UGT8 Promotes Basal-Like Breast Cancer Progression By Regulating Sulfatide-?V?5 Axis

Breast cancer is the most common tumor in women,which can be divided into four subtypes by molecular type,including luminal A,luminal B,basal-like breast cancer(BLBC)and HER2 overexpressed(HER2).BLBC is an especially aggressive subtype that typically afflicts younger and premenopausal women and possesses the worst prognosis of any breast cancer subtypes.BLBC tends to be negative for the expression of estrogen receptor(ER),progesterone receptor(PR),and epidermal growth factor receptor 2(HER2),a situation that lacks effective targeted therapies.Less than 30%patients with metastatic BLBC have a 5-years survival rate despite adjuvant chemotherapy that remains the mainstay of BLBC treatment.Therefore,an in-depth exploration of the pathogenesis and potential therapeutic targets will help us to diagnose and intervene in BLBC early and improve the quality of lifeStudies have shown that in order to meet the demands of cancer cell survival and metastasis,metabolic alterations change almost all metabolic pathways,including amino acid metabolism,carbohydrate metabolism,lipid metabolism,and cellular active molecule metabolism.Breast cancer is closely related to metabolism.However,the metabolic regulation of breast cancer growth and metastasis remains to be further studiesUrine diphosphate-galactose ceramide galactosyltransferase(UGT8)is a key enzyme in the synthesis of sulfatide.We analyzed several publicly available gene expression datasets and found that the expression of UGT8 in BLBC was significantly increased,and predicts poor prognosis in breast cancer patients.Sulfatide is a sphingolipid commonly found on the face of most of eukaryotic cells.Its synthesis is a simple two-step reaction,which UGT8 plays as rate-limiting enzyme in the first step Besides its structural role for the cell membrane,sulfatide is also involved in cell adhesion,modulation of sodium and potassium channels,and cell plasticity.There is growing evidence for the involvement of sulfatide in the regulation of proliferation,differentiation,apoptosis,and senescence of cancer cells.High expression of sulfatide is observed in several human cancer tissues.Significantly elevated expression of sulfatide in ovarian carcinomas and colorectal carcinomas predicts poor prognosis.Our previous research showed that sulfatide was significantly increased in BLBC patients;knockdown or overexpression UGT8 in breast cancer cell lines can reduce or increase the expression of sulfatideMeanwhile,analysis of copy number alteration across cancer genomes reveals that no significant change in TCGA datasets,indicating the involvement of other genetic or epigenetic mediators in UGT8.Sox10,a transcriptional activator,is also highly expressed in BLBC according to TCGA and GSE25066 datasets.And there is a positive correlation between the expression of Sox 10 and UGT8Wo also found that it has been speculated that ZA might be a potential inhibitor of UGT8 through computational modeling.ZA is a marketed drug that has been licensed for the treatment of osteoporosis and bone metastasis.Accumulating evidence indicates that ZA induces apoptosis of cancer cells and suppresses migratory and invasive ability of cancer cells.But the exact mechanism underlying the anticancer property of ZA remains unknownIn view of all results above,we propose several research hypotheses:(1)What effects do UGT8 and sulfatide have on BLBC?And what's the specific mechanism?(2)Is sox 10 a direct transcriptional activator responsible for high UGT8 expression in BLBC?(3)Is ZA a direct inhibitor of UGT8?And what is the effect of ZA on BLBC?Our research designs experiments on the above hypotheses and obtains the following results1.UGT8 is overexpressed in BLBC and promotes tumorigenic and metastatic capacityWe analyzed multiple publicly available gene expression datasets and breast cancer cell line gene expression datasets and we noticed that UGT8 mRNA was remarkably up-regulated in BLBC;Patients with the higher expression of UGT8 have the poorer survival rate,the higher tumor grade and the larger tumor size.We examined the UGT8 level in fresh frozen breast tumor tissues.The expression of UGT8 was up-regulated in triple-negative breast cancer that is mostly also BLBC.We also chose five luminal and