Abstract

   The canonical Wnt/β-catenin pathway is activated in triple-negative
   breast cancer (TNBC). The activation of this pathway leads to the
   expression of specific target genes depending on the cell/tissue
   context. Here, we analyzed the transcriptome of two different TNBC cell
   lines to define a comprehensive list of Wnt target genes. The treatment
   of cells with Wnt3a for 6h up-regulated the expression (fold change >
   1.3) of 59 genes in MDA-MB-468 cells and 241 genes in HCC38 cells.
   Thirty genes were common to both cell lines. Beta-catenin may also be a
   transcriptional repressor and we found that 18 and 166 genes were
   down-regulated in response to Wnt3a treatment for 6h in MDA-MB-468 and
   HCC38 cells, respectively, of which six were common to both cell lines.
   Only half of the activated and the repressed transcripts have been
   previously described as Wnt target genes. Therefore, our study reveals
   137 novel genes that may be positively regulated by Wnt3a and 104 novel
   genes that may be negatively regulated by Wnt3a. These genes are
   involved in the Wnt pathway itself, and also in TGFβ, p53 and Hedgehog
   pathways. Thorough characterization of these novel potential Wnt target
   genes may reveal new regulators of the canonical Wnt pathway. The
   comparison of our list of Wnt target genes with those published in
   other cellular contexts confirms the notion that Wnt target genes are
   tissue-, cell line- and treatment-specific. Genes up-regulated in
   Wnt3a-stimulated cell lines were more strongly expressed in TNBC than
   in luminal A breast cancer samples. These genes were also
   overexpressed, but to a much lesser extent, in HER2+ and luminal B
   tumors. We identified 72 Wnt target genes higher expressed in TNBCs (17
   with a fold change >1.3) which may reflect the chronic activation of
   the canonical Wnt pathway that occurs in TNBC tumors.

Introduction

   Breast cancer is one of the most common tumors in women. It is a
   complex, heterogeneous disease comprising several subgroups of
   pathologies with different patient outcomes [[41]1–[42]3].
   Triple-negative breast cancer (TNBC), closely related to basal-like
   breast cancer (BLBC), is characterized by an absence of estrogen
   receptor (ER) and progesterone receptor (PR) expression and a lack of
   human epidermal growth factor receptor 2 (HER2)
   overexpression/amplification. TNBC itself constitutes a heterogeneous
   group of breast cancer [[43]4–[44]6], which is highly proliferative and
   genetically instable, and associated with a poor prognosis. Unlike
   other breast cancer subtypes, such as luminal (expressing ER and PR)
   and HER2-overexpressing (HER2+) tumors, TNBC cannot be treated with
   targeted therapies, such as tamoxifen or anti-HER2 antibodies. TNBC
   patients are therefore treated exclusively with conventional cytotoxic
   therapies, but about half of them present relapse and metastasis within
   the first three to five years after treatment [[45]7]. Therefore,
   treatment of patients with TNBC remains a major challenge for
   oncologists and alternative treatments to conventional chemotherapies
   are needed to improve their survivals.

