Endothelial SPEN Deficiency Normalizes Tumor Vasculature Via Nucleolar Stress

Background Blood vessels are the most widely distributed organs in the human body,and they play an important role in regulating tissue homeostasis,blood circulation as well as tumor growth,metastasis,and tumor microenvironment.There are currently two generally recognized strategies to target tumor blood vessels,one of which is inhibiting tumor angiogenesis and the other is promoting the normalization of tumor vessels.However,inhibiting tumor angiogenesis alone will result in elevated tumor drug resistance and toxic side effects.Therefore,in recent years,it has been proposed to promote the normalization of tumor vessels at a certain stage of tumor growth,thereby improving the tumor microenvironment and inhibiting tumor growth.Ribosome is an important organelle for protein synthesis in cells.It is composed of ribosomal RNA and proteins.Ribosomal RNA is transcribed from ribosomal DNA in the nucleolus.If the transcription of ribosomal DNA is inhibited,the synthesis of ribosomal DNA is following blocked,and protein synthesis in the cell is therefore affected.Recently,single cell RNA sequencing of tumor endothelium revealed that the expression of ribosomes in tumor endothelium was higher than that in normal endothelium,but the function of ribosome synthesis on tumor endothelium need further investigation.SPEN is a nuclear protein ubiquitously expressed in most kinds of cells,and interacts with multiple pathways such as Wnt and Notch.SPEN also binds to Xist and recruits histone deacetylases to directly participate in the inactivation of the X chromosome.However,the role of SPEN in vascular development and tumor angiogenesis has not been illustrated.Therefore,clarifying the function of SPEN in endothelial cells and its mechanisms will provide insights for novel molecular mechanisms underlying vascular development and new targets for targeted therapy of tumor angiogenesis.Objectives To investigate the role of SPEN in endothelial cells during physiologic and tumor angiogenesis as well as its molecular mechanisms,and to carry out translational medicine research based on SPEN mechanism of action.Methods and Results 1.SPEN is ubiquitously expressed in the nucleus of endothelial cells.Immunofluorescence staining of lung,kidney,brain,heart,liver and retina in C57BL/6J mice showed that SPEN co-localized with endothelial markers in nuclei.The results of real-time PCR demonstrated that the m RNA level of SPEN in endothelial cells was elevated during the development of the retinal vasculature and LLC tumors.2.Endothelial specific SPEN deletion inhibited physiologic angiogenesis.To determine whether SPEN regulates angiogenesis,we assessed retinal vascular development in ec SPEN-/-mice.Examination of ec SPEN-/-retinas by immunofluorescence staining shown a decrease in both tip cell number and vessel density.Staining for Brd U incorporation revealed a reduction of EC proliferation in ec SPEN-/-mice.In the model of matrigel-assisted vessel formation,vessel density in ec SPEN-/-mice was also decreased.3.Endothelial specific SPEN deletion reduced tumor growth and metastasis,as well as promoted the normalization of tumor vessels.LLC or B16F10 cells were inoculated in ec SPEN-/-mice,and reduced tumor growth was observed in both tumor models.Also,in LLC inoculated ec SPEN-/-mice,tumor metastasis was reduced,and the survival time was prolonged.Examination of ec SPEN-/-tumor sections by immunofluorescence staining revealed decreased blood vessel density,increased pericytes and basement membrane coverage,reduced vascular leakage,enhanced perfusion of vessels,and decreased hypoxic degree.Meanwhile,smoother inner walls of the endothelial cells were observed under scanning electron microscope in ec SPEN-/-mice.The endothelial specific SPEN deletion in combination with cisplatin showed increased effects of chemotherapy drugs on tumorigenesis.H&E staining revealed increased necrosis degree in the endothelial specific SPEN deletion combined with the chemotherapy.4.Endothelial specific SPEN deletion improved tumor microenvironment.The expression levels of IFNγ and TNFα within the tumor were increased in ec SPEN-/-mice through qRT-PCR detection of tumor RNA extracts.Flow cytometry analysis revealed a significant increase in the percentage and absolute number of T lymphocytes in ec SPEN-/-mice.FACS analysis and sorting results also showed increased IFNγ-producing CD8+ T cells after endothelial specific SPEN deletion.5.SPEN knockdown inhibited proliferation,migration