GLI1 activation by non-classical pathway integrin αvβ3/ERK1/2 maintains stem cell-like phenotype of multicellular aggregates in gastric cancer peritoneal metastasis

Abstract

Peritoneal metastasis is one of the most important causes of postoperative death in patients with gastric cancer, and the exact mechanism remains unclear. The proliferation of multicellular aggregates of exfoliated malignant gastric cells in the abdominal cavity is the focus of current research. However, the mechanism how gastric cancer multicellular aggregates survive remains unclear. In this study, we demonstrated that multicellular aggregates of exfoliated gastric cancer cells in the abdominal cavity expressed a stem cell-Like phenotype. We found that Integrin α_vβ₃ not only mediated adhesion of gastric cancer multicellular aggregates to form independent functional units, but also maintained their stem cell-like phenotype by the non-classical pathway Integrin α_vβ₃/ERK1/2/GLI1. In addition, ERK1/2 directly regulates the transcriptional activity of GLI1. GLI1 is a key effector of the Integrin α_vβ₃ pathway in regulating stem cell-like phenotype in multicellular aggregates. Our data indicates that although there is a crosstalk between the non-classical Integrin α_vβ₃ pathway and the classical Hedgehog pathway, the activation of GLI1 is almost independent of the Hedgehog pathway in multicellular aggregates of gastric cancer cells. Our study provides a basis for blocking GLI1 activity in the prevention and treatment of peritoneal metastases of gastric cancer.

Fig. 1. Peritoneal MCAs of exfoliated GC cells expressing a stem cell-like phenotype.

a Representative H&E images of peritoneal MCAs and scattered-free cancer cells. Scale bar = 100 μm. b Representative IHC images of CEA and CK19 in peritoneal MCAs and scattered-free cancer cells. Scale bar = 100 μm. c Higher colony forming ability of peritoneal MCAs than scattered-free cancer cells in the control group. d, e IHC and western blottingting showing upregulated stemness-related genes CD44, ALDH1A1, Oct4, Bmi1 and Nanog expressed by peritoneal MCAs compared to scattered-free cancer cells in the control group. Scale bar = 100 μm. f Increased tumor spheres forming ability of peritoneal MCAs compared to scattered-free cancer cells in the control group. Scale bar = 10 μm. g Higher cell viability of peritoneal MCAs after being treated with different concentrations and for different durations with 5-fluorouracil and oxaliplatin compared to scattered-free cancer cells in the control group. h Formation of subcutaneous xenograft tumors by peritoneal MCAs (R) than scattered-free cancer cells (L). i, j Increased number and weight of xenograft tumors formed by peritoneal MCAs compared to scattered-free cancer cells in the control group. Each bar in the figure represents the mean ± SEM of triplicates. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2. GLI1 activation via the non-classical pathway Integrin α_vβ₃/ERK1/2 in MCAs of GC cells.

a Western blottingting showing upregulated Integrin α_vβ₃, p-ERK1/2 and GLI1 in peritoneal MCAs, BGC823 MCAs and SGC7901 MCAs compared to scattered-free cancer cells in the control group. b Western blottingting showing downregulated GLI1 with Integrin α_vβ₃ inhibitor Cilengitide and upregulated GLI1 with co-stimulator ligand RGD compared to the blank control in SGC7901 MCAs and BGC823 MCAs. c Western blottingting showing the most pronounced effect of Integrin β₃ silencing in the shIntegrin β₃#1 group. d Western blottingting showing downregulated GLI1 with Integrin α_vβ₃ co-stimulator ligand RGD plus ERK1/2 inhibitor PD-184161 and upregulated GLI1 with RGD alone compared to the blank control in SGC7901 MCAs and BGC823 MCAs. e Western blottingting showing slightly decreased GLI1 with Hedgehog/Smoothened pathway inhibitor Cyclopamine compared with the blank control group in SGC7901 MCAs and BGC823 MCAs. f, g Decreased luminescence of GLI1 in the group of Integrin α_vβ₃ co-simulator ligand RGD plus ERK1/2 inhibitor PD-184161 or PD-184161 alone compared to the blank control. Each bar in the figure represents the mean ± SEM of triplicates. *p < 0.05; **p < 0.01