   The Wnt signaling pathway mediates biological processes such as cell
   adhesion, migration, proliferation, differentiation and survival
   [[46]8–[47]10]. It consists of two main arms: the canonical
   (Wnt/β-catenin) and the non-canonical pathways, which differ in terms
   of their dependence on β-catenin [[48]11,[49]12]. The activation of the
   canonical Wnt pathway leads to the stabilization of β-catenin which
   translocates to the nucleus and induces the expression of Wnt target
   genes. Besides its function in normal cells/tissues, Wnt signaling can
   become deregulated during human disease. The best documented example is
   the tumorigenesis of colorectal cancer [[50]13]. The Wnt/β-catenin
   pathway is also activated in human breast cancer, in particular in the
   TNBC/BLBC breast cancer subtype that is associated with poor prognosis
   [[51]14]. Indeed, the activated form of β-catenin has been observed in
   breast cancer [[52]15–[53]18], and is frequently found in the TNBC/BLBC
   subtype [[54]14,[55]19–[56]23]. The aberrant activation of the
   Wnt/β-catenin pathway in mice leads to mammary carcinogenesis [[57]24],
   and transgenic mice expressing a constitutively active form of
   β-catenin in the mammary gland develop basal-like tumors [[58]25],
   suggesting a crucial role for the canonical Wnt pathway in TNBC/BLBC
   tumorigenesis. Mutations of genes encoding intracellular components of
   the canonical pathway, including APC (encoding adenomatous polyposis
   coli), CTNNB1 (encoding β-catenin) and AXIN, are frequent in colorectal
   and hepatocellular cancers [[59]11,[60]12,[61]26], but are rare in
   breast cancer [[62]22–[63]24,[64]27–[65]30]. Instead, deregulated
   expression of cell surface components such as LRP6 or FZD7
   transmembrane receptors may be responsible for the activation of the
   Wnt pathway in TNBC/BLBC [[66]14,[67]19–[68]23].

   Nuclear localization of β-catenin and the expression of Wnt target
   genes reflect the activation of the Wnt/β-catenin pathway.
   Nevertheless, the detection of nuclear β-catenin is experimentally
   challenging and dependent on the tissues and the cell lines tested. An
   alternative way to evaluate the activation of the canonical Wnt pathway
   is to measure the expression of Wnt/β-catenin target genes. However,
   Wnt target genes vary substantially depending on the cellular/tissue
   context. The exceptions are AXIN2 and NKD1 which are considered as
   universal Wnt target genes [[69]12]. The role of β-catenin in the
   transcriptional activation of its target genes is well documented.
   However, recent studies have also reported a link between β-catenin and
   transcriptional repression, which is an underestimated aspect of the
   Wnt signaling [[70]31–[71]33]. Several methodological approaches have
   been undertaken to identify Wnt target genes in different cellular or
   tissue contexts: stimulation of cells with Wnt3a or Wnt1 ligand
   (recombinant protein or plasmid) [[72]34,[73]35]; depletion of
   β-catenin (siRNA) [[74]36] or Tcf (dominant negative construct)
   [[75]37]; overexpression of active β-catenin (plasmid) [[76]36];
   evaluation of Wnt signaling activity (nuclear staining of β-catenin
   [[77]38,[78]39], mutations of the CTNNB1 gene [[79]40]); and screening
   to identify binding sites for the Tcf transcription factor in DNA
   sequences [[80]34]. Wnt target genes have been mostly examined in colon
   and also in ovarian and liver cancers [[81]37–[82]39], but are not
   frequently examined in breast cancer. Some Wnt target genes are
   components of the Wnt pathway itself. Such targets are mostly
   inhibitors (e.g., AXIN2 and NKD1) and probably prevent the uncontrolled
   activation of the pathway through negative feedback loops. Therefore,
   the characterization of β-catenin target genes in breast tissue may
   lead to the discovery of new regulators of the Wnt pathway and improve
   our understanding of TNBC tumorigenesis. We thus used microarrays to
   investigate the expression of 19,738 transcripts following Wnt3a
   stimulation in two TNBC cell lines, HCC38 and MDA-MB-468, and we report
   a comprehensive list of genes that are activated or repressed in breast
   cancer by Wnt3a. Pathway analysis revealed that the Wnt target genes
   were mainly associated with the Wnt, TGFβ, p53 and Hedgehog pathways.
   The comparison of our list of Wnt target genes with those previously
   identified in fibroblast and epithelial cell lines confirms that the
   identity of Wnt target genes is highly dependent on the cellular/tissue
   context. We examined the expression level of our lists of Wnt target
   genes in 130 human breast tumors. We found that 72 Wnt target genes (17
   with a fold change > 1.3) may reflect the activation of the canonical
   Wnt pathway in a more chronic situation.