five basal subtype cell lines to test UGT8 expression by western blot,semi quantitative RT-PCR and quantitative RT-PCR.Consistently,UGT8 expression was significantly high in BLBC cell linesWe generated stable transfectants with empty vector or knockdown of UGT8 expression in MDA-MB231 and SUM159 cells and also created stable clones with empty vectors or UGT8 expression in BT549 and HCC1937 cells to examined the effect of UGT8 expression on tumorigenic and metastatic capacities.UGT8 expression induced tumorigenic and metastatic capacities,and knockdown UGT8 dramatically repressed the tumorigenic and metastatic capacities both in vivo and in vitroThe results confirm that UGT8 is highly expressed in BLBC and promotes tumorigenic and metastatic capacities2.UGT8 is a direct target of Sox10We expressed Sox10 in SUM159 and MDA-MB436 cells.And Sox10 up-regulated mRNA and protein levels in all these cell lines.We noticed that UGT8 promoter contained 10 putative consensus Sox10-binding motifs and found that six of them play an important role in up-regulation of UGT8 through promoter-luciferase constructs Also,we performed ChIP assays in MDA-MB231 and HCC1428 cells with endogenous Sox10 expression.A dramatic enrichment of Sox10 in the UGT8 promoter was observed in these cellsThese data suggest that UGT8 is a direct target of Sox103.UGT8 actives ?V?5 signaling via up-regulating sulfatideWe examined the production of GalCer and sulfatide,two downstream metabolites of UGT8 in the sulfatide biosynthetic pathway.Immunoblotting data showed that knockdown of UGT8 expression caused a remarkable decrease,whereas exogenous UGT8 expression resulted in a dramatic increase in both GalCer and sulfatide levels Either UGT8 expression and sulfatide,but not GalCer,markedly induced the migration and invasion of BT549 cells and restored the decreased migration and invasion of MDA-MB231 and SUM159 cells with stable knockdown of UGT8 expression in vitroWe noticed that the most significant pathway was related to the ECM-receptor interaction through KEGG pathway analysis of gene expression in MDA-MB231 cells undergoing knockdown of UGT8.And it has been reported that sulfatide up-regulates ITGAV expression,which is involved in multiple signaling pathway by combining different integrin ? chains.Consistently,UGT8 knockdown or expression led to an obvious change of ITGAV expression in mRNA and protein levels.And sulfatide but not GalCer significantly elevated expression of ITGAVNext,We found that integrin ?V?5 clustering was dramatic increase after UGT8 expression,whereas the knockdown of UGT8 expression resulted a significant decrease Given the tight association of TGF-?signaling and NF-?B pathway with BLBC aggressiveness,we chose them as examples to characterize the regulatory mechanism of UGT8 in BLBC.As expected,UGT8 expression or knockdown of UGT8 can actives or inhibits TGF-? signaling and NF-?B pathwayThese data suggest that UGT8 actives TGF-? signaling and NF-?B pathway via actives ?V?5 signaling by up-regulating sulfatide4.ZAis a direct inhibitor of UGT8 and suppresses metastasis of breast cancerWe examined the effect of ZA on two downstream metabolic of UGT8 and found that ZA dramatically decreased the expression GalCer and sulfatide.And compared genes that were transcriptionally down-regulated in MDA-MB231 cells undergoing knockdown of UGT8 expression to that in MDA-MB231 cells treated with ZA.KEGG pathway analysis of down-regulated genes shows that the pathway terms were almost consistent and the most significant pathway was related to the ECM-receptor interaction,which is same as the knockdown of UGT8 expressionAlso,an in vitro galactosidation assay showed that ZA can strongly block the activity of UGT8.And ZA can remarkably inhibit metastatic both in vivo and in vitroThese data suggest that ZA is a direct inhibitor of UGT8Based on the above results,we reached following conclusionsDue to the transcriptional activation,the expression of UGT8 is significantly increased in BLBC compared with other subtypes of breast cancer;UGT8 promotes the clustering of integrin aV?5 by its downstream metabolite sulfatide,and then activates TGF-? signaling and NF-?B pathway to promote tumorigenic and metastatic capacity At the same time,we found that ZA is a direct inhibitor of UGT8 and can inhibit metastasis of BLBC.