Transcriptomic response of breast cancer cells to anacardic acid

Anacardic acid (AnAc), a potential dietary agent for preventing and treating breast cancer, inhibited the proliferation of estrogen receptor α (ERα) positive MCF-7 and MDA-MB-231 triple negative breast cancer cells. To characterize potential regulators of AnAc action, MCF-7 and MDA-MB-231 cells were treated for 6 h with purified AnAc 24:1n5 congener followed by next generation transcriptomic sequencing (RNA-seq) and network analysis. We reported that AnAc-differentially regulated miRNA transcriptomes in each cell line and now identify AnAc-regulated changes in mRNA and lncRNA transcript expression. In MCF-7 cells, 80 AnAc-responsive genes were identified, including lncRNA MIR22HG. More AnAc-responsive genes (886) were identified in MDA-MB-231 cells. Only six genes were commonly altered by AnAc in both cell lines: SCD, INSIG1, and TGM2 were decreased and PDK4, GPR176, and ZBT20 were increased. Modeling of AnAc-induced gene changes suggests that AnAc inhibits monounsaturated fatty acid biosynthesis in both cell lines and increases endoplasmic reticulum stress in MDA-MB-231 cells. Since modeling of downregulated genes implicated NFκB in MCF-7, we confirmed that AnAc inhibited TNFα-induced NFκB reporter activity in MCF-7 cells. These data identify new targets and pathways that may account for AnAc’s anti-proliferative and pro-apoptotic activity.

. Differentially expressed genes (DEGs). The log2-fold change with zero value in the control conditions were arbitrarily set to one plus the maximum log2-fold change value and those with zero value in the treatment conditions were arbitrarily set to the minimum log2-fold change value minus one. The number of differentially expressed genes in each comparison is shown and the number of upregulated genes indicated with the upward arrow and downregulated genes indicated by downward arrow. Z All Cells is the sum of both cell lines. Y Sum of AnAc treatment and control for each cell line.
AnAc reduced SCD (stearoyl-CoA desaturase, also called SCD1) transcript levels in both MCF-7 and MDA-MB-231 cells, suggesting an ERα-independent effect. However, different mechanisms may be responsible for SCD downregulation by AnAc in each cell line. For example, E 2 stimulates SCD transcription by increasing transcription of SREBP-1C in MCF-7 cells 22 ; thus, it is possible that the ERα-dependent NRAM activity of AnAc 2 in MCF-7 contributes to SCD inhibition. Whereas an ERα-independent activity in MDA-MB-231 cells (or both cell lines) may be involved in the observed decrease in SCD transcript expression. SCD is anchored in the ER where it catalyzes the production of monounsaturated fatty acids (MUFAs, primarily oleic acid, oleate and palmitoleate) that are essential for membrane biogenesis in cancer cell proliferation 20 . Interestingly, oleic acid promotes proliferation in a number of breast cell lines, including MCF-7 and MDA-MB-231 23 . Importantly, oleic acid was also shown to inhibit apoptosis while palmitic acid (a precursor of oleic acid, Fig. 2) increased apoptosis in MDA-MB-231 cells 24 . SCD was also one of the most downregulated genes in primary breast cancer cells treated with 5 µM curcumin, another anticancer phytochemical 25 . SCD protein, not mRNA, was inhibited by cis−9, trans-11 and trans−10, cis−12 conjugated linoleic acid (CLA) isomers (45 μM) in MDA-MB-231 cells, but the mechanism was not identified 26 . A recent study demonstrated that SCD is essential for viability in three out of the four TNBC cell lines studied, including MDA-MB-231, that showed high sensitivity to SCD depletion 27 . Localized and systemic SCD deficiency causes ERS by increasing peroxisome proliferator active receptor ϒ (PPARϒ) Coactivator 1α (PGC-1α) and activates UPR (reviewed in 28 ). "Apoptosis and survival: ERS response pathway" was upregulated by AnAc specifically in MDA-MB-231 cells (Fig. 1).  