HRD1 Inhibits Fatty Acid Oxidation And Tumorigenesis By Ubiquitinating CPT2 In Triple-negative Breast Cancer

Background Triple-negative breast cancer(TNBC)is a heterogeneous group of breast cancers,counting 15-20 % of all breast cancer patients,which characterized by lack of expression of estrogen receptors(ER),progesterone receptors(PR)and human epidermal growth factor receptor 2 gene amplification,making it unresponsive to endocrine therapy and HER2-targeted treatment.Due to the absence of approved targeted therapy,cytotoxic chemotherapy is the current primary established systemic therapy for early and advanced TNBC.Although chemotherapy significantly improves clinical outcomes for TNBC patients,recurrence rates remain relatively high and TNBC tumors often develop resistance to chemotherapeutic agents.Thus,considering the limited treatment options and aggressive phenotypes of TNBC,it is crucial to improve our understanding of TNBC features and discover potential therapeutic targets to aid in the development of effective therapies.Metabolic reprogramming is one of the malignant phenotypes of tumor,since 1921,Otto Warburg first reported tumor cells still preferred to glycolysis for providing ATP for tumor quickly even under aerobic conditions,and then more and more people have been dedicated to the study of tumor in changes of metabolic pathways,including glucose metabolism,lipid metabolism and amino acid metabolism.Metabolic reprogramming is an important feature of tumors that is often accompanied by a rapid increase in glucose and glutamine intake.Study have reported that TNBC shows a unique glutamine-dependent phenotype and FAO accelerated,which means that glutamine supplementation tricarboxylic acid cycle is essential for biomolecule synthesis,energy generation,and glutathione production in TNBC.With the rapid growth of tumors,accelerated glutamine catabolism depletes the local glutamine supply,resulting in glutamine deficiency.Studies have confirmed that glutamine is one of the most depleted metabolites in tumors.However,local glutamine depletion does not inhibit the development of cancer.One of the crucial reasons is that the use of alternative energy sources,such as fatty acids,enables tumor cells to continue to survive and proliferate rapidly in a nutrient-deficient microenvironment.Despite considerable studies,it remains largely unclear how FAO is elevated in TNBC and in particular,whether glutamine deficiency is one of the driving factors of the upregulation of FAO.HMG-Co A reductase degradation protein 1(HRD1)was identified as an E3 ligase that controls cholesterol production by regulating the turnover of the rate-limiting enzyme HMG-COA reductase(HMGCR)in yeast.Subsequent studies have shown that HRD1 is involved in endoplasmic reticulum-related degradation ERAD.HRD1 has been proven to regulate cell processes independent of ERAD.In recent years,numerous studies have found that HRD1 plays important roles in lipid metabolism.HRD1 KO in the liver protects mice against obesity,hyperlipidemia,NAFLD,and insulin resistance induced by a high-fat diet(HFD).HRD1 also participates in the ubiquitylation of VAMP3 to prevent long chain fatty acid(LCFA)uptake.Additionally,HRD1 participates in the regulation of low-density lipoprotein by binding and promoting the ubiquitin degradation of LOX-1 hyperlipidemia,NAFLD,and insulin resistance induced by a high-fat diet(HFD).HRD1 also participates in the ubiquitylation of VAMP3 to prevent long chain fatty acid(LCFA)uptake.Additionally,HRD1 participates in the regulation of low-density lipoprotein by binding and promoting the ubiquitin degradation of LOX-1.In the current study,we demonstrated that HRD1 was downregulated in TNBC tissues and dysregulated FAO by ubiquitinating CPT2,especially under the condition of glutamine deficiency.In addition,we also found that the curative effect of CB-839,a glutaminase(GLS)inhibitor,is more prominent in TNBC cells and tumors with high HRD1 expression.These findings provide insight into HRD1 as a regulator of lipid metabolism and have important implications for TNBC therapeutic targeting.Methods 1.Cancer and para cancer tissues of Ten patients with TNBC were collected from the First Affiliated Hospital of Dalian Medical University between January 1,2019 and June 1,2019.Difference in protein expression of HRD1 in TNBC cancer tissues and para cancer tissues were detected by Western blotting.2.Lentivirus transfection was employed to knock down HRD1 or double knock down HRD1 and CPT2 in MDA-MB-231.3.CCK-8,plate clone,soft agar clone formation experiment and subcutaneous tumor-forming experiment were used to detect the effect of HRD1 knockdown on proliferation.4.The interaction between HRD1 and CPT2 was verified by immunoprecipitation and pulldown assay.5.The expression and ubiquitination levels of HRD1 and CPT2-related proteins were detected by Western Blot.6.Fatty acid related indexes such as carnitine,palmitic acid and stearic acid were detected by LC-MS.7.HRD1 m RNA expression in cancer tissues and para cancer tissues of different subtypes of breast cancer in TCGA database was detected by bioinformatics method.8.SPSS17.0 software was used for statistical analysis.The data of grayscale analysis were expressed as mean ± standard deviation,and at least 3 experiments were repeated independently.Independent sample t test was used to compare the measurement data between the two groups.The data were not normally distributed and compared between groups were analyzed by the ANOVA test.The chi-square test was used to compare the data between the two groups.Univariate survival analysis was performed using Kaplan- Meier method and Log-rank test to calculate P values,and P<0.05 was considered statistically significant.Results Section 1 HRD1 participates in the regulation of tumorigenesis of triple-negative breast cancer.1.HRD1 is low-expressed in TNBC(1)The results of TCGA database showed that the m RNA level of HRD1 was the lowest in TNBC,and the cancer tissue was lower than the paracancerous tissue.