomach leads to inflammation, metaplasia, and dysplasia.

Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H(+)/K(+) ATPase ß promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear ß-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion.

Inflammation and Gli2 suppress gastrin gene expression in a murine model of antral hyperplasia.

Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (Gast⁻/⁻) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1ß expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. LacZ reporter mice for Sonic hedgehog (Shh), Gli1, and Gli2 expression bred onto the Gast⁻/⁻ background revealed reduced Shh and Gli1 expression in the antra compared to wild type controls (WT). Gli2 expression in the Gast⁻/⁻ corpus was unchanged. However in the hyperplastic Gast⁻/⁻ antra, Gli2 expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that Gli2 is differentially regulated in the hyperplastic Gast⁻/⁻ antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1ß and Il-11, which promote gastric epithelial proliferation, were increased in the Gast⁻/⁻ stomach along with Infγ. To test if inflammation could account for elevated epithelial Gli2 expression in the Gast⁻/⁻ antra, the human gastric cell line AGS was treated with IL-1ß and was found to increase GLI2 but decrease GLI1 levels. IL-1ß also repressed human GAST gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the GAST promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed Gast expression but induced Il-1ß gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate Il-1ß expression, repress Gast gene expression and increase proliferation, leading to antral hyperplasia.

Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations.

Uncontrolled Hedgehog (Hh) signaling leads to the development of basal cell carcinoma (BCC), the most common human cancer, but the cell of origin for BCC is unclear. While Hh pathway dysregulation is common to essentially all BCCs, there exist multiple histological subtypes, including superficial and nodular variants, raising the possibility that morphologically distinct BCCs may arise from different cellular compartments in skin. Here we have shown that induction of a major mediator of Hh signaling, GLI2 activator (GLI2ΔN), selectively in stem cells of resting hair follicles in mice, induced nodular BCC development from a small subset of cells in the lower bulge and secondary hair germ compartments. Tumorigenesis was markedly accelerated when GLI2ΔN was induced in growing hair follicles. In contrast, induction of GLI2ΔN in epidermis led to the formation of superficial BCCs. Expression of GLI2ΔN at reduced levels in mice yielded lesions resembling basaloid follicular hamartomas, which have previously been linked to low-level Hh signaling in both mice and humans. Our data show that the cell of origin, tissue context (quiescent versus growing hair follicles), and level of oncogenic signaling can determine the phenotype of Hh/Gli-driven skin tumors, with high-level signaling required for development of superficial BCC-like tumors from interfollicular epidermis and nodular BCC-like tumors from hair follicle stem cells.

The Drosophila Nkx6 homeodomain protein has both activation and repression domains and can activate target gene expression.

Consistent with the common role of Nkx6 family members in specifying motor neuron identity, we show that over-expression of Drosophila Nkx6 results in an increase in the number of Fasiclin II expressing motor neurons in the intersegmental nerve B branch. Our dissection of the regulatory domains of Nkx6 using chimeric cell culture assays revealed the presence of two repression domains and a single activation domain within this transcription factor. As well as its conserved homeodomain, Nkx6 also has a candidate Engrailed homology 1 (Eh1) domain that is conserved amongst all NKx6 family members, through which vertebrate NKx6-type proteins bind the co-repressor, Groucho (Muhr, J., et al., 2001. Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104, 861-73). Paralleling our previous reports that the Eh1 domain of Vnd and Ind are ineffective in Gal4 chimeric assays (Von Ohlen, T., Syu, L.J., Mellerick, D.M., 2007. Conserved properties of the Drosophila homeodomain protein. Ind. Mech. Dev. 124, 925-934; Yu, Z., et al., 2005. Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator. Nucleic Acids Res. 33, 1-12), we found that the Eh1 domain of Nkx6 did not significantly enhance repression in Gal4 chimeric assays. However, when we performed co-immunoprecipitation analyses, we found that Nkx6 can bind Groucho and that binding of Nkx6 to this co-repressor is modulated intra-molecularly. Full length Nkx6 interacted with Groucho poorly, because sequences at the carboxyl terminal of NKx6 interfere with Groucho binding, despite the presence of the Eh1 domain. In contrast, a carboxyl terminal Nkx6 deletion bound Groucho strongly. In keeping with the presence of an activation domain within Nkx6, we also report that Nkx6 can activate reporter expression driven by an Nkx6.1 enhancer that mediates auto-activation in transient transfection assays. The presence of multiple repression domains in Nkx6 supports Nkx6's role as a repressor, potentially using both Groucho-dependent and independent mechanisms. Thus, Nkx6 likely functions as a dual regulator in embryos.

