Abstract

   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.

Introduction

   A number of plants produce anacardic acid (AnAc) which is a mixture of
   6-alkylbenzoic acid congeners^[36]1. Previously, we showed that a
   specific congener, AnAc 24:1n5, acts as a concentration-dependent mixed
   agonist/antagonist of estrogen receptor (ERα)-induced proliferation and
   transcription and inhibits ERα-estrogen response element (ERE) binding
   by interacting with the DNA binding domain (DBD), thus acting as a
   nuclear receptor alternate site modulator (NRAM)^[37]2. AnAc 24:1n5
   also inhibited MDA-MB-231 triple negative breast cancer (TNBC) cell
   proliferation, although at a higher IC[50] and via an unknown
   mechanism^[38]2. We reported that the expression of endogenous
   estrogen-regulated genes, i.e., TFF1, CCND1, and CTSD, was inhibited by
   AnAc 24:1n5 in breast cancer cell lines^[39]2. However, because AnAc
   affects multiple molecular targets (reviewed in^[40]3) and since we
   detected an ERα-independent inhibition of TNBC cell proliferation by
   AnAc 24:1n5, we suspect additional unknown molecular targets,
   independent of ERα, are altered by AnAc in these cells. Gene expression
   profiling is used in drug development to understand and predict the
   activity of novel therapeutic compounds in pre-clinical settings.
   Transcriptome analysis using bioinformatics tools gives an overview of
   biological processes and pathways affected by a ‘drug’; thus providing
   new insights about the potential cellular targets and mechanisms of
   action of that ‘drug’. Identification of such targets using RNA-seq
   would be beneficial in identifying AnAc-regulated pathways and targets
   in both luminal A breast cancer and in TNBC which primarily affects
   premenopausal women with a predominance in women of African and
   Hispanic ancestry^[41]4,[42]5.

   In previous work using RNA-seq analysis of AnAc-treated MCF-7 and
   MDA-MB-231 cells we identified 69 and 37 AnAc-regulated miRNAs,
   respectively^[43]6. MetaCore enrichment analysis revealed that no
   miRNAs were downregulated by AnAc in both cell lines while two miRNAs
   were increased by AnAc in both cell lines: miR-612 and miR-20b with the
   common gene ontology (GO) process “cellular response to inorganic
   substance”^[44]6.

   The goal of the study reported here was to use RNA-seq to identify
   alterations in mRNA target transcript levels in the same representative
   ERα-positive and TNBC breast cancer cell lines after AnAc 24:1n5
   treatment. AnAc up- or down- regulated divergent and common mRNA
   transcripts in MCF-7 and MDA-MB-231 cells. These results provide an
   overview of the processes and targets of AnAc in representative ERα+
   and TNBC breast cancer cells in vitro.

Results and Discussion

RNA-seq analysis of AnAc-regulated RNAs

   To identify primary transcriptome changes in AnAc 24:1n5 (hereafter
   AnAc)-treated MCF-7 (ERα+) and MDA-MB-231 TNBC cells, cells were
   treated with the previously established IC[50] concentrations of AnAc
   for MCF-7 (13.5 µM) and MDA-MB-231 (35.0 µM)^[45]2 prior to RNA
   isolation^[46]6. We note that AnAc has no overt effect on the viability
   of either cell line or cellular bioenergetics at that time^[47]2,[48]7.
   The treatment duration was selected since primary gene targets have
   been identified in MCF-7 cells with a 6 h treatment^[49]8 and because
   the goal was to identify early transcriptome changes in response to
   AnAc in each cell line. For target analysis, only transcripts that
   showed a log2 fold-change greater than 1 (or −1 for repressed mRNAs)
   were included^[50]9.

   Differentially expressed genes (DEGs) were identified for four pairwise
   comparisons (MCF-7 control vs. MCF-7 AnAc-treated; MDA-MB-231 control
   vs. MDA-MB-231 AnAc-treated; MCF-7 and MDA-MB-231 control vs. MCF-7 and
   MDA-MB-231 AnAc-treated; MDA-MB-231 control and AnAc-treated vs. MCF-7
   control and AnAc treated) using cufflinks and
   cuffdiff ^[51]6,[52]10,[53]11. Table [54]1 shows the number of DEGs in
   each comparison. More genes were significantly changed in response to
   AnAc in MDA-MB-231 cells vs MCF-7 cells (Fig. [55]1). These data
   suggest selectivity of AnAc-induced transcriptional perturbations
   between these cell lines.

Table 1.