Materials and Methods

Triple-negative cell lines

   BT20, BT549, HCC38, HCC70, HCC1187, HCC1937, MDA-MB-157, MDA-MB-231 and
   MDA-MB-468 cells were purchased in May 2006 and May 2008 from the
   American Type Culture Collection (LGC Standards, Molsheim, France).
   Cells were characterized by DNA and RNA microarrays [[83]41,[84]42] and
   authenticated in 2013 by short tandem repeat profiling (data not
   shown).

   BT20 cells were cultured in MEM(Eagle) (Sigma, Saint Quentin Fallavier,
   France) containing 1% Glutamax (Invitrogen), 10% fetal bovine serum
   (FBS, Invitrogen, Cergy Pontoise, France), 1.5g/L sodium bicarbonate
   (Invitrogen), 0.1mM non-essential amino-acids (Invitrogen) and 1mM
   sodium pyruvate (Invitrogen). BT549 and MDA-MB-468 cells were
   maintained in RPMI-1640 containing Glutamax (Invitrogen) and
   supplemented with 10% FBS. HCC38, HCC70, HCC1187, HCC1937 cells were
   cultured in RPMI-1640 with Glutamax containing 10% FBS, 1.5g/L sodium
   bicarbonate, 10mM Hepes (Invitrogen) and 1mM sodium pyruvate.
   MDA-MB-157 cells were maintained in Leibovitz's L-15 medium containing
   Glutamax (Invitrogen) and supplemented with 10% FBS and 10mM Hepes.
   MDA-MB-231 cells were cultured in DMEM-F12 with Glutamax (Invitrogen)
   containing 10% FBS. Antibiotics were added to all media (100U/mL
   penicillin and 100μg/mL streptomycin). Cells were cultured at 37°C in a
   5% CO[2] humidified incubator. For all experiments, cells were used up
   to the 15th passage after thawing.

Transfection of plasmid DNA

   The Wnt-responsive element-luciferase reporter WRE was kindly provided
   by the Galapagos company (Romainville, France), the mutant-responsive
   element-luciferase variant MRE by Ron Smits (Rotterdam, The
   Netherlands) [[85]43] and the pRL-TK plasmid by the Servier company
   (Croissy-sur-Seine, France). The construct pRK5-SK-β-cateninΔGSK and
   empty vector were obtained from René Bernards (Ultrecht, The
   Netherlands) [[86]44] and Maria Carla Parrini (Institut Curie Paris,
   France), respectively.

   FuGENE HD reagent (Promega, Charbonnières-les-Bains, France) was used
   to transiently transfect cells with plasmids according to the
   manufacturer’s recommendations.

Compounds

   Recombinant human Wnt3a (R&D Systems, Lille, France) was reconstituted
   at 10μg/mL in PBS containing 0.1% BSA, then used in experiments at a
   final concentration of 100ng/mL. This concentration of Wnt3a is
   routinely used to activate the Wnt pathway in mammary cells, in
   particular in TNBC cell lines [[87]35,[88]45–[89]48]. We have shown
   that this concentration of Wnt3a leads to the activation of the Wnt
   signaling pathway ([90]S1 Fig).

Antibodies

   Primary antibodies used were mouse monoclonal anti-β-catenin (clone
   14/beta-catenin, BD Transduction Laboratories, Le Pont de Claix,
   France; 1:1,000) or anti-active-β-catenin (clone 8E7, Millipore,
   Molsheim, France; 1:500), rabbit monoclonal anti-LRP5 (clone D5G4, Cell
   Signaling Technology, Ozyme, Saint Quentin en Yvelines, France;
   1:1,000), anti-LRP6 (clone C5C7, Cell Signaling Technology; 1:500), and
   mouse monoclonal anti-actin (clone AC-15, Sigma; 1:5,000). The
   secondary antibodies used were horseradish peroxidase-conjugated
   anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch, Interchim,
   Montluçon, France; 1:20,000).

