| According to the latest statistics from the World Health Organization’s International Agency for Research on Cancer(IARC)in 2020,breast cancer has overtaken lung cancer as the most common cancer worldwide,jumping to the first place in terms of cancer incidence and mortality among women worldwide,and in-depth studies on the pathogenesis of breast cancer have become a hot topic in breast cancer research.The phosphatase and tension homolog deleted on chromosome ten(PTEN),an important oncoprotein,is one of the most frequently mutated genes in human cancers,and inhibits phosphatidylinositol 3-kinase(It inhibits the phosphatidyl inositide 3-kinase(PI3K)/Akt oncogenic signalling pathway),thereby inhibiting the growth and proliferation of tumour cells.Mutations in PTEN are rarely seen in breast cancer patients,but reduced PTEN protein expression can be observed in cancerous tissues,suggesting that PTEN is involved in the development of breast cancer.Therefore,an in-depth study of the role of PTEN in breast cancer development and its regulatory mechanism is important for exploring potential drug targets and treatment options for breast cancer.Our collaborators’ lab recently identified PTEN as a novel target of developmental down-regulation protein 8(Nedd8),which is expressed by neural precursor cells and mediates ubiquitin-like modifications of PTEN,leading to over-activation of the PI3K/Akt pathway and thus leads to tumour cell proliferation,which is closely associated with the staging and poor prognosis of breast cancer patients.Through screening of a series of conditions,it was found that high concentrations of glucose could induce ubiquitination-like modifications of PTEN,leading to the nucleation of PTEN in the cytoplasm and promoting tumour cell proliferation and breast cancer development.However,the molecular mechanism by which high concentrations of glucose in breast cancer lead to ubiquitination-like modifications of PTEN is not clear.The ubiquitination-like system consists of an E1 activating enzyme(a heterodimer consisting of UBA3 and NAE1/APP-BP1),two E2-binding enzymes,(UBE2M/Ubc12 and UBE2F),and multiple E3 ligases.Of these,UBA3,one of the E1 activating enzymes,is involved in the occurrence of PTEN-like ubiquitination modifications.However,whether UBA3 is involved in the ubiquitination modification of PTEN induced by high glucose status,and thus in the regulation of breast cancer development,is still a gap in this research field.In order to investigate the molecular mechanism of ubiquitination-like modification of PTEN under high glucose status and its role in breast cancer,human breast cancer cell line MCF-7 and PTEN-deficient human breast cancer cell line BT-549 were selected for this experiment.western blot technique,qPCR technique,CRISPR-Cas9 technique and immunofluorescence technique were applied,combined with CpG-island,UCSC and other databases,we firstly observed the effect of high glucose concentration on UB A3 expression and PTEN ubiquitination modification,and the effect on PTEN subcellular localization after specific knockdown of UBA3.We further applied MNL4924,a small molecule inhibitor of ubiquitin-like activating enzyme E1,to observe the changes in PTEN-like ubiquitination modifications and their pathways in MCF-7 and BT-549,as well as changes in tumour cell behaviour,including cell migration,proliferation and clone formation.The results of the experiments were as follows.1.In MCF-7 cells treated with 25 mM glucose for 24 hrs,UBA3 protein expression levels were significantly upregulated,PTEN(K402)class ubiquitination levels were increased,and pT308 Akt and pS473 Akt phosphorylation levels were also increased,with statistically significant differences compared to the control group(P<0.05).Treatment with 1 μM MLN4924 significantly inhibited PTEN(K402)class ubiquitination levels and decreased pT308 Akt and pS473 Akt phosphorylation levels,with statistically significant differences(P<0.05).2.In MCF-7 cells treated with 5 mM 2-DG for 24 hrs,the mRNA levels and protein expression levels of UBA3 were reduced,and the difference was statistically significant compared to the control group(P<0.05).Immunofluorescence staining showed that 2-DG treatment did not affect the cellular localization of UB A3,with most of it located in the cytoplasm and a small portion in the nucleus.3.In MCF-7 cells,applying CRISPR-Cas9 technology to knockdown UBA3,the protein of cellular UBA3 was not expressed and no PTEN expression was observed in the nucleus,and the difference was statistically significant compared to the control group(P<0.05).4.Application of the CpG-island database and UCSC database sequence analysis showed the presence of a CpG-island in the UBA3 promoter region.application of Human Protein Atlas,GEO and TCGA database analysis showed that UBA3 promoter methylation levels were down-regulated in breast cancer cases and UBA3 protein expression levels were cases were significantly up-regulated.The correlation between UBA3 methylation levels and mRNA expression levels was tested by applying the MethHC database,and linear regression analysis showed a significant negative correlation between UBA3 mRNA levels and promoter methylation levels.5.In BT-549 and MCF-7 cells treated with MLN4924 at concentrations of 0 μM,0.5 μM,1.0 μM,and 1.5 μM for 12 hrs,respectively,the results showed that PTEN-like ubiquitination levels were reduced in MCF-7 cells,and both pT308 Akt and pS473 Akt phosphorylation levels were reduced,compared to the control group.The differences were statistically significant(P<0.05).pT308 Akt and pS473 Akt phosphorylation levels were unchanged in BT-549 cells.6.In MCF-7 cells treated with 0 μM,0.02 μM,0.05 μM and 0.1 μM of MLN4924 for 12 days,the number of clones formed decreased with increasing concentration gradients of MLN4924,with a statistically significant difference compared to the control group(P<0.05).Cell migration capacity decreased with increasing MLN4924 concentration gradient when treated with 0 μM,0.5 μM,1.0 μM and 1.5 μM concentrations for 12 hrs,and the difference was statistically significant compared to the control group(P<0.05).7.No significant change in cell proliferation capacity was observed in BT-549 cells treated with 0.025 μM MLN4924 given for 5 days.No significant change in the number of cell clone formation was observed after 12 days of treatment with 0 μM,0.02 μM,0.05 μM and 0.1 μM concentrations of MLN4924.No significant change in cell migration capacity was observed with increasing concentrations of MLN4924 when treated with 0 μM,0.5 μM,1.0 μM and 1.5 μM for 12 hrs.In summary,high concentrations of glucose can promote breast cancer cell proliferation and migration through activation of the PI3K/Akt pathway by upregulating protein and mRNA levels of UBA3 in MCF-7 cells,leading to ubiquitination-like modifications of PTEN.MLN4924 inhibits the PI3K/Akt pathway in a PTEN-dependent manner to exert anti-tumour effects.As MLN4924 has been evaluated in a series of phase Ⅰ/Ⅱ/Ⅲ clinical trials for antitumour therapy,it has a good safety and tolerability profile and can be used either alone or in combination with chemotherapy/radiotherapy in the treatment of various diseases including oncology.Therefore,this study provides a reliable experimental basis for modulating PTEN-like ubiquitination modifications as a potential target for breast cancer treatment,and also provides ideas for MLN4924 as a potential therapeutic agent for breast cancer. |
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