A SUMOylation-dependent HIF-1?/CLDN6 Negative Feedback Mitigates Hypoxia-induced Breast Cancer Metastasis

Intra-tumoural hypoxia is commonly found in breast cancer due to the rapid growth of tumour and abnormalities in the tumour vasculature,causing a significantly increased risk of breast cancer metastasis.The physiological response to hypoxia is mediated mainly by hypoxia-inducible factor 1?HIF-1?,a heterodimer composed of the oxygen-sensitive subunit HIF-1? and the stable subunit HIF-1?.Under normoxic conditions,HIF-1? is hydroxylated by prolyl hydroxylase domain enzymes?PHDs?and then targeted by the ubiquitin ligase component von Hippel-Lindau?VHL?for degradation.Hypoxia decreases the hydroxylation activity of PHDs and results in HIF-1? protein stabilization and translocation to the nucleus;here,HIF-1? dimerizes with HIF-1? and binds to hypoxia response elements?HREs;5?-A/GCGTG-3??in the genome,leading to the transcriptional activation of hundreds of genes including VEGF,TWIST,SNAIL and GLUT,thus promoting multiple steps within the metastatic cascade.As such,HIF-1? degradation under hypoxic conditions is an essential homeostatic and tumour-suppressing mechanism.Recent data indicate that HIF-1? stabilization is regulated not only by the conventional PHD?VHL system but also by other mechanisms.SUMOylation,the conjugation of small ubiquitin-related modifier protein?SUMO?to a target protein,has been considered to play an essential regulatory role in HIF-1? protein stability.SUMOylation of HIF-1? promotes recruitment of the modified protein to VHL through a PHD-independent mechanism,leading to ubiquitination and proteolysis even under hypoxic conditions.That is,SUMOylation serves as another direct signal of VHL binding for ubiquitin-dependent HIF-1? degradation.SUMOylation is a dynamic process and can be reversed by sentrin/SUMO-specific proteases?SENPs?.It has been reported that SENP1 can remove SUMO-1 from SUMOylated HIF-1? and allows HIF-1? to escape degradation during hypoxia.Tight junctions?TJs?are composed of integral transmembrane and peripheral membrane proteins involved in complex protein-protein interactions.CLDN6 is a 26-kDa TJ protein containing four transmembrane helices with a carboxyl-terminal tail extending into the cytoplasm.The PDZ-domain-binding motif in the carboxy-terminal tail allows CLDN6 to interact with cytoplasmic TJ-associated proteins such as ZO1,?-catenin and cadherins thus regulating various signalling pathways.We found that CLDN6 was transcriptionally upregulated by HIF-1? in three breast cancer cell lines.However,our recent work has shown that CLDN6 may be a tumour suppressor gene in breast cancer.Little is known about the role of CLDN6 in cellular adaptation to hypoxia,whereas the roles of HIF-1? are well understood.Herein,a negative loop involving a SUMOylation-dependent feedback mechanism was identified to explain this seemingly contradictory result.In this study,we demonstrated that HIF-1? accumulation under hypoxia promotes CLDN6 transcription.On the other hand,increased CLDN6 weakens HIF-1? protein stability by reducing SENP1 expression and preventing the deSUMOylation of HIF-1?.This negative feedback loop contributes to oxygen homeostasis and slows down hypoxia-induced breast cancer metastasis.Methods:1.Regulation of hypoxia on CLDN6 expression in breast cancer cells?1?Regulation of hypoxia on CLDN6 expressionBreast cancer cell lines MDA-MB-231,MCF-7,and SkBr-3 were treated with hypoxia culture or chemical hypoxia mimic agent CoCl₂,respectively;RT-PCR was used to detect changes in CLDN6 mRNA expression levels,and Western Blot was used to detect CLDN6 protein expression levels.?2?Mechanism of regulation of hypoxia on CLDN6 expressionHIF-1? knockdown virus was used to transduce MDA-MB-231 cells;Western Blot was used to detect CLDN6 expression under hypoxia;GCBI tool was used to predict the potential HIF-1? binding sequence in the promoter region of CLDN6;luciferase reporter assay was used to detect the effect of HIF-1? on CLDN6 transcription.2.CLDN6 attenuates hypoxia-induced breast cancer metastasis through negative feedback HIF-1??1?Effect of CLDN6 on HIF-1? expression and transcriptional activityA breast cancer MDA-MB-231 cell line stably overexpressing CLDN6 was constructed,and mRNA seq and KEGG were enriched to analyze the downstream pathway of CLDN6;RT-PCR and Western Blot were used to detect the expression