3.1. Global Overview of DHA Impact on MDA-MB-231 Cells Transcriptome
In order to gain further insights into the molecular mechanisms triggered by DHA in breast cancer cells, we have undertaken a transcriptomic analysis of MDA-MB-231 cells treated with 100 µM of DHA, a concentration that has proven its efficiency to reduce proliferation, induce apoptosis, and prevent invasion in this cell line [
37,
38]. The RNA-seq analyses were performed on biological quadruplicates, and the average depth of coverage obtained after sequencing was 37 million pairs of reads. The principal component analysis (PCA) of the samples shows a very high intra-group homogeneity, since more than 90% of the variability of the data is explained by the first two axes (
Figure 1). The samples treated with DHA for 24 h stand out clearly (PC1: 79.5%) from the rest of the samples, which shows that the effect of the treatment appears particularly between 12 and 24 h of treatment.
This obvious change in the pattern of gene expression in treated cells compared to control cells concerns a total of 390 genes differentially expressed in a statistically significant manner (i.e., Log2 fold change ≥1 and
p < 0.001) after 24 h in the presence of DHA. A total number of 23 and 213 genes were upregulated in DHA-treated cells versus controls at 12 h and 24 h time points, respectively (
Table 1 and
Table S2). Among the 23 genes upregulated after 12 h of treatment, 16 were also differentially expressed at 24 h and only seven among them (i.e.,
AKR1C1,
EPGN,
HMOX1,
PLIN2,
SLCO2B1, and two LncRNA/pseudogene) are further upregulated showing a time-dependent effect of DHA (
Figure 2a). By contrast, five genes (i.e.,
AKR1C3,
CPT1A,
GCLM,
HSPA6,
SLC25A20) and two LncRNA (
RP5-875H18.4 and
LINC01363) among those upregulated at 12 h were not differentially expressed at 24 h.
Furthermore, 32 and 177 additional genes were downregulated in DHA-treated cells versus controls at 12 h and 24 h time points, respectively (
Table 2 and
Table S3). Among them, 17 genes were downregulated at 12 h and further decreased at 24 h (
Figure 2b), showing a time-dependent effect of DHA. Besides, the expression level was less decreased at 24 h for three genes (i.e.,
OLR1,
SREBF1,
TMPRSS9) and one LncRNA.
These differentially expressed genes could be categorized in several Gene Ontology classes grouped into eight main groups (
Table S4), including: (1) lipid and sterol metabolism, (2) cell growth/proliferation, (3) apoptosis, (4) cell adhesion, migration and invasion, (5) angiogenesis, (6) ER-stress response, (7) signaling pathways, and (8) miscellaneous. The main unidirectionally modulated pathways are represented in
Figure 3, which show the percentage of genes per category that are up- or downregulated following 24 h of DHA treatment. The cholesterol biosynthesis pathway appears as clearly downregulated, and the ER-stress response pathway as obviously upregulated (
Figure 3).
3.3. DHA Impacted the Regulation of Lipids, Fatty Acid, and Sterol Metabolisms
Although the
CPT1A (Carnitine Palmitoyltransferase 1A), the key enzyme of fatty acid degradation, gene was upregulated at 12 h post-DHA treatment, it was not differentially expressed thereafter. However, most of the genes (i.e., 11 out of 20) that were downregulated by DHA at both 12 h and 24 h are involved in fatty acid and cholesterol metabolisms and their regulation (
Figure 2B,
Table 2 and
Tables S3 and S4). Ten additional genes involved in lipid or lipid-related metabolisms were also downregulated at 24 h, whereas seven genes of such pathways were upregulated. Thus, the number of genes related to lipid metabolism reaches 7% of the differentially expressed genes.