SDS-PAGE and western blotting

   Cells were lysed in Laemmli buffer containing 50mM Tris (pH 6.8), 2%
   sodium dodecyl sulfate (SDS), 5% glycerol, 2mM 1,4-dithio-DL-threitol,
   2.5mM ethylenediaminetetraacetic acid, 2.5mM ethylene glycol
   tetraacetic acid, 2mM sodium orthovanadate, 10mM sodium fluoride and a
   cocktail of protease (Roche) and phosphatase (Pierce, Perbio,
   Brebières, France) inhibitors. Protein concentration in each sample was
   determined with the reducing agent compatible version of the BCA
   Protein Assay kit (Pierce). Equal amounts of total protein were
   fractionated under reducing conditions by SDS–PAGE and then blotted
   onto PVDF membranes (Bio-Rad, Marnes-la-Coquette, France). The
   membranes were blocked with 5% BSA or 10% skimmed milk in TBS
   containing 0.1% Tween 20 (TBS-T), hybridized with the primary antibody
   of interest overnight at 4°C. Membranes were washed in TBS-T and then
   hybridized with the secondary antibody for one hour at room
   temperature. Antibodies were diluted in TBS-T containing 5% BSA or 10%
   skimmed milk. After washing with TBS-T, immune complexes on membranes
   were detected by enhanced chemiluminescence (Amersham, GE Healthcare,
   Orsay, France). Actin was used as loading control.

Beta-catenin reporter assay

   Cells were transiently transfected with either the reporter plasmid WRE
   (containing Tcf-binding sites driving the transcription of the firefly
   luciferase enzyme) or the mutant variant MRE (containing inactive
   Tcf-binding sites) as described above. Co-transfection with pRL-TK,
   which encodes a Renilla luciferase gene downstream from a minimal
   HSV-TK promoter, was systematically performed to normalize for
   transfection efficiency. Eight hours after transfection, cells were
   washed and cultured overnight in culture medium without serum, then
   Wnt3a was added for 3, 6, 9 or 12 hours.

   For experiments in which the construct pRK5-SK-β-cateninΔGSK or pRK5-SK
   was used, cells were co-transfected with WRE and pRL-TK or MRE and
   pRL-TK. Under these conditions, cells were cultured for 6, 12 or 24
   hours.

   After treatment (Wnt3a stimulation or overexpression of active
   β-catenin), cells were lysed, and a luciferase assay was performed with
   the Dual-Luciferase Reporter Assay Kit (Promega), according to the
   manufacturer's instructions.

   Triplicates for each condition were included in the experiment and the
   experiment was repeated at least twice.

Microarray analysis in cell lines

   Cells were seeded in six-well plates, serum starved overnight, and then
   treated with Wnt3a for the indicated times (6, 12 or 24 hours).
   Triplicates for each condition were included in the experiment. Total
   RNA was extracted with the RNeasy Mini Kit from Qiagen (Courtaboeuf,
   France) following the manufacturer’s recommendations. After RNA quality
   and quantity controls, samples were hybridized onto Gene st 1.1
   Affymetrix chips. Samples were processed as described on the website of
   the company. The data were analyzed with the brainarray
   HuGene11stv1_Hs_ENTREZG version 14 custom chipset definition file for
   the HuGene11stv1 affymetrix array [[91]49]. The data were first log2
   transformed and normalized with RMA [[92]50]. A linear model was fitted
   with limma [[93]51] including three factors that were treated as fixed
   effects: cell line (either HCC38 or MDA-MB-468), time (6, 12 or 24
   hours) and treatment (Wnt3a stimulated or control) and all possible
   interaction terms. For each cell line at each time point, the
   significance of differences between Wnt3a-treated and control cells was
   determined and a correction for multiple testing was applied with
   Benjamini & Hochberg’s methodology [[94]52]. Only genes with
   significantly different expression (P < 0.05), with a log2 fold change
   superior to 0.3785 (i.e. fold change > 1.3) or inferior to -0.3785
   (i.e. fold change < 1.3), were selected. Pathway enrichment analysis
   was carried out with GeneTrail [[95]53]. A hypergeometric test,
   corrected for multiple testing with the Benjamini-Hochberg method, was
   used to assess the significance of the over-representation of
   biological annotations among gene lists. The transcriptomic data of
   Wnt3a-treated cells are available in Gene Expression Omnibus (GEO)
   (accession number: [96]GSE65238).