   Differentially expressed genes (DEGs).
                 Comparison                     Cutoff         Number of DEGs
   MCF-7 AnAc vs. control                 P ≤ 0.05           80 (↑36, ↓44)
   MDA-MB-231 AnAc vs. control            P ≤ 0.05           886 (↑508, ↓ 378)
   All Cells AnAc vs. All Cells control^z P ≤ 0.05           25 (↑11, ↓14)
   All MCF-7 vs. All MDA-MB-231^y         Q ≤ 0.01; |FC| ≥ 2 6124 (↑3190, ↓2934)
   [56]Open in a new tab

   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.

   ^ZAll Cells is the sum of both cell lines.

   ^YSum of AnAc treatment and control for each cell line.

Figure 1.

   [57]Figure 1
   [58]Open in a new tab

   Enrichment analysis of RNA-seq data. Differentially expressed genes
   were identified in pairwise comparisons: MCF7 AnAc vs. MDA-MB-231 AnAc
   using the Tuxedo Suite of programs including Cufflink-Cuffdiff2. The
   Venn diagrams show the number of common and differentially expressed
   genes significantly downregulated (A) and upregulated (B). Pathway
   analysis was performed using GeneGo Pathways Software (MetaCore). The
   pathways identified for each comparison are listed in the order
   provided by MetaCore analysis.

   DEGs for each comparison were used for further analysis of enriched
   GO:BP^[59]12,[60]13 and KEGG Pathways^[61]14 using
   CategoryCompare^[62]15. Table [63]2 and Supplementary Tables [64]1 and
   [65]2 list the top enriched GO:BP terms with p-value cutoff 0.001 for
   each of the four pairwise comparisons of DEGs while Supplementary
   Table [66]3 lists the top enriched KEGG pathways identified in
   AnAc-treated vs. control for each cell line. None of the top five GO
   terms for DEGs from (MCF-7 control and AnAc-treated) vs. (MDA-MB-231
   control and AnAc-treated) (Supplementary Table [67]1) overlapped with
   those previously identified using Agilent microarrays to identify
   differential gene expression between non-treated MCF-7 vs. MDA-MB-231
   cells^[68]16. The difference in these results may reflect changes in
   the rank order of differentially expressed genes of cell lines treated
   with AnAc or may reflect a difference in methodological approaches to
   analyze transcriptomes.

Table 2.

   Top enriched GO:BP terms for DEGs from MCF-7 and MDA-MB-231 AnAc vs.
   MCF-7 and MDA-MB-231 control using CategoryCompare.
    GO term                    Description                   Gene# P value
   GO:0008203 Cholesterol metabolic process                  3     0.00016
   GO:0016125 Sterol metabolic process                       3     0.00022
   GO:0046165 Alcohol biosynthetic process                   3     0.00025
   GO:0006066 Alcohol metabolic process                      4     0.00032
   GO:1901617 Organic hydroxyl compound biosynthetic process 3     0.00062
   [69]Open in a new tab

   For MCF-7 cells, only one GO term was identified for DEGs in AnAc
   cells: “Cellular response to acid chemical” with four genes in that
   pathway (Supplementary Table [70]2). In contrast, for AnAc-treated
   MDA-MB-231 cells five GO:BP terms were identified with 15–27
   genes/GO:BP term and GO:BP terms related to the endoplasmic reticulum
   (ER) stress (ERS) and the unfolded protein response (UPR) as well as
   cholesterol and sterol biosynthetic responses (Supplementary
   Table [71]2). Since AnAc 24:1n5 inhibits cell proliferation in both
   cell lines after 24 h (18 h longer than the treatment here)^[72]2 these
   gene changes/pathways suggest mechanisms by which AnAc achieves its
   anti-proliferative effects differ between the two cell lines.
   Cholesterol and sterol biosynthesis take place in the ER and thus, the
   identification of these GO terms suggest that AnAc targets the ERS
   signaling pathway that is a survival factor in cancer^[73]17,[74]18.
   Others reported that targeting MAPK-activation of the ERS response in
   TNBC cells, including MDA-MB-231, induces apoptosis^[75]19. We reported
   that 24 h treatment with 10–25 µM AnAc stimulates basal oxygen
   consumption and proton leak and reduces mitochondrial reserve in both
   MCF-7 and MDA-MB-231 cells, hallmarks of the apoptotic response^[76]7.

   MetaCorenetwork enrichment analysis of the DEGs identified in
   AnAc-treated MCF-7 vs. MDA-MB-231 cells identified both cell
   line-specific and common enrichment pathways (Fig. [77]1) and GO
   processes (Supplementary Figure [78]1). MetaCore shortest direct
   pathways analysis of AnAc-regulated genes in MCF-7 cells suggests that
   increased JNK (MAPK8–10) is associated with higher ERV6 (TEL1) and
   decreased STIM1 associating with reduced EGR1 that associates with
   lower TGM2 (Supplementary Figure [79]2). Further discussion of these
   genes follows.