As it could be expected, the incorporation of DHA into the cells [
23,
37] resulted in the inhibition of the biosynthesis pathway of long-chain fatty acid as shown by the decreased expression of
Acyl-CoA thioesterase 1 (
ACOT1),
Fatty Acid Elongase 3 (
ELOVL3),
Stearoyl-CoA Desaturase (
SCD) and moreover long-chain polyunsaturated fatty acids (LCPUFAs) by decreasing the expression of the delta-6 desaturase
Fatty Acid Desaturase 2 (
FADS2). Twenty-four hours of DHA treatment also decreased by 2.3-fold the expression of the
Fatty Acid Synthase (
FASN), and even if
Oleoyl-ACP Hydrolase (
OLAH) gene expression was four-fold increased. Besides, the
PNPLA3 gene, which encodes a triacylglycerol lipase, is two-fold decreased. Altogether, these data indicate that fatty acid metabolism and then energy metabolism may be strongly affected by DHA treatment, which may explain in part its antiproliferative effect. Besides, DHA was shown to inhibit the increase of lipogenic activity and gene expression (especially that of
FASN) in
HER2-overexpressing breast cancer cells, which was independent of the mTOR and PPARγ pathways [
52], suggesting that DHA effect is also independent of these pathways.
In addition, ten genes involved in cholesterol metabolism were differentially expressed after DHA treatment. Nine genes involved in the cholesterol biosynthesis pathway (i.e.,
HMGCS1,
HMGCR,
MVK,
IDI1,
FDFT1,
LSS,
MSMO1,
DHCR24, and
DHCR7) are downregulated (
Figure 5), whereas one gene (
CH25H) coding an enzyme involved in cholesterol catabolism was upregulated indicating that cholesterol may be dramatically affected by DHA treatment as previously observed [
53,
54].
In addition, some genes involved directly or indirectly in fatty acid and cholesterol metabolism regulation by the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1/SREBP1) or Peroxisome Proliferator-Activated Receptor alpha (PPAR-α) were downregulated. A three-fold decrease of
SREBF1 gene itself was observed at 12 h post-DHA treatment. This gene encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor that binds to the sterol regulatory element-1 and then regulates transcription of the
LDL receptor (
LDLR) gene, as well as the fatty acid and to a lesser degree the cholesterol synthesis pathway. Indeed, the
LDLR gene was also downregulated by about three-fold following DHA treatment. The
INSIG1 gene was dramatically downregulated by DHA both after 12 h (about four-fold) and 24 h (about five-fold) of treatment (
Figure 2b). This gene encodes a transmembrane protein of the endoplasmic reticulum (ER) that regulates cholesterol metabolism, lipogenesis, and glucose homeostasis [
55]. The INSIG1 protein binds the sterol-sensing domains of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), and is essential for the sterol-mediated trafficking of these two proteins [
56]. In the same way, the
PDK4 gene, which encodes a pyruvate dehydrogenase kinase that plays a key role in the regulation of glucose and fatty acid metabolism and homeostasis via phosphorylation of the two pyruvate dehydrogenase subunits [
57], is upregulated by 5.5- and four-fold at 12 h and 24 h, respectively (
Figure 2a). By contrast, the
ANKRD1 gene is only early downregulated at 12 h but not at 24 h. The encoded protein may be a transcription factor involved in the regulation of lipid metabolism by PPAR-α [
58].
The lipoprotein metabolism is also affected by the downregulation of the
LIPG gene, which encodes a triglyceride lipase involved in high-density lipoproteins hydrolysis. The
Oxidized Low-Density Lipoprotein Receptor 1 (
OLR1) gene expression is also reduced, at both time points (
Figure 2b). However, the impact in cell cultures should rather be due to the role of OLR1 in the regulation of Fas-induced apoptosis [
59].
By contrast, several genes involved in eicosanoid, lipophilic hormones, and vitamins are upregulated. This includes the aldo-keto reductases genes
AKR1B10,
AKR1C1, and
AKR1C3, which are involved in retinoids, progesterone, and prostaglandin metabolisms, respectively. In addition, the
ALOX5AP gene encoding a 5-lipoxygenase activating protein required for leukotriene synthesis is also upregulated, as well as the
PTGS2 gene encoding Prostaglandin-Endoperoxide Synthase 2 (or cyclooxygenase-2), the key enzyme of prostaglandin synthesis. This suggests that increasing PUFA content leads to the stimulation of storage and use paths. In agreement with this suggestion, the
Perilipin (
PLIN2) gene, which encodes a protein involved in the coating of intracellular lipid droplets [
60,
61], is strongly upregulated at both time points (
Figure 2a). Finally, the
GPCPD1 gene that encodes Glycero-phosphocholine Phosphodiesterase 1 is also upregulated at 24 h (
Tables S2 and S4).
