| Objective(s): Colon cancer is one of the malignant tumors that severely endanger human health.The morbidity and mortality of colon cancer remain top among various malignant tumors.Hypoxia is one of the characteristics of tumor microenvironment,hypoxia and hypoxia inducible factor 1 subunit alpha(HIF-1α)play an important role in the tumorigenesis and development of colon cancer.However,the exploration of targeted therapies for hypoxic microenvironments and HIF signaling pathways is still in early stages,and it is urgent to find a new molecular therapeutic strategy.In previous studies,we found that tumor suppressor protein 14-3-3σ can inhibit hypoxia-associated glycolysis,which drives us to explore the effect of 14-3-3σ protein on the hypoxic tumor microenvironment of colon cancer.In addition,we further explored whether the 14-3-3σ protein regulates hypoxia-induced colon cancer metastasis and angiogenesis,and analyze its underlying mechanism.Methods:1.In the first part of this study,bioinformatics was used to analyze the 14-3-3σ m RNA of colon cancer patients and the correlation between 14-3-3σ and colon cancer Overall survival(OS)from the public databases of The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO).Immunohistochemistry was further used to analyze the expression of 14-3-3σ protein in colon cancer patients on tumor microarray(TMA)chips and its correlation with OS in patients.Meanwhile,Cox univariate and multivariate analysis were used to further confirm the relationship between 14-3-3σprotein expression and prognosis in patients with colon cancer.Finally,the Chi-square test was used to analyze the relationship between the expression level of 14-3-3σ and the clinicopathologic features of colon cancer patients.2.In the second part of this study,we established 14-3-3σ knockdown colon cancer cell lines.Transwell assay was used to detect the effects of 14-3-3σ on colon cancer migration and invasion at different oxygen concentrations.The effects of knockdown or overexpression of 14-3-3σ on VEGF m RNA level and secretory VEGF protein in colon cancer cells under different oxygen concentrations were detected by RT-q PCR and ELISA.Finally,the effect of 14-3-3σ on hypoxia-induced colon cancer angiogenesis was examined by in vitro angiogenesis experiment.3.In the third part of this study,we first established 14-3-3σ and HIF-1α doubleknockdown colon cancer cell lines,and in vitro Transwell assay and in vivo colon cancer lung metastasis mouse model were used to verify whether 14-3-3σ affects hypoxia-induced colon cancer migration and metastasis through HIF-1α.In mechanistic part,the post-transcriptional regulation of 14-3-3σ on HIF-1α was detected by western blotting,co-immunoprecipitation,RT-q PCR and protein stability assay.The translocation to nucleus regulation of 14-3-3σ on HIF-1α was detected by immunofluorescence and cell lysate fractionation analysis.The effect of 14-3-3σ on HIF-1α transcriptional activity was detected by RT-q PCR.Finally,the regulation of 14-3-3σ on HIF-1α ubiquitination was detected by protein ubiquitin purification experiment.4.In the fourth part of this study,the 14-3-3σ-binding protein NEDD4 L was identified by co-immunoprecipitation combined with mass spectrometry,and whether NEDD4 L was an E3 ligase targeting HIF-1α was verified by western blotting,coimmunoprecipitation,protein stability tests and protein ubiquitin purification tests.The effect of 14-3-3σ on the binding of HIF-1α to NEDD4 L was detected by competitive binding immune-coprecipitate and colloid filtration.The effect of the 14-3-3σ-NEDD4 L complex on HIF-1α expression was detected by western blotting.Finally,since 14-3-3σ generally binds to target proteins containing specific motifs,we searched for the 14-3-3σ-binding site on NEDD4 L and observed the effect of 14-3-3σ-NEDD4 L complex on HIF-1α expression and ubiquitination by western blotting after mutation of the binding site.5.In the fifth part of this study,a colon cancer xenograft mouse model was established to observe the effects of bevacizumab alone or in combination with 14-3-3σ expression on the growth of colon cancer transplanted tumor.In addition,to verify the mechanism of 14-3-3σ promoting bevacizumab sensitivity,we used immunohistochemistry to detect the expressions of Ki67 and CD31 proteins in transplanted tumors.Finally,we detected the expression of 14-3-3σ and HIF-1α in colon cancer TMA,and further analyzed the relationship between the combined expression of 14-3-3σ and HIF-1α and the overall survival of colon cancer patients using TMA.Results:1.Bioinformatics combined with TMA immunohistochemistry found that 14-3-3σ was low expressed in colon cancer,and low expression of 14-3-3σ was associated with poor survival prognosis.14-3-3σ expression is a protective factor for colon cancer and 14-3-3σ expression level is an independent prognostic factor for colon cancer.There was no significant correlation between the expression level of 14-3-3σ and gender,age,pathological grade,clinical stage,tumor size,lymph node metastasis,and the presence of distant metastasis in colon cancer patients.2.14-3-3σ can inhibit tumor migration and invasion induced by hypoxia.By inhibiting VEGF gene expression and VEGF protein secretion in colon cancer cells,14-3-3σinhibits hypoxia mediated colon cancer angiogenesis.3.The rescue experiment confirmed that 14-3-3σ inhibits the in vitro and in vivo metastasis of colon cancer by inhibiting HIF-1α.Mechanism studies have shown that14-3-3σ can directly bind to HIF-1α and inhibit the protein stability of HIF-1α by promoting its ubiquitination degradation.14-3-3σ does not affect the HIF-1α entry into the nucleus,but inhibits the HIF-1α expression in the cytoplasm,ultimately affecting its transcriptional activity.Importantly,14-3-3σ mediates ubiquitination degradation of HIF-1α in a VHL-independent manner.4.Further mechanism studies showed that E3 ubiquitin ligase NEDD4 L is the binding protein of 14-3-3σ and HIF-1α is the substrate of NEDD4 L.14-3-3σ can mediate ubiquitination and protein degradation of HIF-1α by promoting the binding of NEDD4 L and HIF-1α.448-phosphorylated serine of NEDD4 L is the binding site of 14-3-3σ,mutation of this site can abolish the binding of 14-3-3σ-NEDD4 L,and ultimately weaken the inhibition of 14-3-3σ-NEDD4 L complex on HIF-1α.5.In vivo transplantation experiments have confirmed that 14-3-3σ can inhibit colon cancer tumor proliferation by inhibiting hypoxia-associated angiogenesis,and based on this mechanism,14-3-3σ can enhance bevacizumab sensitivity.By analyzing the expression levels of 14-3-3σ and HIF-1α in TMA,we also found that colon cancer patients with high expression of 14-3-3σ and low expression of HIF-1α had a better prognosis.Conclusion(s): Our results revealed that 14-3-3σ enhances HIF-1α poly-ubiquitination and subsequent proteasome-mediated degradation by promoting the binding of S448 phosphorylated NEDD4 L to HIF-1α,thereby inhibiting hypoxia-induced colon cancer metastasis and angiogenesis and ultimately affecting the development of colon cancer. |
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