and angiogenic capacity in endothelial cells.HUVECs were infected with sh SPEN lentivirus,and a significant increase in the volume of endothelial cells was observed.Staining for Ed U incorporation and FACS analysis for cell cycle revealed that the proliferation ability was inhibited in sh SPEN endothelial cells.Meanwhile,SPEN knockdown inhibited cell migration,as shown in scratch wound and Transwell assay.Live-cell imaging was also analyzed for the movement,and HUVECs infected with sh SPEN lentivirus had reduced motility.Immunofluorescence staining of HUVECs revealed reduced formation of lamellipodia and increased level of F-actin after SPEN knockdown.Impaired endothelial sprouting and tube formation was also observed in fibrin beads assay and tube formation assay.6.P53 is a central component of SPEN functions in ECs.To gain insight into the underlying mechanisms for SPEN fuctions,we performed transcriptome analysis of HUVECs infected with sh SPEN lentivirus and tumor endothelial cells isolated in ec SPEN-/-mice using anti-CD31 microbeads.Bioinformatics analysis revealed an enrichment of P53 signaling in genes induced by SPEN interference,and these results were verified by qRT-PCR,western blotting and reporter assay.In HUVECs infected with sh SPEN lentivirus combined with sh P53 or sh P21 lentivirus,impaired proliferation,migration,movement and angiogenic capacity of HUVECs after SPEN knowdown were rescued.In endothelial specific SPEN deletion combined with P53 haplo-insufficient mice,the phenotypes of ec SPEN-/-mice were also rescued,such as reduced tumor growth,promoted normalization of tumor vessels and improved tumor microenvironment.7.SPEN deletion in endothelial cells triggered nucleolar stress,which in turn activated P53 signaling pathway.GSEA analysis of HUVECs transcriptome and qRT-PCR results revealed that the expression of ribosome genes were consistently reduced after SPEN knockdown.HUVECs were immuno-stained with nucleolus markers,observation under superresolution microscopy revealed that SPEN knockdown abrogated the phase separation of NPM1 and resulted in UBF and RPA40 relocation to the nucleolar periphery,and the morphology was reminiscent of “nucleolar necklaces”.After the treatment with CHX,P53 protein in sh SPEN group was more stable and degraded more slowly.Also,western blotting revealed that P53 nuclear translocation was elevated after SPEN knockdown,and IP showed SPEN knockdown disrupted P53-MDM2 interaction.8.SPEN knockdown changed the chromatin modification of r DNA locus and reduced r RNA synthesis.Results of qRT-PCR showed that SPEN knockdown reduced the expression level of pre-r RNA and mature ribosomal RNA,such as 18 S r RNA,28 S r RNA and 5.8S r RNA.Moreover,the reduction in pre-r RNA level occurred before the activation of P53 signaling pathway.CHIP and qRT-PCR revealed that histone modifications associated with transcriptional activation were decreased at gene promoter of r DNA locus after SPEN knockdown and repressive modifications were elevated.In addition,the binding of RNA polymerase I transcription preinitiation complex on r DNA locus was also found decreased by CHIP analysis with RPA194 and UBF antibody.The above phenomena could be understood as a result of elevated p RNA after SPEN knockdown.9.RNA polymerase I inhibitor CX5461 could promote tumor vessel normalization under certain conditions.The results of real-time PCR demonstrated that the expression level of pre-r RNA in endothelial cells was elevated during the development of the LLC tumors.Tumor vessels could be normalized in C57BL/6J mice received an oral gavage of CX5461 under certain conditions.Moreover,when combined with cisplatin or anti-PD-1 antibody administration in C57BL/6J mice,CX5461 administration could promote T lymphocyte infiltration and improve the efficacy of chemotherapy drugs and anti-PD-1 therapeutic antibody.Conclusions Endothelial cell SPEN deletion could induce nucleolar stress by inhibiting the transcription of ribosomal DNA,therefore activated P53 signaling pathway and reduced the proliferation of endothelial cells,inhibited physiological angiogenesis and promoted tumor vessel normalization,and then enhanced the infiltration of T lymphocytes and IFNγ production in CD8+ T cells.Based on these mechanisms,it is found that the RNA polymerase I inhibitor CX5461 could promote tumor vessel normalization under certain conditions as well as promote the infiltration of T lymphocytes.Moreover,CX5461 administration could be combined with cisplatin and anti-PD-1 antibody to promote therapeutic efficacy.