The Drosophila homeodomain transcription factor, Vnd, associates with a variety of co-factors, is extensively phosphorylated and forms multiple complexes in embryos.

Vnd is a dual transcriptional regulator that is essential for Drosophila dorsal-ventral patterning. Yet, our understanding of the biochemical basis for its regulatory activity is limited. Consistent with Vnd's ability to repress target expression in embryos, endogenously expressed Vnd physically associates with the co-repressor, Groucho, in Drosophila Kc167 cells. Vnd exists as a single complex in Kc167 cells, in contrast with embryonic Vnd, which forms multiple high-molecular-weight complexes. Unlike its vertebrate homolog, Nkx2.2, full-length Vnd can bind its target in electrophoretic mobility shift assay, suggesting that co-factor availability may influence Vnd's weak regulatory activity in transient transfections. We identify the high mobility group 1-type protein, D1, and the novel helix-loop-helix protein, Olig, as novel Vnd-interacting proteins using co-immunoprecipitation assays. Furthermore, we demonstrate that both D1 and Olig are co-expressed with Vnd during Drosophila embryogenesis, consistent with a biological basis for this interaction. We also suggest that the phosphorylation state of Vnd influences its ability to interact with co-factors, because Vnd is extensively phosphorylated in embryos and can be phosphorylated by activated mitogen-activated protein kinase in vitro. These results highlight the complexities of Vnd-mediated regulation.

Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/beta3-catenin signaling.

Constitutive Hedgehog (Hh) signaling underlies several human tumors, including basal cell carcinoma (BCC) and basaloid follicular hamartoma in skin. Intriguingly, superficial BCCs arise as de novo epithelial buds resembling embryonic hair germs, collections of epidermal cells whose development is regulated by canonical Wnt/beta-catenin signaling. Similar to embryonic hair germs, human BCC buds showed increased levels of cytoplasmic and nuclear beta-catenin and expressed early hair follicle lineage markers. We also detected canonical Wnt/ beta-catenin signaling in epithelial buds and hamartomas from mice expressing an oncogene, M2SMO, leading to constitutive Hh signaling in skin. Conditional overexpression of the Wnt pathway antagonist Dkk1 in M2SMO-expressing mice potently inhibited epithelial bud and hamartoma development without affecting Hh signaling. Our findings uncover a hitherto unknown requirement for ligand-driven, canonical Wnt/ beta-catenin signaling for Hh pathway-driven tumorigenesis, identify a new pharmacological target for these neoplasms and establish the molecular basis for the well-known similarity between early superficial BCCs and embryonic hair germs.

The Nk-2 box of the Drosophila homeodomain protein, Vnd, contributes to its repression activity in a Groucho-dependent manner.

The transcription factor, Vnd, is a dual regulator that specifies ventral neuroblast identity in Drosophila by both repressing and activating target genes. Vnd and its homologues have a conserved amino acid sequence, the Nk-2 box or Nk specific domain, as well a conserved DNA-binding homeodomain and an EhI-type Groucho interaction domain. However, the function of the conserved Nk-2 box has not been fully defined. To explore its function, we deleted the Nk-2 box and compared the regulatory activity of mutant Vnd in transgenic over-expression assays to that of the wild-type protein. We were unable to assign regulatory activity to the Nk-2 box using an over-expression assay, because the mutant protein activated expression of endogenous Vnd, masking a requirement for the Nk-2 box. However, in transgenic rescue assays, Vnd lacking the Nk-2 box repressed ind expression at 30% lower levels than the wild-type protein. Moreover, in transient transfection assays using Gal4 DNA-binding domain-Vnd chimeras, the repression activity of Vnd lacking the Nk-2 box was compromised. Because Vnd represses target gene expression in conjunction with Groucho, we asked whether the Nk-2 box affects Vnd's ability to interact with this co-repressor. Vnd lacking the Nk-2 box binds Groucho 30% less efficiently than wild-type Vnd in co-immunoprecipitations. These data suggest that the Nk-2 box contributes to the repression activity of Vnd by stabilizing its interaction with the co-repressor, Groucho.

Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator.

At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861-873; Weiss et al. (1998) Genes Dev., 12, 3591-3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165-1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions.