Loss Of ABAT-Mediated GABAergic System Promotes Basal-Like Breast Cancer Progression By Activating Ca²⁺-NFAT1 Axis

Basal-like breast cancer(BLBC)is the most aggressive subtype of breast cancer,which comprised of four major molecular subtypes,including luminal A,luminal B,HER2/ERBB2,and basal-like.BLBC subtype accounts for approximately 10-20%of breast cancers and frequently occurs in younger patients.BLBC tumors are usually of larger size and higher grade with a tendency for recurrence and metastasis and have a poor response to chemotherapy.Furthermore,BLBC has an increased propensity to metastasize to the brain and lungs,which often leading to a fatal clinical outcome.There is a critical need to elucidate the underlying molecular mechanisms and identify molecular targets in BLBCGamma-aminobutyric acid(GABA)is a major inhibitory neurotransmitter in the mammalian central nervous system.The biological effects of GABA are mainly mediated by its receptors,which exist in cell membrane,rising the intracellular calcium ion concentration In addition to being a neurotransmitter,studies have shown that GABA is closely related to the development of tumor.GABA has inhibitory effects of cancer growth and metastasis on colon cancer,gastric cancer,and hepatocellular carcinoma.GABA has also been shown to promote pancreatic cancer growth through upregulation of the GABA receptor.These findings suggest a critical role for the GABAergic system in cancer and enforce the need for further evaluation of this pathway in distinct types of cancerWe analyzed gene expression profiles of breast cancer in several publicly available cDNA microarray datasets.Notably,in contrast to other subtypes,Gamma-aminobutyrate aminotransferase(ABAT)expression was markedly downregulated in BLBC.ABAT,a key enzyme responsible for the catabolism of GABA,catalyzes the transfer of the amino group of GABA to a-ketoglutarate,producing succinic semialdehyde and L-glutamate.Next,we analyzed the association of GABA with aggressive breast cancer using the previous metabolomics data and found that the GABA level was significantly elevated in BLBC and ER-negative breast cancerWhile investigating the expression of ABAT in BLBC,we noticed the copy number variants on ABAT expression and inverse expression patterns of ABAT and Snail in breast cancer.Snail,the first proven EMT-inducing factor,is a transcriptional repressor that regulates EMT during the formation and transfer of embryos.Its gene sequence is highly conserved from invertebrates to vertebrates and even humans,especially its DNA binding domain,and it is highly homogeneous among different species.Snail expression is related to tumor grade and the metastasis of tumor.Patients with high expression of Snail often have a poor prognosis.As a key inducer of EMT,Snail plays a key role in proliferation,differentiation,invasion and metastasis of tumor cells.On the promoter of ABAT,we found that it contains four conserved Snail binding sites,E-box(CAGGTG)Based on the Above,we speculate that loss of ABAT,which inhibits by Snail,can drive BLBC progression by activating related cellular pathways through GABAergic system1.ABAT is downregulated in BLBC subtype,and suppresses tumorigenicity and metastasis of breast cancerWe found that ABAT was significantly downregulated in BLBC by analyzing multiple gene expression profiles of breast cancer,including GSE1456,GSE25066,NKI295,TCGA and MEBTABRIC.We collected freshly frozen tumor tissues from 30 cases to confirm this observation.Meanwhile,we evaluated ABAT expression in gene expression datasets of breast cancer cell lines,including GSE12777,GSE10890,E-TABM-157 and E-MTAB-181 Consistently,loss of ABAT was correlated with the basal subtype of breast cancer cell lines We confirmed these findings by western bolt,semi-quantitative RT-PCR and quantitative real-time PCR in a panel of breast cancer cell lines containing 5 luminal and 5 BLBC cell lines.And all experimental results were consistent with the analysis aboveAlso,the datasets show that a significant association between small tumor size and ABAT expression.Less ABAT expression was present in higher histological grades of tumor And tumor with low ABAT expression had a higher probability of developing metastasis,less survival and chemotherapy resistance.We generated stable clones with the ABAT expression vector or the empty vector in MDA-MB231 and SUM159 cells.Both in vivo and in vitro experiments showed that ABAT expression inhibited tumorigenicity and metastasisThese results confirmed that loss of ABAT expression was primarily restricted to BLBC,and ABAT expression suppressed tumorigenicity and metastasis of breast cancer2.ABAT expression is downregulated due to deleted ABAT copy number and Snail-mediated repression of ABATTo determine the copy number variants on ABAT,we analyzed two publicly available datasets.We found that about 5%of primary breast tumors having deletions in ABAT and about 70%of ABAT copy number deletions fell into BLBC subtypes.Consistently,analysis of breast cancer cell lines showed that 30%of cell lines hve ABAT copy number deletions,about 85%of which belonged to BLBC subtypes.These data strongly associate ABAT copy number deletions with loss of ABAT expression and BLBC subtypeWe expressed Snail in three luminal breast cancer cell lines,BT483,HCC1428,and MCF7.We found that Snail expression significantly downregulated ABAT expression by western blot and quantitative real-time PCR.Meanwhile,we cloned the human ABAT promoter-luciferase constructs,and found that the E-box,the one closed to TSS,was required for Snail-induced transcriptional repression.And the chromatin immunoprecipitation(ChIP)assays in three cells above confirmed that Snail directly bound to the ABAT promoterThese results demonstrate that ABAT is a direct target of Snail3.loss of ABAT expression activates Ca2+-NFAT1 axisIn MDA-MB231,SUM159 and BT549 cells,we generated stable clones with ABAT expression or empty vectors.Analysis showed that ABAT expression caused a remarkable decrease in GABA level.Next,we found that ABAT expression reduced the intracellular Ca2+ concentration in MDA-MB231 and BT549 cells.And Ca2+ concentration was much higher in BLBC cells without ABAT expression than in luminal cells with endogenous ABAT-expression.We used Ca2+ image and found that GABA mainly rise intracellular Ca2+concentration by activating GABA-A-mediated signalingCa2+ signaling functions in cancer cells through upregulating oncogenes and/or downregulating tumor suppressors.Study showed that NFAT1 was constitutively activated in triple-negative breast cancer and promoted tumorigenesis and metastasis.And NFAT1 is an important Ca2+ sensor;upon stimulation by Ca2+,it is dephosphorylated and translocated to the nucleus where it is transcriptionally active.Our results showed that ABAT expression led to a significant decrease in nuclear translocation of NEAT1 by immunostaining-confocal analysis and western blot in MDA-MB231 and SUM159 cellsBased on the above,we reached the following conclusions1.ABAT expression is specifically down-regulated in BLBC and correlates with poor prognosis in breast cancer patients2.ABAT is a direct target of Snail.Due to the high expression of Snail in BLBC,ABAT expression is lower in BLBC than other breast cancer subtypes3.Due to the low expression of ABAT in BLBC,the GABA concentration rises.It mainly actives GABA-A receptors to increase the intracellular Ca2+ concentration,thereby activating NFAT1 to promote tumor progression.