AnAc-downregulated genes in common to MCF-7 and MDA-MB-231 cells

   AnAc treatment downregulated three genes (SCD, INSIG1, and TGM2) in
   both MCF-7 and MDA-MB-231 cells (Fig. [80]1). Hence, we would expect
   this downregulation to be ERα-independent. The third of the top 10
   common downregulated pathways was “Regulation of lipid metabolism”
   (Fig. [81]1), which relates to SCD and INSIG1. The top GO processes
   identified were “response to fatty acid, triglyceride metabolic
   process”, and “regulation of steroid metabolic processes”
   (Supplementary Figure [82]1). Aberrant activation of lipid biosynthesis
   is involved in the early stages of breast cancer development (reviewed
   in^[83]20). Further, cell migration, invasion, and angiogenesis are all
   associated with increased SREBP-coordinated lipid biosynthesis^[84]20,
   results which may help to explain the more general, i.e.,
   ERα-independent, breast cancer cell inhibition demonstrated by AnAc. We
   modeled the roles of the three AnAc-downregulated genes (SCD, INSIG1,
   and TGM2) and one of the three commonly AnAc-upregulated genes (PDK4)
   in lipid biosynthesis in Fig. [85]2. Each gene is discussed
   individually below. Supporting this model, ginkgolic acid (an AnAc
   congener from Ginkgo biloba) that suppresses pancreatic cancer cell
   viability, colony formation, migration, and invasion while increasing
   apoptosis, was reported to inhibit expression of enzyme targets
   involved in lipid biogenesis^[86]21.

Figure 2.

   [87]Figure 2
   [88]Open in a new tab

   Modeling roles of four AnAc-regulated genes in MCF-7 and MDA-MB-231
   cells. AnAc treatment reduced SCD, INSIG1, and TGM2 and 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. SCD-1 (SCD, stearoyl-CoA desaturase-1) is a key
   rate-limiting enzyme for the synthesis of monounsaturated fatty acids.
   Endogenously synthesized monounsaturated fatty acids are metabolized by
   diacylglycerol acyltransferase (DGAT) to synthesize triglycerides (TG)
   or by acyl-CoA:cholesterol acyltransferase (ACAT) for cholesterol
   esters (CE) synthesis. INSIG1 anchors sterol regulatory element-binding
   protein (SREBP)/cleavage-activating protein (SCAP) in the endoplasmic
   reticulum (ER) membrane. SREBP-1 upregulates SCD and FASN
   transcription. TGM2 (transglutaminase 2) has various functions
   described in the text including activation of NFκB, which in turn
   regulates TGM2 expression. NFκB and proinflammatory cytokines, elevated
   in breast cancer, activate ER stress and SREBP-1.

   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^[89]22; thus, it is possible that the
   ERα-dependent NRAM activity of AnAc^[90]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^[91]20. Interestingly, oleic acid promotes
   proliferation in a number of breast cell lines, including MCF-7 and
   MDA-MB-231^[92]23. Importantly, oleic acid was also shown to inhibit
   apoptosis while palmitic acid (a precursor of oleic acid, Fig. [93]2)
   increased apoptosis in MDA-MB-231 cells^[94]24. SCD was also one of the
   most downregulated genes in primary breast cancer cells treated with
   5 µM curcumin, another anticancer phytochemical^[95]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^[96]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^[97]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^[98]28).
   “Apoptosis and survival: ERS response pathway” was upregulated by AnAc
   specifically in MDA-MB-231 cells (Fig. [99]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^[100]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^[101]29. We used
   BreastMark^[102]30 and KM plotter^[103]31 to examine the correlation of
   SCD transcript expression and DFS in breast tumors (Supplementary
   Fig. [104]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. [105]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^[106]32, possible targets of AnAc action.
   Although 13 miRNAs were predicted to target the 3-UTR^[107]33, few have
   been experimentally validated. miRNAs downregulating SCD by direct
   interaction with its 3′UTR include miR-125b^[108]34,
   miR-199a-3p^[109]35, miR-212-5p^[110]36, and miR-27a^[111]37. None of
   these miRNAs were upregulated by AnAc with a 6 h treatment of MCF-7 or
   MDA-MB-231 cells^[112]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. [113]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^[114]38. INSIG1 is a direct
   target of SREBP^[115]39. Supporting a role for INSIG1 in cell
   viability, knockdown of INSIG1 inhibited ZR-75-1 and MDA-MB-468 breast
   cancer and MCF-10A immortalized normal breast epithelial cell
   viability^[116]40. A methanol extract of black cohash (40 µg/ml) first
   stimulated (6 h) and then inhibited (24 h) INSIG1 transcript expression
   in MDA-MB-453 breast cancer cells^[117]41. In contrast, gemcitabine, a
   nucleoside analog used to treat breast cancer, stimulated INSIG1
   expression in MCF-7 and MDA-MB-231 cells with MCF-7 cells showing
   higher INSIG1 than MDA-MB-231 cells^[118]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^[119]43,[120]44. TGM2 has intrinsic and
   Ca^2+dependent kinase activity and phosphorylates target proteins
   involved in cell proliferation and/or apoptosis^[121]45. TGM2 results
   in constitutive activation of NFκB via the noncannonical pathway,
   creating a feedback loop where NFκB upregulates TGM2
   expression^[122]46. The increased NFκB and TGM2 results in
   drug-resistance and increased cancer stemness^[123]47. Knockdown of
   TGM2 in MDA-MB-231 cells reversed epithelial to mesenchymal transition
   (EMT) and stimulated doxetaxel-induced apoptosis^[124]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^[125]49. Interestingly, we reported that
   AnAc stimulates basal OCR in both MCF-7 and MDA-MB-231 cells^[126]7, a
   result correlating with the reduction in TGM2 transcript detected here.