Quantitative real time reverse transcription-polymerase chain reaction
(qRT-PCR)

   Cells were seeded in six-well plates, serum starved overnight, and then
   treated with Wnt3a for 3, 6, 9 or 12 hours. For experiments in which
   cells were transfected with the construct pRK5-SK-β-cateninΔGSK or the
   empty vector pRK5-SK, cells were cultured for 12, 24 or 48 hours.
   Triplicates for each condition were included in the experiment and the
   experiment was repeated at least three times.

   Total RNA was extracted with the RNeasy Mini Kit (Qiagen). For each
   reaction, 50 to 100ng RNA was combined with components of KAPA SYBR
   FAST One-Step qRT-PCR Kit (KapaBiosystems, Clinisciences, Nanterre,
   France) and the Quantitect primers (Qiagen) to a final volume of 20μL,
   according to instructions of the manufacturers. The reaction mix was
   subjected to qRT-PCR performed with the 7900HT apparatus and SDS2.4
   software (Applied Biosystems) with the following settings: 5 min at
   42°C (RT step), 5 min incubation at 95°C followed by a three-step
   cycling program with 40 cycles of 15 sec at 95°C, 30 sec at 60°C and 30
   sec at 72°C (PCR step). A post-PCR dissociation analysis step was
   included according to instrument guidelines to distinguish specific
   from non-specific amplification products. All data were normalized to
   endogenous actin expression.

Tissue samples and microarray data

   The human samples used in this study have been previously described
   [[97]41,[98]42]. RNA microarray (Affymetrix U133 Plus 2.0) performed on
   41 TNBC, 30 HER2^+, 30 luminal B (LB), 29 luminal A (LA) and 11 healthy
   tissue breast samples, have also been previously described
   [[99]41,[100]42] (GEO accession number: [101]GSE65216).

Analysis of the enrichment of the up- and down-regulated Wnt target genes in
human breast tumor samples

   We analyzed whether the Wnt target genes we identified were enriched in
   human tumor samples. We compared the gene expression data obtained in
   TNBC cell lines stimulated with Wnt3a with that obtained in our cohort
   of 130 human breast cancer samples [[102]41,[103]42]. The two
   experiments were not done with the same arrays: the cell line (Gene st
   1.1, Affymetrix) and the tumor (U133 Plus 2.0, Affymetrix) experiments
   contained 19738 and 11543 genes, respectively. We restricted our study
   to the 11262 genes present on both arrays. Of note, AXIN2 was not found
   in our tumor dataset. We applied a FDR (False Discovery Rate) cut-off
   of 0.05 and a log-2 fold change threshold of 0.

   We used the Fisher exact test to assess the significance of the
   intersection between the genes that were up- or down-regulated upon the
   stimulation of cell lines with Wnt3a, and those that were more strongly
   or more poorly expressed in tumors (TNBC, HER2+, LB) than in LA
   samples. Several gene sets were considered. For the cell line
   experiment, we considered the up- or down-regulated genes, the
   different time points (6, 12 and 24 hours) and the two TNBC cell lines.
   For the breast tumors, we considered the genes that were more strongly
   or more poorly expressed in the three different subgroups of tumors
   (TNBC, HER2+ and LB) than in LA samples. Each subgroup was considered
   separately and all comparisons between the different breast cancer
   subtypes were performed.

   The comparisons between tumors and cell lines were performed with data
   obtained in HCC38 and MDA-MB-468 cell lines. However, there was no
   significant overlap between the MDA-MB-468 and the tumor datasets,
   possibly because of the very low number of genes found to be
   differentially expressed in Wnt3a-stimulated MDA-MB-468 cells.
   Therefore, we focused only on the data obtained with HCC38 cells.