of HIF-1? after overexpression of CLDN6;RT-PCR was used to detect HIF-1? downstream target genes Glut1,EPO and SOX2 expression after overexpression of CLDN6.?2?CLDN6 inhibits hypoxia induced breast cancer metastasis by down regulating HIF-1?HIF-1? was overexpressed in MDA-MB-231/CLDN6 and SKBR-3/CLDN6;cell migration was detected by scratch test;cell invasion was detected by Transwell test;the expression of N-cadherin,vimentin and E-cadherin was detected by Western Blot.?3?Mechanism of CLDN6 inhibition of HIF-1?? Whether CLDN6 inhibits HIF-1? through the classical PHD/VHL pathway: breast cancer cells were treated with a nascent protein synthesis inhibitor CHX,and the half-life of HIF-1? protein was detected by Western Blot;then cells were treated with the proteasome inhibitor MG-132,and HIF-1? protein expression was detected by Western Blot to determine the pathway by which CLDN6 promotes HIF-1? degradation;RT-PCR and Western Blot were used to detect the effect of CLDN6 on PHD/VHL pathway expression.? The mechanism of CLDN6 inhibiting HIF-1? through SENP1: RT-PCR and Western Blot detected the effect of CLDN6 on breast cancer cell lines MDA-MB-231,MCF-7 and SkBr-3 expression;RT-PCR and Western Blot were used to detect the expression of SENP1 after CLDN6 was knocked down in HBL-100;overexpression of SENP1 was detected in MDA-MB-231/CLDN6 cells,and the expression of HIF-1? was detected by Western Blot under hypoxia;the level of global SUMOylation of proteins in breast cancer cells was tested by Western Blot;the effect of CLDN6 on the SUMO-1 conjugation level of HIF-1? protein was tested by IP experiments;overexpression of SENP1,Western Blot was used to detect the level of global SUMOylation;the Co-IP experiment were used to detect the SUMO-1 conjugation level of HIF-1? protein;Western Blot was used to detect the effect of CLDN6 on the expression of HIF-1? SUMO site mutant plasmid.? The mechanism of CLDN6 inhibition of SENP1: ChIP test was used to verify the binding of ?-catenin protein to the predicted site of SENP1 promoter region;Western Blot detected the effect of CLDN6 on ?-catenin expression;RT-PCR and Western Blot were used to detect SENP1 expression after overexpression of ?-catenin in MDA-MB-231/CLDN6 cells;CHX-treated cells were used to detect the effect of CLDN6 on ?-catenin protein half-life;Co-IP was used to detect the binding of CLDN6 and ?-catenin;Duolink PLA test was used to observe CLDN6 and ?-Catenin binding site in breast cancer cells;Western Blot was used to detecte the effect of CLDN6 on ?-catenin expression in plasma and nucleoproteins.3.Animal experiments to detect the effects of CLDN6/SENP1/HIF-1? on breast cancer metastasis28 female BALB/c nude mice were randomized into four groups;the MDA-MB-231/Vec,MDA-MB-231/CLDN6,MDA-MB-231/CLDN6+HIF-1? or MDA-MB-231/CLDN6+SENP1 cells were injected into the mice via the tail vein.After four weeks bioluminescence imaging was performed;then the mice were euthanized,the lung metastatic nodules were examined macroscopically and subjected to H&E staining;genomic DNA was extracted and measured by qPCR assays with primers for human HK2 gene and mouse 18 S rRNA to reflect the amount of circulating tumor cells.Results:1.Regulation of hypoxia on CLDN6 expression in breast cancer cells?1?Regulation of hypoxia on CLDN6 expressionCompared with the control group,the hypoxia culture or CoCl₂ treatment both significantly increased the mRNA and protein expression of CLDN6.?2?Mechanism of regulation of hypoxia on CLDN6 expressionCompared with the control group,the expression of CLDN6 in MDA-MB-231/shHIF-1? cells was significantly reduced under hypoxia;ChIP experiments showed that HIF-1? binds to the HRE sequence in the CLDN6 promoter region;in contrast,the fluorescence activity of the HIF-1? plasmid group was increased under hypoxia or transfection.Those results indicate that CLDN6 is a target gene of HIF-1?.2.CLDN6 attenuates hypoxia-induced breast cancer metastasis through negative feedback HIF-1??1?CLDN6 inhibits HIF-1? and downstream target gene expressionKEGG analysis showed that overexpression of CLDN6 significantly affected the expression of HIF-1 pathway,suggesting that CLDN6 may feedback regulate HIF-1 pathway;in both MDA-MB-231 and SkBr-3 breast cancer cell lines,overexpression of CLDN6 significantly reduced the deficiency hypoxia-induced accumulation of HIF-1? protein;CLDN6 significantly reduced