It should be noted that the effect of DHA on breast cancer cells may be different in other cell lines due to their mesenchymal
versus epithelial status. Indeed, the breast cancer epithelial cell model MCF7, with respect to the MDA-MB-231 mesenchymal model, displays increased lipogenic activity as highlighted by the higher level of FASN and other lipogenic enzymes whose expression is controlled transcriptionally [
62]. Therefore, in further work, the transcriptomic response to DHA should also be investigated in MCF-7 cells.
3.4. Antiproliferative Effect and Induction of Apoptosis
Several genes involved in the control of cell growth and the proliferation, and cell death by apoptosis or autophagy were found as differentially expressed, as highlighted in
Table S4. Many genes contribute to both cell growth and cell death pathways, as well as other biological processes. In addition, some of these gene products act in an opposite way depending on the cell type or condition. For example,
NUPR1 may act as a negative regulator of apoptosis as well as a facilitator of apoptosis [
63,
64] or
FGFR3, which may promote apoptosis in chondrocytes, but can also promote cancer cell proliferation [
65,
66]. Therefore, it is not surprising to find these genes either down- or upregulated in the same category. However, some clear trends could be highlighted from
Table S4.
Genes identified as negative regulators of cell proliferation such as
DLEC1, which is the highest upregulated gene (about 30-fold increase),
KLF4,
HMOX1,
IL12A,
NUPR1,
TRIB1 are rather upregulate, whereas positive regulators of cell proliferation are downregulated such as
BMP4,
CDKN2C,
ERBB3,
FGFR3,
TGFB3, and
WT1. Interestingly, the
PLK1 gene is also downregulated in DHA-treated cells. It encodes a CDC5/Polo-Like Kinase that acts as a negative regulator of p53 family members and then as a critical regulator of cell cycle progression, mitosis, and cytokinesis [
67,
68]. The INSIG1 metabolic regulator, whose expression is downregulated in DHA-treated cells (see above), may also play a regulatory role during the G0/G1 transition of cell growth [
69]. Genes that are involved in the mitotic process, such as
ASPM and
SAPCD2 may also play a role in the positive regulation of cell proliferation, and tumor cell growth [
70,
71,
72,
73,
74], the DHA-induced decrease of their expression is then consistent with the antiproliferative effect of DHA.
However, the most striking finding is the upregulation of 25% of the genes involved in the apoptotic signaling pathway in response to ER-stress, namely
ATF4,
CEBPB (or
NF-IL6),
CHAC1,
DDIT3 (also known as
CHOP),
ERN1 (
IRE1-α),
PPP1R15A (
GADD34),
TRIB3, and
XBP1 (
Table S4,
Figure 3 and
Figure 6) [
75,
76]. The upregulation of
INHBE is also a marker of the ER-stress induction in DHA-treated cells and may act on cell growth inhibition [
77]. The triggering of ER-stress-induced apoptosis by DHA agrees with previous studies using colon, colorectal, and glioma cancer cells [
54,
78,
79,
80] and even in non-alcoholic steatohepatitis patients [
81]. In addition, the Glutathione-Specific Gamma-Glutamyl-cyclo-transferase encoded by the
CHAC1 gene, which is also upregulated, induces a glutathione depletion that is an important factor for apoptosis initiation and execution, and thus, acts as a pro-apoptotic component of the unfolded protein response pathway by mediating the pro-apoptotic effects of the ATF4-DDIT3/CHOP cascade [