Upregulation of SCD in B16F10 mouse melanoma cells contributed to tumor formation and metastasis in vivo and CAY10566, a selective SCD inhibitor (IC 50 ~7 nM), reduced lung metastasis in vivo 29 . That paper reported high SCD was associated with shorter disease free survival (DFS) in skin cutaneous and uveal melanoma, renal clear cell carcinoma, and pancreatic adenocarcinoma 29 . We used BreastMark 30 and KM plotter 31 to examine the correlation of SCD transcript expression and DFS in breast tumors ( Supplementary Fig. 2). These analyses reveal that high SCD correlates with lower DFS in all breast and luminal A tumors, but does not reach statistical significance in TNBC, perhaps due to a lower number of tumor samples analyzed ( Supplementary Fig. 2C). While the mechanism of AnAc inhibition of SCD expression reported here is unknown, the SCD promoter binds and is upregulated by AP1, C/EBPα, LXR, TR, SREBP1, NF1, NFY, SP1, C/EBPα, PPARα and PPARγ 32 , possible targets of AnAc action. Although 13 miRNAs were predicted to target the 3-UTR 33 , few have been experimentally validated. miRNAs downregulating SCD by direct interaction with its 3′UTR include miR-125b 34 , miR-199a-3p 35 , miR-212-5p 36 , and miR-27a 37 . None of these miRNAs were upregulated by AnAc with a 6 h treatment of MCF-7 or MDA-MB-231 cells 6 . Further studies will be necessary to delineate the mechanism for SCD downregulation in both cell lines.
AnAc inhibited INSIG1 (Insulin Induced Gene 1) expression in MCF-7 and MDA-MB-231 cells ( Fig. 1), again implying an ERα-independent mechanism. INSIG-1 anchors sterol regulatory element-binding protein (SREBP)/ cleavage-activating protein (SCAP) in the ER membrane prior to its glycosylation or cholesterol binding which reduces its affinity to INSIG-1 allowing movement of SCAP/SREBP to the Golgi. Subsequent proteolytic activation of SREBP leads to its nuclear localization and upregulation of genes important in the uptake and synthesis of fatty acids, cholesterol, and phospholipids 38 42 .
AnAc reduced TGM2 (transglutaminase 2) transcript levels in MCF-7 and MDA-MB-231 cells. TGM2 is a tumor and stem cell survival factor in breast and other cancers 43,44 . TGM2 has intrinsic and Ca 2+ dependent kinase activity and phosphorylates target proteins involved in cell proliferation and/or apoptosis 45 . TGM2 results in constitutive activation of NFκB via the noncannonical pathway, creating a feedback loop where NFκB upregulates TGM2 expression 46 . The increased NFκB and TGM2 results in drug-resistance and increased cancer stemness 47 . Knockdown of TGM2 in MDA-MB-231 cells reversed epithelial to mesenchymal transition (EMT) and stimulated doxetaxel-induced apoptosis 48 . Overexpression of TGM2 in MCF-10A cells inhibited basal oxygen consumption rate (OCR) and stimulated glycolysis as measured by extracellular acidification (ECAR) whereas TGM2 knockdown in MCF-7 cells had the opposite effect 49 . Interestingly, we reported that AnAc stimulates basal OCR in both MCF-7 and MDA-MB-231 cells 7 , a result correlating with the reduction in TGM2 transcript detected here.  Table 2). The canonical network analysis of the 19 genes downregulated by AnAc in MCF-7 generated by pathway enrichment analysis in MetaCore is shown in Fig. 3. The pathways and GO processes identified by MetaCore in the AnAc-downregulated genes in MCF-7 cells are shown in Fig. 1 and the pathway enrichment analysis of networks associated with DEGs in MCF-7 is shown in Supplementary Fig. 3. The top network for AnAc-downregulated genes centers on Acyl-CoA synthetase, ACSL6, APBECH3, CDIP, and EGR1 ( Supplementary Fig. 4). MetaCore transcription factor network analysis identified 30 transcription factors in the DEGs in MCF-7 cells including CREB, p53, ESR1 (ERα), and RelA/NFκB ( Supplementary Fig. 5). AnAc was previously reported to inhibit NFκB activation in KBM-5 cells 50 .

