Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment.Experimental Design: We used a combination of pharmacologic inhibition, gain-and loss-of-function genetic experiments, cyto-metry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC.Results: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression.Conclusions: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

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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.

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.Previous studies have demonstrated that insulin stimulation of the insulin receptor tyrosine kinase results in Ras activation and subsequent downstream stimulation of the Raf/MEK/ ERK 1 pathway (1-3). The activation of Ras occurs predominantly through the tyrosine phosphorylation of Shc followed by the association with the Grb2-SOS complex (4, 5). However, Ras activation is transient and rapidly returns to the inactive state despite continuous activation of the insulin receptor tyrosine kinase and prolonged Shc tyrosine phosphorylation (6, 7). Since insulin does not affect Ras-GTPase activating protein activity and/or targeting (8, 9), the mechanism responsible for the desensitization of Ras has remained obscure.Recently it has been reported that stimulation of several cell types with growth factors and other mitogenic agents results in the serine/threonine phosphorylation of SOS (10, 11). In addition, SOS phosphorylation precedes an insulin-dependent disassociation of the Grb2-SOS complex (7, 12). The insulin time dependence of the SOS phosphorylation and uncoupling of Grb2 from SOS was consistent with the desensitization phase of Ras inactivation. To determine whether the ERK pathway is involved in this event, we used two independent approaches to inhibit MEK activity and, hence, ERK activation. In this study we demonstrate that prevention of insulin-stimulated SOS phosphorylation and subsequent disassociation of the Grb2-SOS complex results in a prolongation of Ras activation. EXPERIMENTAL PROCEDURESCell Culture-Chinese hamster ovary cells expressing the human insulin receptor (CHO/IR) and 3T3L1 adipocytes were isolated and cultured as described previously (7). Cells were incubated for 16 h in serum-free media and then pretreated for 1 h with vehicle (0.5% dimethyl sulfoxide) or 100 M PD98059. The cells were then incubated with and without 100 nM insulin for various times, followed by lysis in 50 mM Hepes, pH 7.8, 1% Triton X-100, 2.5 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 2 M pepstatin, 0.5 trypsin inhibitory unit of aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 10 M le...

Tumor cells expressing programmed cell death ligand 1 (PD-L1) interact with PD-1 on CD8+ cytotoxic T lymphocytes (CTLs) to inhibit CTL effector function. In gastric cancer, the mechanism regulating PD-L1 is unclear. The Hedgehog (Hh) signaling pathway is reactivated in various cancers including gastric. Here we tested the hypothesis that Hh-induced PD-L1 inactivates effector T cell function and allows gastric cancer cell proliferation. Mouse organoids were generated from tumors of a triple-transgenic mouse model engineered to express an activated GLI2 allele, GLI2A, in Lgr5-expressing stem cells, (mTGOs) or normal mouse stomachs (mGOs). Bone marrow-derived dendritic cells (DCs) were pulsed with conditioned media collected from normal (mGOCM) or cancer (mTGOCM) organoids. Pulsed DCs and CTLs were then co-cultured with either mGOs or mTGOs in the presence of PD-L1 neutralizing antibody (PD-L1Ab). Human-derived gastric cancer organoids (huTGOs) were used in drug and xenograft assays. Hh/Gli inhibitor, GANT-61 significantly reduced the expression of PD-L1 and tumor cell proliferation both in vivo and in vitro. PD-L1Ab treatment induced tumor cell apoptosis in mTGO/immune cell co-cultures. GANT-61 treatment sensitized huTGOs to standard-of-care chemotherapeutic drugs both in vivo and in vitro. Thus, Hh signaling mediates PD-L1 expression in gastric cancer cells and subsequently promotes tumor proliferation.