Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression.

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST.

The glypican 3 hepatocellular carcinoma marker regulates human hepatic stellate cells via Hedgehog signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) frequently represents two diseases as it often arises in the setting of cirrhosis caused by the proliferation and activation of hepatic stellate cells (HSCs). Previously, we identified that Hedgehog (Hh) signaling regulates HSC viability and fibrinogenesis, as well as HCC tumorigenesis. Although it is increasingly recognized that HSCs and HCCs communicate via paracrine signaling, Hh's role in this process is just emerging. We hypothesized that a secreted HCC tumor marker and Hh mediator, glypican 3 (GPC3), may regulate HSC. METHODS: Using three human HCC lines (Hep3B, PLC/PRF/5 and SK-Hep-1) and one Hh-responsive human HSC line (LX-2), we developed two in vitro models of HCC-to-HSC paracrine signaling using a Transwell coculture system and HCC-conditioned media. We then evaluated the effects of these models, as well as GPC3, on HSC viability and gene expression. RESULTS: Using our coculture and conditioned media models, we demonstrate that the three HCC lines decrease HSC viability. Furthermore, we demonstrate that recombinant GPC3 dose-dependently decreases the LX-2 viability while inhibiting the expression of Hh target genes that regulate HSC viability. Finally, GPC3's inhibitory effects on cell viability and Hh target gene expression are partially abrogated by heparin, a competitor for GPC3 binding. CONCLUSIONS: For the first time, we show that GPC3, an HCC biomarker and Hh mediator, regulates human HSC viability by regulating Hh signaling. This expands on existing data suggesting a role for tumor-stroma interactions in the liver and suggests that GPC3 plays a role in this process.