Genes uniquely inhibited by AnAc in MCF-7 cells downstream of NFκB

   Of the 44 gene transcripts identified as downregulated by AnAc in MCF-7
   cells, 19 were matched to genes, 12 were protein-coding genes, and 13
   are unannotated (Table [127]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. [128]3. The pathways and GO
   processes identified by MetaCore in the AnAc-downregulated genes in
   MCF-7 cells are shown in Fig. [129]1 and the pathway enrichment
   analysis of networks associated with DEGs in MCF-7 is shown in
   Supplementary Fig. [130]3. The top network for AnAc-downregulated genes
   centers on Acyl-CoA synthetase, ACSL6, APBECH3, CDIP, and EGR1
   (Supplementary Fig. [131]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. [132]5). AnAc was previously reported to inhibit NFκB activation
   in KBM-5 cells^[133]50.

Figure 3.

   [134]Figure 3
   [135]Open in a new tab

   AnAc downregulated genes canonical pathway map for MCF-7 cells
   generated by MetaCore. Networks identified were: 1) SCD, LXRα, Insulin,
   Norepinephrine, IGF-1; 2) miR-22, CDIP; 3)MALL, NCOA2, E2 cytoplasm,
   hyaluronic acid extracellular, ESR1 (nuclear); 4) MRLC, CaMK II, STIM1,
   CARACM1, Ca; 5) uPAR, fibrinogen, BDKRB2, C2b, alpha-X/beta-2 integrin.
   All objects with the blue circle are downregulated by AnAc. The lines
   are connections that have been documented in the literature with green
   lines indicating canonical pathways.

AnAc inhibits tumor necrosis factor α (TNFα)-stimulated NFκB in MCF-7 cells

   The pathway enrichment analysis of networks associated with
   downregulated genes in AnAc-treated MCF-7 cells (Supplementary
   Fig. [136]6) suggests involvement of NFκB. MCF-7 cells have low NFκB
   activity^[137]51. We examined if AnAc would inhibit TNFα-stimulated
   NFκB luciferase reporter activity in transiently transfected MCF-7
   cells (Fig. [138]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. [139]2), AnAc inhibited TNFα-stimulated NFκB luciferase reporter
   activity (Fig. [140]4). We reported that AnAc inhibits NFκB target gene
   CCND1 expression in MCF-7 cells^[141]2 and AnAc reduced CCND1 in
   MDA-MB-231 cells (Supplementary Table [142]5), results in agreement
   with the antiproliferative activity of AnAc.

Figure 4.

   Figure 4
   [143]Open in a new tab

   AnAc inhibits TNFα-induced NFκB luciferase reporter activity in
   transiently transfected MCF-7 cells. MCF-7 cells were transfected with
   a NFκB response element luciferase reporter and a Renilla reporter for
   48 h. Cells were treated with 10 ng/ml TNFα EtOH (vehicle control, open
   bar, and 1–40 µM AnAc for 6 h before performing dual luciferase assay.
   Values are the average of three separate wells in one experiment ± SEM.
   *p < 0.01 versus EtOH control (open bar).

