| Background: Tumor cells adapt to complex microenvironments through metabolic reprogramming,which plays an important role in tumor malignant progression,drug resistance and immune escape.In the absence of oxygen,tumor cells promote the transcriptional expression of pyruvate dehydrogenase kinase 1(PDK1)through up-regulation of hypoxia-inducible factor 1-alpha(HIF-1α).PDK1 phosphorylation deactivates pyruvate dehydrogenase complex(PDC),inhibits mitochondrial oxidative phosphorylation,promotes glycolysis,and makes cells undergo metabolic remodeling to adapt to anoxic microenvironment.Previous studies have shown that PDK1 exists nuclear localization in breast cancer cells,and the aggregation of PDK1 nuclei increases significantly under hypoxia conditions.Previous studies were mostly limited to the role of PDK1 in tumor metabolism remodeling,while the function and mechanism of PDK1 in the malignant process of breast cancer are still not fully understood.By deeply exploring the function of PDK1 in the malignant process of breast cancer,this study elucidates the interaction and molecular mechanism between PDK1 and HIF-1α in cytoplasm and nucleus,enriching our understanding of the promotion of malignant process of breast cancer by oncogene PDK1.Methods: 1)The difference of PDK1 expression in breast cancer tissues was detected by database analysis and clinical immunohistochemistry.PDK1-knockout cell line MCF7-sh and PDK1-knockout cell line BT-549-KO were constructed.The effects of PDK1 on proliferation,migration and anti-apoptosis of breast cancer cells were evaluated in vitro by plate cloning,flow cytometry and wound healing.2)A subcutaneous tumor model was constructed in nude mice to detect the effect of PDK1 on tumorigenic ability of breast cancer cells.The subcutaneous tumor tissues were embedded and sliced for immunohistochemical staining to detect the expression levels of Ki-67,CD31 and HIF-1α.In vivo experiments were conducted to verify the effects of PDK1 on cell proliferation,angiogenesis and HIF-1α pathway in vivo.3)The effects of PDK1 on HIF-1α protein level and ubiquitination level were detected by WB,qRT-PCR and Co-IP experiments.The interaction between PDK1 and HIF-1α was detected by immunofluorescence assay and Co-IP assay.According to the molecular structure of PDK1,truncated mutant plasmid of PDK1 was constructed to explore the structure of the interaction between PDK1 and HIF-1α.Secondly,the inactivation mutation of PDK1 D318 was simulated to construct the inactivation mutant constitution of PDK1 kinase.The effect of PDK1 kinase activity on HIF-1α protein level was detected by WB assay.Finally,HIF-1α samples enriched by IP before and after PDK1 knockout were detected by phosphorylation modification mass spectrometry to identify the key phosphorylation sites of HIF-1α regulated by PDK1,and the transcriptional activity of HIF-1α was detected by qRT-PCR,double luciferase reporting assay and Ch IP assay.Results: Based on existing literature reports,PDK1 was summarized as an oncogene in Cancer Mine database,which plays a role in promoting cancer.Further analysis of GSE data set found that PDK1 was significantly higher expressed in breast cancer tissues compared with normal tissues.Combined with immunohistochemistry of clinical tissue samples,the expression of PDK1 in breast cancer tissue was significantly higher than that in paracancer tissue and normal breast tissue.In vitro experiments,knocking down PDK1 level in breast cancer cells can significantly reduce cell proliferation,anti-apoptotic ability and migration ability.Moreover,the tumorigenic ability of breast cancer cells decreased after PDK1 knockdown,and HIF-1α pathway was significantly inhibited.IP experiment showed that PDK1 could reduce the ubiquitization level of HIF-1α and promote the stability of HIF-1α protein.The regulation of PDK1 on HIF-1α protein level depends on its kinase activity.PDK1 mediated HIF-1α Ser 451 phosphorylation,which disappeared when PDK1 was knocked out,and HIF-1α Ser 451 phosphorylation stabilized HIF-1α protein levels in cytoplasm by inhibiting its interaction with PHD/VHL.Under hypoxia conditions,the localization of PDK1 in breast cancer nucleus was significantly increased,and the expression of PDK1 could significantly promote the interaction between HIF-1α and transcription cofactor p300,and enhance the transcription activity of HIF-1α,especially the transcription level of angiogenesis related genes.Conclusions: In this study,we found that glycolytic enzyme PDK1 is distributed in the cytoplasm and nucleus of breast cancer cells,and hypoxia treatment can promote the increase of the overall level of PDK1 and the increase of intracellular localization.In cytoplasm,PDK1 phosphorylates HIF-1α Ser451 with kinase activity,reduces its ubiquitination level,and enhances the stability of HIF-1α protein.PDK1 enhances the binding ability of HIF-1α to p300 and promotes the transcriptional activity of HIF-1α.In conclusion,HIF-1α can induce the expression of PDK1,which in turn enhances the protein stability and transcriptional activity of HIF-1α,forming a positive feedback loop to promote the progression of breast cancer. |
