A Study On The Maintenance Of Prostate Epithelial Features And Underlying Molecular Mechanisms

Objective:The prostatic epithelium is mainly comprised of two different types of epithelial cells,basal cells and luminal cells.They display similarities but possess respective specific attributes which are regulated and sustained by distinct molecular mechanisms.This study aims to:(1)explore the mechanisms and key factors in controlling the stem/progenitor cell functions of prostatic basal epithelial cells,(2)illustrate the underlying molecular mechanisms orchestrating cell-cell junctions,cell polarity and mitotic spindle positioning of the prostatic luminal epithelium.Methods:The expression pattern and biological functions of epithelial-to-mesenchymal transition transcriptional factors were assessed in vitro and in vivo.Signaling pathways enriched in Zeb1~+epithelial cells were investigated utilizing high-throughout single cell RNA sequencing and in vivo experiments to maintain stemness and EMT features of the basal stem cell subpopulation and ensure the normal development.The expression patterns of cell-cell adherens junction protein and phenotypes of E-cadherin knockout mice were evaluated in vitro and in vivo.The interactions among cell-cell adherens junctions protein E-cadherin,Scribble polarity complex and mitotic spindle orientation determinant LGN complex were determined by RNA sequencing and biochemical approaches.All statistical tests were two-sided.Results:(1)The core epithelial-to-mesenchymal transition inducer Zeb1 is exclusively expressed in a prostate basal cell subpopulation,which shares gene expression signatures with both epithelial and mesenchymal cells.The Zeb1~+prostatic basal cells were more frequently detected in the urethra-proximal region relative to the distal region,the location where prostate stem cells were suggested to reside.(2)Zeb1~+basal cells were detected both in prostates of Hi-myc mice,a spontaneous prostate cancer mouse model,and human samples.Intriguingly,Zeb1~+CK8~+luminal cells were barely detected in non-BPH but could be observed occasionally in BPH specimens and more frequently in prostate cancer specimens.(3)Compared to Zeb1~-basal cells,Zeb1~+basal cells possessed greater serial organoid forming and serial kidney capsule transplantation capacities,which suggested that Zeb1~+basal cells were multipotent prostate stem cells and possessed the capacity of generating functional prostates.(4)In vitro and in vivo Zeb1 deletion caused marked decrease of basal cells and impeded the normal development of prostate basal cell compartment.However,Zeb1 loss had minimal effect on the normal development of prostate luminal cells and neuroendocrine cells.(5)Wnt signaling pathway played a positive role on the expansion of Zeb1~+basal cells and contributed to sustain specific phenotypes of prostate basal cells.(6)E-cadherin was more predominantly expressed in the lateral cell membrane of tightly-contacted prostatic luminal cells than scattered basal cells over time.(7)At different time points during prostate development,growth and castration-regeneration stages,E-cadherin deficiency led to a hyperproliferative phenotype of prostatic luminal cells,promotion of carcinogenesis and invasion of epithelial cells into the surrounding prostatic stroma due to disruption of the basement membrane in the aged mice.(8)In vitro and in vivo experiments confirmed that horizontal division of prostate luminal cells was randomized by lack of E-cadherin.Meanwhile,E-cadherin deficiency impaired cellular distribution and association of key spindle positioning proteins LGN and NuMA as well as cell polarity proteins complexes,PAR and SCRIB.(9)Genes regulating cellular biological processes including cell proliferation,cell adhesion and cell migration were the most transcriptionally altered genes.Additional qRT-PCR confirmation experiments showed that genes which act to promote cell proliferation and cell migration were more preferentially expressed in the E-cadherin null prostate epithelial cells,whereas molecules that facilitate cell junctions were significantly downregulated in E-cadherin knockout prostate epithelia.Moreover,the transcriptional profile of E-cadherin knockout mouse prostates resembled the human prostate cancer from the TCGA human prostate cancer database.(10)Mechanistically,Co-IP assay showed endogenous interactions among E-cadherin,SCRIB and LGN in RWPE-1 cells.Endogenous SCRIB interacted with LGN,which can be suppressed by E-cadherin knockdown.Furthermore,the association between LGN and E-cadherin was markedly attenuated by SCRIB knockdown.The fragment containing the PDZ domain of SCRIB directly bound to both the E-cadherin and LGN.Conclusion:(1)The core EMT inducer Zeb1 is exclusively expressed in a small prostate basal cell subpopulation.The Zeb1~+prostate epithelial cells are multipotent prostate basal stem cells(PBSCs)that can self-renew and generate functional prostatic glandular structures with all three epithelial cell types.Meanwhile,The Zeb1~+epithelial cells play an indispensable role on normal prostate basal cell development.(2)The prostate luminal cell adherens junctions,cell polarity and mitotic spindle positioning are tightly regulated to maintain luminal epithelial integrity and to prevent carcinogenesis in vivo.E-cadherin and the spindle positioning determinant LGN interact with the PDZ domain of cell polarity protein SCRIB and form a ternary protein complex to bridge cell polarity and cell division orientation.Scientific significances:The discovery and functional studies of Zeb1~+epithelial cells unveil multipotent basal stem/progenitor cells in normal development of the prostatic epithelium and provide new clues for the cell of origin for prostate cancers.Uncovering the E-cadherin/SCRIB/LGN complex in prostate luminal cells provides an important molecular link to orchestrate cellular adherens junctions,cell polarity and mitotic spindle positioning which are essential in maintain the proper luminal epithelial architecture.These findings have important implications for our understanding of prostate development and regeneration and for tumorigenesis.