AnAc inhibits tumor necrosis factor α (TNFα)-stimulated NFκB in MCF-7 cells. The path-
way enrichment analysis of networks associated with downregulated genes in AnAc-treated MCF-7 cells ( Supplementary Fig. 6) suggests involvement of NFκB. MCF-7 cells have low NFκB activity 51 . We examined if AnAc would inhibit TNFα-stimulated NFκB luciferase reporter activity in transiently transfected MCF-7 cells (Fig. 4). Consistent with the DEGs identified in RNA-seq analysis of AnAc-treated MCF-7 cells and with the AnAc inhibition of TGM2 that stimulates NFκB expression and activity (modeled in Fig. 2), AnAc inhibited TNFα-stimulated NFκB luciferase reporter activity (Fig. 4). We reported that AnAc inhibits NFκB target gene CCND1 expression in MCF-7 cells 2 and AnAc reduced CCND1 in MDA-MB-231 cells (Supplementary Table 5), results in agreement with the antiproliferative activity of AnAc.

Genes downregulated in MCF-7 cells by AnAc.
We hypothesized that the ERα antagonist activity of AnAc 6 might be involved in the decrease of selected gene transcripts in AnAc-treated MCF-7 cells and not in MDA-MB-231 cells. Based on our data and the literature reports cited below, we suggest that this hypothesis may support the downregulation of ZNF462, MALL (BENE), and EGR1 transcript expression by AnAc in MCF-7 cells.
AnAc inhibited ZNF462 expression in MCF-7 cells (Table 3). ZNF462 was identified as a putative target of miR-210 which is upregulated by HIF-1α in pancreatic cancer 52 . A search in the NURSA Transcriptomine  database 53 revealed that both E 2 and 4-OHT increase transcript levels of ZNF462 in MCF-7 cells. Thus, the NRAM activity of AnAc with ERα 2 may be responsible for the observed decrease in ZNF462 expression.
AnAc inhibited MALL (BENE) expression in MCF-7 cells (Table 3). MALL is a member of the proteolipid family that localizes in glycolipid-and cholesterol-enriched membrane rafts and it interacts with CAV-1. A search in the NURSA Transcriptomine database 53 revealed that both E 2 (100 nM, 12 h) and fulvestrant (100 nM, 12 h) inhibited MALL transcript expression in MCF-7 cells, a result that seems contradictory for an ERα-mediated response, but not one mediated by GPER1 that binds E 2 and fulvestrant as agonists with Kd = 3-6 and 10-100 nM, respectively 54 . No references regarding the regulation or function of MALL in breast or other cancers were found in PubMed.
AnAc inhibited EGR1 expression in MCF-7 cells (Table 3). EGR1 is a member of the immediate-early gene group of transcription factors whose transcription is rapidly increased by E 2 in MCF-7 cells 55 and deleted in ERαbreast tumors where it is thought to be a tumor suppressor 56 . 4-OHT suppresses E 2 -simulated EGR1 transcription in MCF-7 cells 57 . Thus, the repression of EGR1 expression by AnAc in MCF-7 cells may reflect its ability to block E 2 -induced gene transcription by inhibiting ERα-DNA binding 2 .
AnAc had the greatest inhibitory activity on RBMS1 (also called MSSP, MSSP-1) transcript expression in MCF-7 cells (Table 3). RBMBS1/MSSP1 has been implicated in DNA replication, gene transcription, cell cycle progression and apoptosis 58 . Increased RBMBS1/MSSP-1 was associated with cisplatin resistance in ovarian cancer cells 59 . A search in the NURSA Transcriptomine database 53 revealed that E 2 inhibited RBMS1 expression in MCF-7 cells. Another dietary anticancer agent, bromelain, a mixture of proteolytic enzymes found in pineapples (reviewed in 60 ) also downregulated RBMS1 expression in MCF-7 cells 61 . Thus, two dietary phytochemicals inhibit RBMS1 expression in MCF-7 cells. RBMS1 is downregulated by miR-383 62 , but miR-383 was not regulated by AnAc treatment of MCF-7 cells 6 . It is unknown how RBMS1 downregulation by AnAc in MCF-7 cells contributes to AnAc's antiproliferative/pro-apoptotic activity in these cells.
