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.
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California; Faculty of Medicine and Psychology, Azienda Ospedaliera Sant'Andrea, Sapienza-Università di Roma, Rome, Italy.
Paolo
P
Magistri
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California.
Stephanie Y
SY
Leonard
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California.
Chih-Min
CM
Tang
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California.
Jonathan C
JC
Chan
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California.
Tracy E
TE
Lee
Division of Surgical Oncology, Department of Surgery, Moores UCSD Cancer Center, University of California, San Diego, California. Electronic address: jsicklick@ucsd.edu.
Jason K
JK
Sicklick
D000925:Anticoagulants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism; D053673:Glypicans / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D055166:Hepatic Stellate Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology
D000925:Anticoagulants; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; C105988:EIF3A protein, human; D039621:Eukaryotic Initiation Factor-3; C105937:GPC3 protein, human; D053673:Glypicans; C117596:HHIP protein, human; D008562:Membrane Glycoproteins; D006493:Heparin
Epithelial-mesenchymal interactions; GPC3; HCC; HSC; Hedgehog-interacting protein; Hepatoma; Tumor–stroma interactions