Study Of PLEC,a Critical Target Of Notch Signaling,in Regulating Squamous Epithelial Homeostasis And Participating In Carcinogenesis Of Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is a common malignant tumor of digestive system,with high malignancy and poor prognosis.Occurrence of ESCC is a result of interactions of environmental and genetic factors.Environmental factors mainly include exposure to chemical carcinogens,smoking and drinking.In terms of genetics,several high-throughput whole-genome or whole-exon sequencing revealed some importantly altered genes in ESCC,such as TP53,Notch1,CDKN2A and PIK3CA.These genes involve multiple driving events including genomic instability,differentiation,proliferation,cell cycle and epigenetic changes,of which differentiation plays an extremely important role,but its molecular mechanisms have not yet been fully elucidated.Notch signaling is one of the key pathways in regulating squamous epithelial differentiation,of which Notchl is the dominant factor.Notch 1 is also involved in the regulation of various differentiation-related cytoskeletal proteins and cell morphology.In squamous cell carcinoma(SCC),Notch signaling plays a�double-edged sword' role.Notch functions as a tumor suppressor gene,however,Notch signaling can also be activated to accelerate the development of SCC.Plectin(PLEC)is a cytoskeletal crosslinker that interacts with three major cytoskeletal systems,including intermediate filaments(IFs),microtubules and actin filaments,and participates in regulation of cell morphology and mechanical dynamic properties of them.Several studies have reported that PLEC mutations are linked with pathogenesis of malignant tumors.However,the role of PLEC in ESCC remains unclear,and the relationship of it,as an important cytoskeletal crosslinker,with differentiation-homeostasis of squamous epithelium and Notch signaling has not yet been confirmed.In this study,bioinformatic analysis identified that PLEC showed high frequency of mutations in human ESCC tissues,and it may be related to Notch signaling.Subsequently,our study confirmed that in differentiation homeostasis of esophageal epithelium,PLEC was a downstream target of Notch signaling using differentiation model induced by calcium in vitro.In differentiated epithelium,plectin displayed significant membranous and partial cytoplasmic localization,and interacted with cytokeratins and desmoplakin to maintain the morphology and function of epithelial cells.Knockdown of PLEC by lentiviral shRNAs induced alteration of cell morphology and impaired epithelial homeostasis.Additionally,using three-dimensional organoid system derived from rat esophageal epithelial cells,we demonstrated that PLEC deficiency significantly inhibited organoid formation,thus illustrating the importance of plectin in normal epithelium.Afterwards,our study investigated the role of plectin in tumorigenesis and development of ESCC.We revealed that PLEC showed different expression patterns in ESCC samples by qRT-PCR and tissue microarray-immunohistochemistry assays,and its expression was positively correlated with Notchl expression.By functional assays,our study uncovered that PLEC exerted a dual role in suppressing or promoting ESCC,and its differential functions were associated with genetic background and localization of PLEC as well as tumor differentiation.In ESCC with Notch or PLEC mutations,aberrant function or decreased expression of PLEC contributed to carcinogenesis;in well differentiated ESCC with wild-type PLEC,PLEC expression was mainly involved in differentiation and inhibits tumorigenesis,however,for poorly differentiated ESCC with wild-type PLEC,high expression of PLEC played a role in promoting cancer.Taken together,PLEC,as a critical target gene of Notch signaling,participates in the maintenance of differentiation homeostasis of esophageal epithelium,and exerts an important role in carcinogenesis and development of ESCC that will provide novel guidance and direction for improvement of ESCC pathogenesis and exploration of therapeutic targets.