AnAc inhibited STIM1 and LAM2 expression in MCF-7 cells (Table 3). STIM1 is an ER Ca 2+ sensor that triggers Ca 2+ influx by activating store-operated calcium entry and is involved in the TGF-β-induced suppression of cell proliferation 66 . Inhibition of STIM1 expression by TGF-β in MCF-7 and MDA-MB-231 cells inhibited cell proliferation 66 . LAMC2 is a subunit of the basement membrane protein laminin-332 that interacts with CD44 on the membrane of breast cancer cells, stimulates cell migration, and is regarded as a typical cancer invasion marker corresponding with poor patient prognosis 67 . Thus, the inhibition of LAMC2 by AnAc provides a potential mechanism for the anti-proliferative activity of AnAc in breast cancer cells in vitro.
AnAc inhibited SAMD9 expression in MCF-7 cells (Table 3). SAMD9 has antiproliferative activity in H1299 lung adenocarcinoma cells in vitro and in tumor xenografts in vivo 68 . Further, SAMD9 expression is lower in breast tumors than normal breast 69 . A search in the NURSA Transcriptomine database 53 revealed that knockdown of ERα coactivator SRC-1/NCOA1 increased SAMD9 in LY2 endocrine-resistant cells derived from MCF-7 cells 70 . However, we did not detect any change in NCOA1 transcript expression in our RNA-seq data of AnAc-treated MCF-7 cells. The inhibition of putative tumor suppressor SAMD9 expression by AnAc seems to conflict with AnAc's anti-cancer activity in MCF-7 cells. However, this may be a time-dependent effect since we analyzed gene expression after only 6 h of AnAc treatment.

Genes downregulated by AnAc in MDA-MB-231 cells. AnAc inhibited the expression of 378 genes in
MDA-MB-231 cells (Table 1, Supplementary Table 4). The top 10 downregulated pathways identified by MetaCore enrichment analysis is shown in Fig. 1. Not surprisingly since AnAc inhibits MDA-MB-231 cell proliferation 2 , the top two pathways involve cell cycle regulation. Intriguingly, third on the list is "Transcription: Ligand-dependent activation of the ESR1/SP pathway". Although MDA-MB-231 cells are ERα-, they were reported to express ERβ protein 76 . Recent studies indicate that activation of peroxisome proliferator activated receptor (PPAR) δ and inhibition of PPARγ stimulate ERα expression in ERα-mouse mammary tumors 77,78 . We did not detect ESR1 or any of the PPAR genes among those regulated by AnAc in MDA-MB-231 cells. A PubMed search for RNA-seq studies in MDA-MB-231 cells treated with an 'anti-cancer drug' , found only one report 79

Three genes upregulated by AnAc in both MCF-7 and MDA-MB-231 cells. AnAc stimulated
ZBTB20, PDK4, and GPR176 expression in MCF-7 and MDA-MB-231 cells (Fig. 1). We would expect these increases to be ERα-independent. MetaCore analysis identified only one pathway in common for these three upregulated genes: "Transcription Sirtuin6 (SIRT6) regulation and function". SIRT6, an established chromatin regulatory protein, is a tumor suppressor that has three enzymatic activities: deacetylase, ADP-ribosyltransferase, and de-fatty-acylase 80 . SIRT6 overexpression inhibited breast cancer stem cell biogenesis in cells with a PI3K mutation and murine PyMT mammary tumor progression in vivo 81 . Hence if ZBTB20, PDK4, and GPR176 indeed stimulate SIRT6 function, e.g., by increasing transcription, stabilizing the protein, or increasing its activity by increasing NAD+ and free FAs 80 , this could provide a mechanism by which AnAc inhibits MCF-7 and MDA-MB-231 viability. Further study will be needed to examine AnAc-mediated metabolic changes in these cells. Figure 6 is model of the potential cellular functions of the three AnAc-upregulated genes common to MCF-7 and MDA-MB-231 breast cancer cells. ZBTB20 (also called DPZF, HOF, and ZNF288) is a member of the POK (POZ and