the expression of downstream target genes of HIF-1??Glut1,EPO and SOX2?under hypoxia.?2?CLDN6 inhibits hypoxia induced breast cancer metastasis by down regulating HIF-1?Overexpression of CLDN6 inhibited N-cadherin and Vimentin expression and promotes E-cadherin expression,while restoring HIF-1? levels significantly restored the suppressed EMT phenotype.CLDN6 significantly reduced the migration rate and the number of invasive cells in breast cancer cells.Restoring of HIF-1? significantly increased the migration rate and number of invasive cells in breast cancer cells.The above results indicate that CLDN6 inhibits the migration and invasion of breast cancer cells by down-regulating HIF-1?.?3?Mechanism of CLDN6 inhibition of HIF-1?? CLDN6 does not depend on the classical PHD/VHL pathway to inhibit HIF-1?: Compared with the control group,overexpressing CLDN6 does not significantly affect the expression of HIF-1? mRNA under normal or hypoxia conditions,but significantly increases the degradation rate of HIF-1?;HIF-1? expression was increased in the both MDA-MB-231/Vec and MDA-MB-231/CLDN6 cells after MG-132 treated cells,but there was no statistical difference in HIF-1? levels between the two groups,indicating that CLDN6 promotes HIF-1? degradation via the ubiquitin-protease pathway;CLDN6 didn't affect the mRNA and protein levels of PHD/VHL pathway proteins,indicating that the regulatory effect of CLDN6 on HIF-1? may be mediated by other non-classical pathways.? CLDN6 inhibits HIF-1? through SENP1: Transcriptome sequencing analysis found that CLDN6 inhibits the expression of SENP1;in breast cancer cells,over-expressing CLDN6 significantly inhibited the expression of SENP1,the mRNA and protein expression of SENP1 increased significantly after knocking down CLDN6 in the cell line HBL-100;overexpression of SENP1 in MDA-MB-231/CLDN6 cells significantly restored the protein accumulation of HIF-1? under hypoxia,indicating that CLDN6 inhibited HIF-1? via SENP1.CLDN6 significantly increased SUMO-1 conjugation of HIF-1? protein.Overexpression of SENP1 in MDA-MB-231/CLDN6 cells restored increased SUMO-1 conjugation of HIF-1? protein;CLDN6 significantly inhibited HIF-1? ^WTprotein rather than HIF-1? ^K391,477Rprotein,indicating that the SUMO site is necessary for CLDN6 to regulate HIF-1?.The above results indicate that CLDN6 increases the SUMOylation level of HIF-1? through SENP1.? CLDN6 inhibits SENP1 through ?-catenin: ChIP experiments show that ?-catenin binds to the predicted site of SENP1,indicating that ?-catenin may be one of the transcription factors of SENP1;CLDN6 significantly down-regulated ?-catenin expression in MDA-MB-231 cells;transfection of ?-catenin significantly increase SENP1 expression at mRNA and protein levels in MDA-MB-231/CLDN6 cells,indicating that CLDN6 down-regulates the expression of SENP1 by inhibiting ?-catenin;over-expressing CLDN6 significantly promotes the degradation of ?-catenin and shortens its protein half-life;,CLDN6 and ?-catenin bind to each other in breast cancer cells;compared with the MDA-MB-231/Vec,the expression level of ?-catenin in cytoplasmic protein was not statistically different,while the expression level of ?-catenin in nuclear protein was significantly reduced in MDA-MB-231/CLDN6 cells.The above results suggest that CLDN6 inhibited the nuclear translocation of ?-catenin and the expression of the ?-catenin downstream gene SENP1.3.Effects of CLDN6/SENP1/HIF-1? on breast cancer metastasis in vivoCompared with the MDA-MB-231/Vec group,the fluorescence value of the lungs of the nude mice in the MDA-MB-231/CLDN6 group was significantly reduced,the number of peripheral blood circulating tumor cells was significantly reduced,and the number and area of lung metastases were significantly reduced;while in the MDA-MB-231/CLDN6+HIF-1? and MDA-MB-231/CLDN6+SENP1 groups The lung fluorescence value,the number of peripheral blood circulating tumor cells,and the number and area of lung metastases were significantly higher than those in the CLDN6 group.The above results indicate that CLDN6 inhibits breast cancer metastasis through the SENP1/HIF-1? axis.Conclusions:1.The transcription of CLDN6 was up regulated by HIF-1? under hypoxia.2.There is a previously unrecognized negative feedback mechanism between CLDN6 and HIF-1? under hypoxia.3.CLDN6 attenuates hypoxia induced breast cancer metastasis by regulating HIF-1? Sumoylation.