Fig. 3. The Integrin α_vβ₃/ERK1/2/GLI1 pathway maintains the stem cell-like phenotype in MCAs of GC cells.

a Decreased tumor spheres forming ability in the group of Integrin α_vβ₃ inhibitor Cilengitide or ERK1/2 inhibitor PD-184161 or GLI1 inhibitor GANT61 compared to the blank control in SGC7901 MCAs and BGC823 MCAs. Scale bar = 10μm. b Lower colony forming ability in the group of Integrin α_vβ₃ inhibitor Cilengitide or ERK1/2 inhibitor PD-184161 or GLI1 inhibitor GANT61 compared to the blank control in SGC7901 MCAs and BGC823 MCAs. c Western blottingting showing downregulated stemness-related genes CD44, ALDH1A1, Oct4, Bmi1 and Nanog in the group of Integrin α_vβ₃ inhibitor Cilengitide or ERK1/2 inhibitor PD-184161 or GLI1 inhibitor GANT61 compared to the blank control in SGC7901 MCAs and BGC823 MCAs. d, e Decreased volume and weight of xenograft tumors in the group of Integrinα_vβ₃ inhibitor Cilengitide or ERK1/2 inhibitor PD-184161 or GLI1 inhibitor GANT61 compared to the blank control in SGC7901 MCAs and BGC823 MCAs. Each bar in the figure represents the mean ± SEM of triplicates. *p < 0.05; **p < 0.01

Fig. 4. The key effector GLI1 of Integrin α_vβ₃/ERK1/2 pathway regulates the stem cell-like phenotype in MCAs of GC cells.

a Western blottingting showing the most pronounced effect of GLI1 silencing in the shGLI1#1 group in BGC823MCAs and SGC7901MCAs. b Western blottingting showing evident overexpression of GLI1 in BGC823MCAs and SGC7901MCAs transduced with overGLI1 lentivirus. c Increased tumor spheres forming ability in the overGLI1 group and decreased formating ability in the shGLI1#1 group compared to the control group (WT) in SGC7901 MCAs and BGC823 MCAs. Scale bar = 10 μm. d Higher colony forming ability in the overGLI1 group and lower colony forming ability in the shGLI1#1 group compared to the control group in SGC7901 MCAs and BGC823 MCAs. e Western blottingting showing downregulated stemness-related genes in the shGLI1#1 group but upregulated genes CD44, ALDH1A1, Oct4, Bmi1 and Nanog in the overGLI1 group compared to the control group in SGC7901 MCAs and BGC823 MCAs. f, g Decreased volume and weight of xenograft tumors in the shGLI1#1 group and increased volume and weight in the overGLI1 group compared to the control group in SGC7901 MCAs and BGC823 MCAs. Each bar in the figure represents the mean ± SEM of triplicates. *p < 0.05; **p < 0.01

Fig. 5. In vivo validation of the role of the Integrin α_vβ₃/ERK1/2/GLI1 pathway on the peritoneal metastasis of GC.

a, b Higher abdominal cavity tumor nodules formed by overGLI1 and lower nodules formed by shIntegrin β₃#1 and shGLI1#1 compared to the control group transduced SGC7901 MCAs and BGC823 MCAs. Arrows indicate intraperitoreal nodules. c Western blottingting showing higher levels of Integrin α_vβ₃, p-ERK1/2, and GLI1 in fresh surgical specimens of GC peritoneal metastatic compared to paired primary tumors. Each bar in the figure represents the mean ± SEM of triplicates. *p < 0.05; **p < 0.01

Fig. 6

Integrin α_vβ₃ mediates intercellular adhesion in MCAs of GC cells to form an independent functional unit and activates GLI1 through the non-classic ERK1/2 pathway and crosstalk with the classic Hedgehog pathway to maintain cancer stem cell-like phenotype