Krüppel) family of transcriptional repressors 82 . ZBTB20 is upregulated in HCC 82 and non-small cell lung cancer (NSCLC) 83 , but downregulated in primary prostate cancer samples and metastases 84 . ZBTB20 was among the genes downregulated in  primary breast tumors from patients treated with aromatase inhibitors, either letrozole or anastrozole for 2 weeks prior to surgery 85 . In NSCLC, ZBTB20 downregulated FOXO1 by binding to its 5′ promoter 83 . Although FOXO1 was not among the AnAc-regulated genes in MCF-7 cells, FOXO1 was upregulated by AnAc in MDA-MB-231 cells (Supplementary Table 5), a result opposite of what might be anticipated if an AnAc-mediated increase in ZBTB20 inhibits FOXO1 expression. However, SIRT6 deacetylates FOXO1 leading to its export from the nucleus to the cytoplasm and hence inhibiting its transcriptional activity, as well as glycolysis, the pentose phosphate pathway, lipid metabolism, and breast cancer stem cell biogenesis 81,86 . ZBTB20 was recently identified as a tumor suppressor that cooperates with PTEN to prevent malignant progression in prostate cancer 84 . We did not detect PTEN among the AnAc-regulated genes in either cell line. Together these studies suggest that an increase in tumor suppressor ZBTB20 may contribute to the anti-proliferative activity of AnAc in breast cancer cells. When active, PDK4 phosphorylates and inhibits pyruvate dehydrogenase (PDH) which converts pyruvate to acetyl-CoA for the TCA cycle or fatty acid biosynthesis. Thus, an increase in PDK4 would decrease glucose carbon flux into the TCA cycle and lipid biosynthesis, consistent with the three common downregulated genes modeled in Fig. 2. Estrogen-related receptor gamma (ERRγ, ESRRG) is a major activator of PDK4 87,88 , but we did not find ESRRG among the AnAc-regulated genes in either cell line. PDK4 expression was higher in TAM-resistant MCF-7 cells than parental MCF-7 cells and siPDK4 sensitized the cells to growth inhibition by fulvestrant 89 . Increased PDK4 results in an increase in mitochondrial ROS 88 and ERS 90 , findings commensurate with MetaCore-identified upregulated pathway 2: "Apoptosis and Survival: ERS response" in MDA-MB-231 cells (Fig. 1).
AnAc increased GPR176, orphan G-protein coupled receptor (GPCR), in both MCF-7 and MDA-MB-231 cells. GPR176 is an evolutionarily conserved, vertebrate class A orphan GPCR that acts in a ligand-independent manner and can repress adenyl cyclase 91 . A sequence-structure based alignment of known GPCRs to identify putative ligand associations for orphan GPRs posited free fatty acids as ligand for GPR176 92 . This association raises an interesting speculation that AnAc may activate GPR176. Since an increase in active GPR176 would be expected to decrease cAMP, and hence decrease active PKA, we wondered if cAMP regulates SIRT6. Indeed an increase in cAMP-activated PKA reduced SIRT6 by promoting its ubiquitin-proteasome-mediated degradation 93 . This is modeled in Fig. 6. Taken together, the observed common gene changes in response to AnAc correspond with the observed ability of AnAc to inhibit the proliferation of these two breast cancer cell lines. Table 1, Fig. 1), 12 were protein-coding genes that were not affected by AnAc in MDA-MB-231 cells (Table 4). Only 2 genes have been reported to be regulated by ERα; hence, both ERα and ERα-independent mechanisms are likely involved in the AnAc-regulated changes in their expression specifically in MCF-7 cells. MetaCore network analysis of these AnAc-upregulated genes identified only one direct interaction between JNK and ETV6 (Supplementary Fig. 7). When analyzed by pathway enrichment using two steps, MetaCore calculated 'hub proteins' including Vimentin, JNK, PKA, and GSK3beta ( Supplementary Fig. 8).

