Study On The Mechanism Of SCRIB Low Expression Fibroblasts To Promote Lung Cancer Cell Invasion

Background:Lung cancer is the most common cause of cancer-related death in the world.Most of these patients are non-small cell lung cancer(NSCLC).Squamous cell carcinoma and adenocarcinoma are the most common subtypes in NSCLC.Smoking,air pollution,and chronic inflammation are all causes of lung cancer.Oncogene activation and tumor suppressor gene deletion result to increase tumor cell proliferation and reduce apoptosis.Changes in tumor cell adhesion,cytoskeletal dynamics,and enhanced extracellular matrix(ECM)degradation promote tumor cells to break through the basement membrane and infiltrate into tissues.At the same time,tumor cells can also migrate into blood vessels and lymphatic vessels and be transferred to other organs of the body for colonization and growth.Tumor microenvironment(TME)is the external environment for tumor cell growth and plays an important role in the formation and development of tumors.Tumor-associated fibroblasts(CAFs)are an important component of tumor stroma.CAFs can directly affect tumor growth,invasion,and metastasis by secreting cytokines and reconstructing ECM,which also indirectly affect tumor progression by affecting tumor-associated immune cells and vascular endothelial cells.Single cell transcriptome sequencing for NSCLC revealed six CAFs subtypes,which are rare in the adjacent normal lung tissue.CAFs can be used as a predictor of the recurrence and survival prognosis of NSCLC.Elucidating the specific phenotype and function of CAFs in NSCLC will provide new therapeutic targets for NSCLC.CAFs are a group of activated fibroblasts located in the tumor stroma,which are long spindle-shaped and lack specific molecular markers.CAFs are derived from normal fibroblasts(NAFs),bone marrow-derived mesenchymal stem cells,pericytes,and epithelial-mesenchymal transition(EMT).Compared with NAFs,CAFs secrete a large number of cytokines,ECM components,and their ability to contract and invade is significantly enhanced.Tumor cells induce interstitial fibroblasts to aquire the phenotype and function of CAFs.For example,lung cancer cells educate NAFs to express a series of inflammatory factors expressed which are known expressed on CAFs after co-culture,the upregulated inflammatory factors have important roles in lung tumor growth.However,the function of CAFs is heterogeneous.For example,the absence of caveolin-1 expression in CAFs is related to the poor prognosis of triplenegative breast cancer.Another study found that the expression of caveolin-1 in CAFs promotes the invasion and metastasis of breast cancer.Therefore,identifying of the specific molecular characteristics of CAFs and the molecular mechanisms of how those molecular contribute to tumor progression is critical.SCRIB protein contains multiple domains,mainly including 16 LRR domains and 4 PDZ domains.They can bind multiple types of proteins such as cell polarity proteins,adhesion molecules,signaling proteins,kinases,etc.Therefore,SCRIB participates in a variety of cell biological processes such as cell polarity,migration,proliferation,apoptosis,invasion,etc.SCRIB can directly bind to cell signaling molecules or be associate with other proteins to indirectly regulate cell signaling pathway activities such as affecting PI3 K / AKT,Ras / MAPK,Hippo,WNT and other signaling pathways.The earliest studies in Drosophila melanogaster organisms show that the absence of SCRIB induces excessive proliferation of epithelial cells,thus SCRIB is considered as a tumor suppressor gene.In mammals,the loss of SCRIB expression in breast,prostate,and lung epithelial cells is also associated with disorders of epithelial structure and tissue.In lung epithelial cells,the absence of SCRIB combined with the KRAS mutation significantly promoted the growth of lung adenocarcinoma,increased tumor fibrosis and macrophage infiltration,suggesting the SCRIB has anti-tumor effect.However,the character of SCRIB on CAFs is not investigated yet.Therefore,in this study,we explored the functions of SCRIB on fibroblasts and its effect on lung tumor cells.Purpose:This study mainly explored the correlation between SCRIB expression in NSCLCderived CAFs and tumor progression,as well as the underlying mechanism of how SCRIB regulate the function of fibroblasts on lung tumor cell invasion.Methods:1)Immunohistochemical detection of SCRIB expression in CAFs of clinical lung squamous cell carcinoma and adenocarcinoma,and statistical analysis of its association with clinical features of patients.2)The mass spectrometry was used to detect the protein profile in co-cultured fibroblasts with lung cancer cell.QRT-PCR was used to detect the expression of SCRIB in co-cultured fibroblasts.3)Incu Cyte was used to observe the proliferation and migration ability of SCRIB knockdown and control fibroblasts;Transwell invasion assay was used to detect the invasion ability of SCRIB knockdown and control fibroblasts.4)Western blot was used to detect the signalling pathways activation in SCRIB knockdown and control fibroblasts;Transwell invasion test was used to detect the effect of inhibiting the corresponding signalling pathway in SCRIB knockdown and control fibroblasts on their invasion ability.5)Incu Cyte was used to detect the LLC proliferation when was co-cultured with SCRIB knockdown and control fibroblasts;Transwell migration and invasion assay was used to detect the migration and invasion ability of LLC when was co-cultured with SCRIB knockdown or control fibroblasts.6)Subcutaneous co-injection xenograft model was used to observe the tumor growth of LLC when was co-injected with SCRIB knockdown or control fibroblasts;hematoxylin-eosin staining(HE)was used to detect the muscle infiltration of tumor cells at the tumor margin in early tumor stage.7)Transcriptome sequencing was used to analyze differentially expressed genes between SCRIB knockdown and control fibroblasts,and q PCR and western blot were used to detect the asporin expression in SCRIB knockdown and control fibroblasts.8)si RNA was used to inhibit the ASPN expression of SCRIB knockdown fibroblasts,and Transwell invasion assay was used to observe the effect of inhibition of ASPN in SCRIB knockdown fibroblasts on LLC invasion.Results:In this study,we found that the low expression of SCRIB in CAFs of human lung squamous cell carcinoma was associated with late tumor stage and poor survival prognosis.The expression of SCRIB in human lung adenocarcinoma CAFs was not associated with tumor stage,lymph node metastasis,and clinical stage.The expression of SCRIB in fibroblasts co-cultured with lung cancer LLC cells was reduced.The proliferation of SCRIB knock-down fibroblasts was slightly weakened,and invasive ability was significantly enhanced.Inhibition of the PI3 K / AKT signaling pathway could attenuate SCRIB knockdown fibroblasts invasion.SCRIB knockdown fibroblasts had no significant effect on LLC proliferation,but significantly promoted their migration and invasion.SCRIB knockdown of fibroblasts had no significant effect on the early growth of LLC tumors,and promoted tumor cell to infiltrate into adjacent muscle layers.Transcriptomics analysis revealed that knockdown of SCRIB increased the expression of ASPN in fibroblasts,and inhibition of ASPN reduced its ability to promote the invasion of LLC cells in vitro.Using a database for predictive analysis of ASPN transcription factors,STAT5 A is a potential transcription factor that regulates ASPN expression.Knockdown of SCRIB increased activation of STAT5 signaling pathway in fibroblasts.Conclusion:This study indicated that fibroblasts with low expression of SCRIB promoted their own invasion through the PI3 K / AKT signaling pathway,and could promote the invasion of lung cancer cells in vitro by increasing the secretion of ASPN.