82]. Although it is always difficult to generalize results from one cancer lineage to another, previous studies in different cancer cell lines may provide some suggestions of the mechanisms by which DHA might act in MDA-MB-231 breast cancer cells to induce ER-stress and then apoptosis. In colon cancer cell lines, DHA enhanced lipid peroxidation followed by the increased levels of phosphorylated eIF2a, an early hallmark of ER-stress [
54]. Prolonged ER-stress may lead to apoptosis through the activation of the ATF-4 transcription factor and DDIT3/CHOP [
54], which agrees with our present results. In addition to being the principal site for protein synthesis and folding, ER is also the major site of Ca
++ storage and signaling. In colon cancer cells, Jacobsen et al. have shown that DHA treatment mobilizes Ca
++ from ER into the cytosol by an unknown mechanism, and then this Ca
++ perturbation may promote ER-stress [
54]. The perturbation of biosynthesis of fatty acids and cholesterol may also induce ER-stress. However, DHA reduced the cholesterol synthesis pathway in colon cancer cells [
54], contrasting with our transcriptomic results in breast cancer cells (see above). The results of Shin et al., using the cisplatin-resistant gastric cancer cell line SNU601/cis2, suggest that increased ROS generation by DHA could induce ER stress via direct action on eIF2α kinases, working upstream of the inositol 1,4,5-triphosphate receptor and Ca
++-mediated induction of the ER-stress response and apoptosis [
80]. They also highlighted the involvement of the G-protein coupled receptor 120 (GPR120), a receptor of long-chain fatty acid [
80], but this receptor does not appear to be expressed in MDA-MB-231 cells contrarily to MCF-7 breast cancer cells [
83].
In addition, some other pro-apoptotic pathways may be stimulated by DHA through the up-regulation of
CEBPG,
DDIT4,
G0S2,
PPP1R15A,
UNC5B,
PTPRH,
SESN2, and
TNFSF15 (
Table 1 and
Table S2). The CEBPG (CCAAT Enhancer Binding Protein Gamma) bZIP transcription factor may stimulate the Akt-dependent apoptotic pathway through its cooperation with FOS [
84]. The G0/G1 Switch 2 (G0S2) protein promotes apoptosis by binding to BCL2, hence preventing the formation of protective BCL2-BAX heterodimers, and is related to the regulation of lipid metabolism by PPAR-α [
85,
86]. Besides, the
PPP1R15A gene transcript levels are increased as previously reported following stressful growth arrest conditions and treatment with DNA-damaging agents [
87]. The level of the PPP1R15A (Protein Phosphatase 1 Regulatory Subunit 15A) protein, also known as GADD34, is correlated with apoptosis through the recruitment of the serine/threonine-protein phosphatase PP1. Then, PPP1R15A downregulates the TGF-β signaling pathway by promoting dephosphorylation of TGFB1 by PP1 and apoptosis by inducing TP53 phosphorylation on Ser-15 [
88,
89]. The DDIT4 proteins also regulate p53/TP53-mediated apoptosis in response to DNA damage, but via its effect on mTORC1 activity [
90]. The mTORC1 pathway may also be inhibited by Sestrin-2 (SESN2), which is known as a stress-inducible metabolic regulator stimulated, among other stresses, by ER-stress [
91]. However, the
CASTOR3 and
Sestrin-3 (
SES3) genes for mTORC1 regulators were found downregulated (
Table 2 and
Table S3). Finally, Sestrin-2, the expression of which is upregulated in DHA-treated cells, may also positively regulate the transcription by NFE2L2 of genes involved in response to oxidative stress by facilitating the SQSTM1-mediated autophagic degradation of KEAP1 [
92]. The expression of the
SQSTM1 gene being also upregulated in DHA-treated cells (
Table S2).