MCF-7 cells (
The mixed agonist/antagonist activity of AnAc in MCF-7 cells 2 may play a role in the increase in transcript level of VIM (vimentin), TMC5, a multi-pass membrane protein, ETV6, and MAPK10 (Table 5). This suggestion is based on data in the NURSA Transcriptomine database 53 that revealed E 2 inhibited VIM, TMC5, ETV6, and MAPK10 expression in MCF-7 cells. We reported that AnAc reduced miR-378g in MCF-7 cells 6 and note that miR-378 targets VIM 94 , suggesting a possible mechanism for the increase in VIM by relief of repression. ETV6 is a dominant-acting cancer gene that appears to be a site of frequent genomic rearrangements in human breast tumors, but is not amplified 95 . The mechanism for AnAc regulation of ETV6 and MAPK10 will require further investigation.

Genes uniquely increased by AnAc in MDA-MB-231 cells. AnAc uniquely upregulated 503 genes in
MDA-MB-231 and not in MCF-7 cells (Table 1, Fig. 1, and Supplementary Table 5). The top 10 upregulated pathways identified by MetaCore enrichment analysis are shown in Fig. 1 and the top 10 GO processes in Fig. 2. Notably, the ERS response pathway was increased by AnAc in MDA-MB-231 cells. Network analysis of the top 50 upregulated genes using Dijkstra's shortest paths algorithm calculating the shortest direct paths with one step shows connections of a number of upregulated genes to each other and to STAT3, STAT5, NFκB, AP1, and AR in MDA-MB-231 cells (Fig. 7). The top 10 genes upregulated uniquely by AnAc in MDA-MB-231 cells and their roles in the ER stress response are summarized in Supplementary Table 7.
When cellular stresses perturb energy levels, the redox state, or Ca 2+ concentrations, unfolded proteins accumulate triggering the unfolded protein response (UPR) and proteins aggregate, contributing to ERS 96 Table 7). The UPR inhibits protein translation, induces expression of chaperones, and exports misfolded proteins to the cytosol for degradation. If the UPR fails to relieve the stress, the function of the UPR switches from promoting cell survival to promoting cell death 17,97 , which we speculate may be involved in the observed AnAc-mediated inhibition of cell viability and increased apoptosis 2 .

AnAc regulated lncRNAs in MDA-MB-231 cells. Data from the Encyclopedia of DNA Elements
(ENCODE) Project Consortium indicate that the human genome encodes >28,000 long noncoding RNAs (lncR-NAs) that are transcribed by RNA pol II, capped and polyadenylated and expressed in a tissue-specific manner (reviewed in 98 ). lncRNAs are involved in regulating numerous biological processes including roles as scaffolds, decoys or signals, cis-and trans-regulation of transcription, antisense interference, imprinting genomic loci, shaping chromosome conformation and allosterically regulating enzymatic activity (reviewed in 99 (Table 4). These changes are likely due to changes in the entire transcriptome in response to AnAc. MetaCore enrichment analysis only included MIR24-2, reflecting the paucity of information included in this database on lncR-NAs. The GO Processes and Diseases identified by MetaCore analysis are shown in Supplementary Fig. 9A and B. Only three of the lncRNAs (all downregulated by AnAc) were found in PubMed: CFLAR-AS1, UBL7-AS1, and MIR210HG (Table 4). Future studies will be needed to address the roles of these lncRNAs in mediating MDA-MB-231 cellular responses to AnAc.    Fig. 1, MetaCore analysis identified only one pathway for these three common upregulated genes: Transcription Sirtuin6 (SIRT6) regulation and function. AnAc increased GPR176 in both cell lines. GPR176 is a GZ-coupled receptor that decreases cAMP, which would be expected to decrease PKA, both of which would maintain SIRT6 protein by preventing its ubiquitinylation and proteasome-mediated degradation (dashed arrow,+). AnAc increased ZPTB20, a transcriptional repressor that inhibits FOXO1 transcription. SIRT6 deacetylates FOXO1 which decreases its nuclear localization, hence reducing glycolysis, pentose phosphate pathway, lipid metabolism, and cancer stem cell biogenesis. AnAc increased PDK4 in both MCF-7 and MDA-MB-231 cells. PDK4 phosphorylates and inhibits pyruvate dehydrogenase (PDH), which would be expected to decrease acetyl CoA, possibly inhibiting the TCA cycle, oxidative phosphorylation, and FA biosynthesis. Taken together, the observed gene changes are commensurate with the observed ability of AnAc to inhibit the proliferation of these two breast cancer cell lines.