Genes downregulated in MCF-7 cells by AnAc

   We hypothesized that the ERα antagonist activity of AnAc^[144]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 [145]3). ZNF462
   was identified as a putative target of miR-210 which is upregulated by
   HIF-1α in pancreatic cancer^[146]52. A search in the NURSA
   Transcriptomine database^[147]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^[148]2 may be responsible for the observed
   decrease in ZNF462 expression.

Table 3.

   Genes significantly inhibited in MCF-7 cells after 6 h. of 13.5 µM
   anacardic acid (AnAc) treatment.
   Gene Control AnAc P value Description Top 3 GO terms
   RBMS1 35.69 15.14 5.00E-05 RNA Binding Motif Single Stranded
   Interacting Protein GO:0006396: RNA processing; GO:0006260: DNA
   replication; GO:0003697: single-stranded DNA binding
   SCD 400.61 234.01 0.001 stearoyl-CoA desaturase GO:0004768:
   stearoyl-CoA 9-desaturase activity;GO:0006633: fatty acid biosynthetic
   process;GO:0005506: iron ion binding
   RNU4-2 104.59 27.41 0.0016 U4 Small Nuclear 2
   TSPAN33 104.59 27.41 0.0034 Tetraspanin 33 GO:0016021; integral to
   membrane
   STIM1 25.05 10.56 0.0038 Stromal Interaction Molecule 1 GO:0006812:
   cation transport; GO:0043234: protein complex; GO:0005515: protein
   binding
   GATA6-AS1 1.19 0.19 0.0047 GATA6 Antisense RNA 1 (Head To Head)
   TGM2 3.82 2.27 0.0069 Transglutaminase 2 GO:0043277: apoptotic cell
   clearance; GO:0018149: peptide cross-linking; GO:0060662: salivary
   gland cavitation
   CD22 1.04 0.57 0.0161 Sialic Acid Binding Ig-Like Lectin 2
   GO:0009897:external side of plasma membrane; GO:0005887: integral to
   plasma membrane; GO:0005515:protein binding
   RN7SL389P 1.15 0.00 0.02285 RNA, 7SL, Cytoplasmic 389, Pseudogene
   CDIP1 1.25 0.61 0.0229 Cell Death-Inducing P53 Target 1 GO:0042771:
   intrinsic apoptotic signaling pathway in response to DNA damage by p53
   class mediator; GO:0033209: tumor necrosis factor-mediated signaling
   pathway; GO:0006915:apoptotic process
   INSIG1 86.16 64.07 0.0285 Insulin Induced Gene 1 GO:1901303: negative
   regulation of cargo loading into COPII-coated vesicle; GO:0032937:
   SREBP-SCAP-Insig complex GO:0032933: SREBP signaling pathway
   LAMC2 5.68 3.17 0.03285 Laminin Subunit Gamma 2 GO:0005610: laminin-5
   complex; GO:0034329: cell junction assembly; GO:0031581: hemidesmosome
   assembly
   SAMD9 2.16 1.44 0.0353 SAM Domain-Containing Protein 9 GO:0043231:
   intracellular membrane-bounded organelle; GO:0005737: cytoplasm;
   GO:0005515: protein binding
   ZNF462 1.35 0.67 0.0413 Zinc Finger Protein 462 GO:0006325: chromatin
   organization; GO:0043392: negative regulation of DNA binding;
   GO:0003677: DNA binding
   MIR22HG 12.49 5.52 0.0421 MIR22 Host Gene
   MALL 15.93 10.98 0.04315 Mal, T-Cell Differentiation Protein-L; Protein
   BENE GO:0030136: clathrin-coated vesicle; GO:0016023: cytoplasmic
   membrane-bounded vesicle; GO:0042632: cholesterol homeostasis
   RNU5B-1 76.15 25.32 0.0453 U5B Small Nuclear 1
   RNU4-1 57.57 26.33 0.0467 U4 Small Nuclear 1
   EGR1 17.13 13.03 0.0474 Early Growth Response 1 GO:0072303: positive
   regulation of glomerular metanephric mesangial cell proliferation
   GO:0072110: glomerular mesangial cell proliferation; GO:0071873:
   response to norepinephrine
   [149]Open in a new tab

   Genes are arranged from the most to least statistical significance.
   Values are FKPM. All values are significantly different, P < 0.05. The
   GO terms are listed in the order provided by DEG analysis and
   subsequent network analysis in MetaCore Gene Ontology (GO) algorithm to
   characterize the biological pathways altered by AnAc.

   AnAc inhibited MALL (BENE) expression in MCF-7 cells (Table [150]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^[151]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^[152]54. No references regarding the regulation