The Role And Mechanism Of GLI1 Activation By Integrin α_vβ₃/ERK1/2 Maintains Stem Cell-like Phenotype Of Multicellular Aggregates In Gastric Cancer Peritoneal Metastasis

Background:Gastric cancer（GC）is the third leading cause of cancer-related deaths worldwide.Peritoneal metastasis is the most common metastasis observed in GC patients after surgery.According to the classic―seed and soil‖theory,the survival of exfoliated cancer cells from the primary site to the abdominal cavity is an important step of GC peritoneal metastasis.Two common types of exfoliated cancer cells are scattered-free cancer cells and multiple exfoliated cancer cells.The latter forms multicellular aggregates/spheroids（MCAs/MCSs）.Scattered-free cancer cells often undergo anoikis when nutrition is relatively scarce,while MCAs can be suspended and grown in the abdominal microenvironment.MCAs of GC cells are the major seeding cells of peritoneal metastases,but the survival mechanism of MCAs remains unclear.In our study,we found that MCAs of GC cells possessed several cancer stem cell-like phenotypes,including self-renewal,cancer stem cell marker gene expression and tumorigenesis in vivo.Therefore,we investigated the mechanism of maintaining cancer stem cell-like phenotype.The classical Hedgehog-GLI signaling pathway plays an important role in the regulation of the stemness of cancer cells.GLI1 is not only a downstream key effector of the classical Hedgehog（Hh）ligands-PTCH1-SMO axis,but also has crosstalk with the non-classical PI3K/AKT,TNF-α/mTOR,and MAPK/ERK1/2 pathways.Our results showed that the mRNA expression of stemness-related markers in peritoneal MCAs of exfoliated GC cells was slightly decreased after treatment with inhibitors of the classical Hedgehog pathway,whereas the addition of GLI1 or ERK1/2 inhibitors resulted in a significant decrease.These results suggest that the stem cell-like phenotype of gastric cancer MCAs may be regulated by the activation of GLI1 via the ERK1/2 pathway.The specific regulatory pathway needs to be studied further.Integrin is a heterodimer formed byαandβsubunits.Integrins are distributed and function differently in different tissues.Theβ₃ subunit mediates tumor cell aggregation and cell viability.Our results indicated that Integrinα_vβ₃ mediates the aggregation of exfoliated GC cells to form MCAs in the abdominal cavity.GLI1 often functions as a downstream regulatory molecule of the Integrin signaling pathway.Objective:1.To verify that GLI1 is activated via the non-classical pathway Integrinα_vβ₃/ERK1/2 in MCAs of GC cells.2.To investigate how the Integrinα_vβ₃/ERK1/2/GLI1 pathway regulates the stem cell-like characteristics of gastric cancer MCAs.Materials and methods:1.Collect MCAs in ascites/peritoneal lavage fluid from patients who suffered from peritoneal metastasis after gastric cancer surgery to identify the cancer stem cell-like biological characteristics of peritoneal MCAs of exfoliated GC cells,including self-renewal,proliferation,drug resistance,stemness-related markers and tumorigenesis.2.Establish serum-free suspension culture SGC7901 MCAs and BGC823 MCAs.Use Integrinα_vβ₃ inhibitor Cilengitide,co-stimulator ligand RGD,ERK inhibitor PD-184161,Hedgehog/Smoothened pathway inhibitor Cyclopamine,lentivirus to silence Integrinβ₃ or Smo,the luciferase reporter gene vector containing the GLI1 promoter region to verify GLI1is regulated through the non-classical pathway Integrinα_vβ₃/ERK1/2 in MCAs of gastric cancer cells.3.Integrinα_vβ₃ inhibitor Cilengitide,ERK inhibitor PD-184161 and GLI1 inhibitor GANT61 were added into BGC823 MCAs and SGC7901 MCAs.The stem cell-like characteristics of each experimental group were examined for self-renewal,proliferation,the protein levels of stemness-related markers,and tumorigenesis to investigate whether the Integrinα_vβ₃/ERK1/2/GLI1 pathway regulates the stem cell-like characteristics of gastric cancer MCAs.4.BGC823 MCAs and SGC7901 MCAs were transduced with either GLI1 silencing virus or GLI1 overexpressing vectors,respectively.Then the stem cell-like characteristics of each experimental group was examined for self-renewal,proliferation,the protein levels of stemness-related markers,and tumorigenesis.To verify GLI1 is the key molecule that regulates the stem cell-like biological characteristics of gastric cancer MCAs through the Integrinα_vβ₃/ERK1/2/GLI1 pathway.5.Use scattered cells from BGC823 MCAs and SGC7901 MCAs transduced with either shIntegrinβ₃#1,shGLI1#1,overexpression vector of GLI1 to construct a model of peritoneal metastasis of gastric cancer in nude mice to further verify the role of the non-classical pathway Integrinα_vβ₃/ERK1/2/GLI1 in the peritoneal metastasis of gastric cancer in vivo.Results:1.Compared with scattered-free cancer cells,peritoneal MCAs of exfoliated GC cells had increased colony forming efficiency,higher levels of stemness-related genes,including CD44,ALDH1A1,Oct4,Bmi1 and Nanog,larger number of tumor spheres forming efficiency,stronger drug resistance over time for 5-fluorouracil and oxaliplatin,larger number of the xenograft tumors.2.Integrinα_vβ₃ can regulate GLI1 in gastric cancer MCAs through ERK1/2 pathway.The Hh ligands-PTCH1-SMO axis,which is the classical pathway of GLI1 activation,regulates GLI1 slightly.3.In contrast to the blank control group,the stemness characteristics of GC MCAs in the group of Integrinα_vβ₃ inhibitor Cilengitide,ERK inhibitor PD-184161,GLI1 inhibitor GANT61 were significantly reduced.And the reduction in the stem cell-like phenotype was the most profound in the GLI1 inhibitor group.4.The stem cell-like biological characteristics in the shGLI1#1 group were significantly reduced compared with the control group.In contrast,the stem cell-like biological characteristics in the overGLI1 group significantly increased.5.The number of xenograft tumors in the abdominal cavity of nude mice in the group of shIntegrinβ₃#1 and shGLI1#1 were significantly decreased,while the number in the overGLI1 group was significantly increased.Conclusions:1.Compared with scattered-free cancer cells,peritoneal MCAs of exfoliated GC cells have a stronger capacity for self-renewal,proliferation,drug resistance,stemness-related markers and tumorigenesis,and express stem cell-like characteristics.2.Although crosstalk with the classic Hedgehog-GLI signaling pathway exists,GLI1activation occurs mainly through the non-classical pathway Integrinα_vβ₃/ERK1/2 in MCAs of GC cells.3.Integrinα_vβ₃/ERK1/2/GLI1 pathway regulates the stem cell-like characteristics of gastric cancer MCAs.The inhibition of any molecule in the Integrinα_vβ₃/ERK1/2/GLI1pathway can downregulate the stem cell-like characteristics in MCAs of gastric cancer cells.4.GLI1 is the key molecule that regulates the stem cell-like biological characteristics of gastric cancer MCAs through the Integrinα_vβ₃/ERK1/2/GLI1 pathway.5、Further validates the potential role of the Integrinα_vβ₃/ERK1/2/GLI1 pathway in the peritoneal metastasis of gastric cancer.