Conclusions
The major goal of this study was to identify the global effects of AnAc on the RNA transcriptome of two well-characterized cell lines representing luminal A, ERα+ (MCF-7) and TNBC (MDA-MB-231) breast cancer. We have provided the comprehensive mRNA and lncRNA sets for each cell line and defined their common and cell-specific expression. Notably, AnAc regulated more transcripts in MDA-MB-231 than MCF-7 cells. Only three genes were commonly down-and up-regulated, respectively, by AnAc in both cell lines. The cell-specific and common up-and down-regulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. Top among the downregulated enrichment pathways were Development, Unsaturated fatty acid biosynthesis, and Immune response in MCF-7 and Cell cycle and Transcription: ligand-dependent activation of the ESR1/SP pathway in MDA-MB-231 cells, respectively. ERα-dependent and independent pathways are suggested to be involved in the AnAc-mediated transcriptome responses. Top among the upregulated enrichment pathways were Neurophysiological process and Immune response: MIF-JAB1 signaling in MCF-7 cells and PDE4 regulation of cyto/chemokine expression in arthritis and apoptosis and survival: Endoplasmic reticulum stress response pathway in MDA-MB-231 cells. Only one pathway was identified for the three common upregulated genes: Transcription Sirtuin6 regulation and function. qPCR confirmed AnAc regulation of seven genes. Our results suggest that AnAc regulates common and different pathways in ERα+ MCF-7 and MDA-MB-231 TNBC cells.

Methods
Materials. AnAc 24:1n5 was purified to greater than 95% as previously reported 2,101 . AnAc 24:1n5 was dissolved in ethanol (EtOH); thus, EtOH was used as a vehicle control. For mRNA RNA-seq. RNA was isolated from three separate experiments for each cell line and treatment as previously reported 6 . The Truseq Stranded mRNA kit (Illumina) was used to prepare mRNA libraries from 1 µg  Differential Gene Expression Analysis. The analysis was similar to that used to identify miRNAs regulated by AnAc in these cell lines 6 and that data analysis pipeline is shown in Supplementary Figure 10. The number of raw reads, number of reads after trimming, and number of reads successfully aligned for each of the samples is provided in Supplementary  In silico MetaCore network analysis. Pathway and network analysis of differentially expressed genes was performed in MetaCore version 6.27 (GeneGO, Thomson Reuters, New York, N.Y.) 103 .
Luciferase assay. To analyze NFκB activity, MCF-7 cells were transiently transfected with pGL4.32[luc2P/ NF-κB-RE/Hygro] (Promega, Madison, WI) containing five copies of a NFκB response element and pGL4-hRluc-TK (Renilla, Promega) for 48 h and treated with 10 ng/ml TNFα and 0-40 µM AnAc 24:1n5 for 6 h before performing dual luciferase assay (Promega). Firefly luciferase was normalized by Renilla luciferase. Values are the average of three separate wells in one experiment ± SEM. RNA isolation, RT-PCR and quantitative real-time PCR (qPCR) was performed essentially as described previously in MCF-7 and MDA-MB-231 cells treated with vehicle control (EtOH) or AnAc 24:1n5 (13.5 and 35 µM, respectively) for 6 h 6 . PCR Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and sequences used were are listed in Supplementary Table 9. GAPDH was used as a reference for normalization 104 . qPCR was performed in triplicate using ABI Viia 7 (LifeTechnologies). Fold change relative to vehicle-treated, control cells was estimated by the comparative threshold cycle (Ct) method (2 −ΔΔCT ) 105 .  Data availability statement. Raw sequencing data files obtained from our analysis are available at GEO: accession number GSE78011. All data analyzed during this study are included in this published article (and its Supplementary Information files).