3.5. Reduction of Migration and Invasion
Migration and invasion of MBA-MD-231 cells were reduced by DHA-treatment as previously reported [
37], but the mechanism of this still remains unknown. From the present RNA-seq analysis, no clear scheme could be drawn, and any specific pathway appears really modulated. However, several genes known, as involved in cell migration and invasion, are differentially expressed (
Table S4). The protein encoded by the
TMPRSS9 gene is a membrane-bound type II serine polyprotease that is cleaved to release three different proteases. Two of the proteases are active and can be inhibited by serine protease inhibitors, and one is thought to be catalytically inactive. The
TMPRSS9 expression was reported to enhance the invasive capability of pancreatic cancer cells [
93] and may be involved in cancer progression. Therefore, the two-fold downregulation of
TMPRSS9 may account, at least in part, for the reduction of the invasive phenotype of MDA-MB-231 cells. Similarly, the 2.5-fold downregulation of
ADAMTS4, which encodes a protein of the “disintegrin and metalloproteinase with thrombospondin motifs” family responsible for the degradation of aggrecan, a major proteoglycan of cartilage, and brevican, a brain-specific extracellular matrix protein [
94,
95], may also account from some reduction of the invasive phenotype.
Maybe more relevant is the 2.3-fold downregulation of
ASAP3, which encodes a member of a subfamily of ADP-ribosylation factor (Arf) GTPase-activating proteins. Indeed, the reduction of
ASAP3 expression levels slowed cell migration and invasion of hepatocellular carcinoma HepG2 and MDA-MB-231 breast cancer cells [
96]. The 2.7-fold decrease of
CDC42BPG gene expression, which is known as a key regulator of cell migration and cancer dissemination [
97], is also a potential explanation of the reduction of MDA-MB-231 cell migration and invasion induced by DHA. The
CEMIP (
KIAA1190) gene was previously shown to play an important role in breast tumor growth and invasiveness [
98,
99], but, by contrast with previous genes, its early downregulation after 12 h of DHA-treatment was lost at 24 h (
Table S3). The two-fold downregulation of the
CSTK gene, encoding the Cathepsin-K lysosomal cysteine proteinase, which is initially involved in bone remodeling and resorption, is also of interest since Cathepsin-K is obviously involved in migration, invasion, and finally, bone metastasis of breast cancer cells [
100,
101].
In addition, the strong increase of
ANGPTL4 gene expression (12- and eight-fold at 12 h and 24 h, respectively—
Table 1) can also be involved in the reduction of invasion. Indeed,
ANGPTL4 encodes a glycosylated, secreted protein containing a C-terminal fibrinogen domain, which is induced by peroxisome proliferation activators and functions as a serum hormone that mediates inactivation of the lipoprotein lipase LPL and, thereby, regulates glucose homeostasis, lipid metabolism, and insulin sensitivity [
102]. However, this protein can also act as an apoptosis survival factor for vascular endothelial cells [
103] and can prevent metastasis by inhibiting vascular growth and tumor cell invasion [
103,
104,
105] although opposite effects can be observed under certain conditions or different cell types [
106,
107,
108].
The
SERPINE1 gene is also upregulated. SERPINE-1 (previously known as PAI-1) is the principal inhibitor of tissue plasminogen activator (PLAT or tPA) and urokinase (PLAU or uPA), and hence, is an inhibitor of fibrinolysis, and also of breast cancer cell migration and invasion [
109,
110]. It is noteworthy that the
PLAT gene is downregulated after 24 h of DHA treatment (
Table S3). Altogether, the increase of
SERPINE-1 and the decrease of
PLAT and
MMP11, which act as SERPIN inhibitors [
111], expression may lead to an important inhibitory effect on migration and invasion of DHA-treated cells, as previously observed [
37].
Some other genes that are more or less directly involved in cell adhesion and migration are downregulated, such as
CD34,
CLDN2,
COL1A1,
COL9A3,
FAM20C, and
NDNF (
Table S3). However, some inconsistencies should be noted such as the downregulation of the known negative regulator of breast cancer invasion or epithelial-mesenchymal transition
KLF17 [
112,
113,
114] or the upregulation of extracellular matrix protein 2 (ECM2) gene and the collagen-induced receptor tyrosine kinase DDR2 gene, which is positively involved in breast tumor metastasis [
115]. Similarly, the 2.4-fold upregulation of
calcireticulin (
CALR) and
S100P genes, both of which encode calcium-binding proteins involved in the increased migration and invasion of breast cancer cells [
116,
117,
118,
119], is surprising and further research is needed to explain these apparent contradictions.