(2)Western blot results of 10 paired TNBC patients showed that the protein level of HRD1 was lower in cancer than that in adjacent normal breast tissue.2.The soft agar cloning experiment results showed that knocking down HRD1 promoted the formation of MDA-MB-231 three-dimensional cloning.3.Mouse model experiments confirmed that HRD1 knockdown promoted tumor formation in vivo of TNBC.4.HRD1 knockdown had no effect on the proliferation and plate clone formation of MDA-MB-231 cells under normal culture or glucose deficient culture conditions.5.HRD1 knockdown significantly promoted the proliferation and plate clone formation of MDA-MB-231 cells under the condition of glutamine deprivation.6.The protein level of HRD1 is down regulated in a time-and concentration-dependent manner during glutamine starvation.Section 2 The mechanism of HRD1 inhibiting TNBC fatty acid oxidation and tumorigenesis through ubiquitination of CPT2 1.Under glutamine starvation occurred,the ratio of C2 carnitine /C0 carnitine in MDA-MB-231 cells increased significantly,that is FAO accelerated.The ratio of C2 carnitine /(C16 carnitine +C18:1 carnitine)was increased,while the ratio of C16 carnitine +C18 carnitine /C0 carnitine was not significantly changed,indicating that the activity of CPT2 in MDA-MB-231 cells was significantly up-regulated after glutamine deprivation,while the activity of CPT1 was not significantly changed.2.HRD1 knockdown significantly increased the ratio of C2 carnitine to C0 carnitine under glutamine deprivation.3.HRD1 significantly decreased palmitic acid and stearic acid contents under glutamine deprivation.4.Under glutamine deprivation,HRD1 knockdown significantly increased the carnitine C2 carnitine /(C16 carnitine +C18:1 carnitine)ratio,but did not affect the carnitine(C16 carnitine +C18 carnitine)/C0 carnitine ratio,indicating that HRD1 knockdown significantly increased the activity of CPT2.CPT1 activity was not affected under glutamine deprivation.5.Knocking down HRD1 up-regulated the protein level of CPT2 without affecting the m RNA level.6.The results of immunoprecipitation and Pulldown showed that HRD1 interacted with in TNBC.7.Knocking down HRD1 prolonged the half-life of CPT2.8.HRD1 polyubiquitinates CPT2 through the K-48(1)Endogenic ubiquitination of MDA-MB-231 cells stable knockdown HRD1 showed that knockdown of HRD1 could reduce the ubiquitination level of CPT2.(2)Exogenous ubiquitination experiments based on HEK-293 T cells showed that over expression of Myc-HRD1 increased the K-48 ubiquitination chain mediated polyubiquitination of CPT2.9.The results of TMA immunohistochemistry showed a negative correlation between the protein expression levels of HRD1 and CPT2 in TNBC tissues(R=--0.224,P=0.047).10.HRD1 knockdown promoted fatty acid oxidation and cell proliferation of TNBC through CPT2 Further knockdown of CPT2 on the basis of HRD1,the enhanced ability of clone formation,accelerated tumor proliferation in vivo and accelerated fatty acid oxidation mediated by knockdown of HRD1 could all be reversed by the loss of CPT2.Section 3 HRD1 is a potential predictor of the efficacy of glutaminase inhibitors.1.HRD1 is a potential predictive marker for the efficacy of glutaminase inhibitor(CB-839).1)CCK-8 results showed that CB-839 could significantly inhibit the proliferation of MDA-MB-231 cells in the control group compared with HRD1 knockdown group.2)In vivo experimental results showed that compared with HRD1 knockdown group,CB-839 significantly inhibited subcutaneous tumor-forming ability of MDA-MB-231 cells in the control group.Conclusion In this study,we investigated and validated HRD1,a key regulatory protein of lipid metabolism in TNBC cells,and found it may be a potential predictive marker of CB-839 therapy.Section 1 HRD1 is involved in the regulation of tumorigenesis of triple-negative breast cancer.1.HRD1 was low expression of in TNBC;2.HRD1 is a TNBC tumor suppressor;3.HRD1 knockdown can promote TNBC proliferation in vitro Under the condition of glutamine deprivation,Section 2 The mechanism of HRD1 inhibiting TNBC fatty acid oxidation and tumor genesis through ubiquitination of CPT2 1.Knockdown of HRD1 promotes the fatty acid oxidation of TNBC under the condition of glutamine deprivation.2.HRD1 interacts with CPT2 and reduce the stability of CPT2.3.HRD1 ubiquitin CPT2 through K48 polyubiquitination and target proteasome pathway.4.Knockdown of HRD1 increases the FAO level of TNBC by upregulating the activity of CPT2 after glutamine deprivation.Section 3 HRD1 is a potential predictor of the efficacy of glutaminase inhibitors1.HRD1 may be a predictor of the efficacy of the glutaminase inhibitor CB-839,that is,tumors with high expression of HRD1 are more sensitive to CB-839.