   The gene lists were as follows:
     * MDA-MB-468 cells: 6h up-regulated (66 genes), 12h up-regulated (74
       genes), 24h up-regulated (20 genes), 6h down-regulated (34 genes),
       12h down-regulated (43 genes), 24h down-regulated (12 genes).
     * HCC38 cells: 6h up-regulated (419 genes), 12h up-regulated (907
       genes), 24h up-regulated (653 genes), 6h down-regulated (256
       genes), 12h down-regulated (707 genes), 24h down-regulated (530
       genes).
     * Tumor samples (relative to LA): TNBC up-regulated (3497 genes),
       HER2+ up-regulated (2110 genes), LB up-regulated (1587 genes), TNBC
       down-regulated (2971 genes), HER2+ down-regulated (2001 genes), LB
       down-regulated (1184 genes).
     * Other lists of genes up- or down-regulated in tumors: TNBC vs HER2+
       (up-regulated: 2242 genes; down-regulated: 1865 genes), TNBC vs LB
       (up-regulated: 2775 genes; down-regulated: 2725 genes), HER2+ vs LB
       (up-regulated: 581 genes; down-regulated: 581 genes).

   In addition, we selected the Wnt target genes that were up-regulated at
   both the earliest (6h) and the latest time (24h) point after Wnt3a
   stimulation, to identify potential up-regulated Wnt target genes that
   could reflect the chronic activation of the Wnt pathway in human
   cancer. The analysis was performed with the 133 Wnt target genes
   up-regulated in HCC38 cells, which displayed a higher number of
   up-regulated genes than in MDA-MB-468 cells (only 13 genes were
   up-regulated at both 6h and 24h). Of these 133 genes that were
   up-regulated in HCC38 cells at both 6h and 24h after Wnt3a stimulation,
   72 were more strongly expressed in TNBC than in LA tumors. We applied a
   fold change of > 1.3, which was defined for the cell line experiment,
   to select the most differently expressed Wnt target genes. Seventeen
   out of 72 genes were more strongly expressed in TNBC than in LA tumors,
   at this fold change. We generated a heatmap of the genes ordered by
   their P value in the t-test.

   We used the Fisher exact test to assess the significance of the overlap
   between two gene lists. For all analyses comparing cell lines and
   tumors, we used an adjusted (Benjamini Hochberg) P value cut off of 5%
   and no fold change threshold.

Statistical analyses for in vitro experiments

   Data are presented as the mean ± standard deviation (SD). Differences
   between groups were determined with Student's t-test. Results were
   considered significant at a P value lower than 0.05.

Results and Discussion

The canonical Wnt pathway is activated in triple-negative breast cancer cell
lines

   We sought to evaluate Wnt activity in TNBC cell lines; therefore, we
   measured the abundance of total β-catenin and its unphosphorylated,
   active form in TNBC cell lines under resting conditions ([104]Fig 1).
   The abundance of the total and active forms of β-catenin varied in the
   different cell lines ([105]Fig 1). Except for MDA-MB-231, the canonical
   Wnt pathway was active in all tested TNBC cell lines in unstimulated
   conditions, and was most active in HCC38 cells ([106]Fig 1).

Fig 1. Expression of β-catenin, LRP5 and LRP5 in TNBC cell lines.

   [107]Fig 1
   [108]Open in a new tab

   The abundance of LRP5, LRP6 and β-catenin (total and active forms) was
   evaluated in different TNBC cell lines by western blotting. Actin was
   used as a loading control: actin (a) for LRP5 and beta-catenin; actin
   (b) for LRP6 and active beta-catenin.

   We examined whether Wnt3a, a Wnt ligand commonly used to stimulate the
   canonical Wnt pathway, could stimulate Wnt/β-catenin signaling beyond
   basal levels observed in HCC38 and MDA-MB-468 cell lines, which
   displayed differences in their basal Wnt activity ([109]Fig 1).
   Tcf-mediated transcriptional activity was measured with a reporter
   plasmid that expresses the luciferase gene under the control of several
   Tcf binding sites (see [110]Material and Methods section). For both
   cell lines, Wnt/β-catenin activity was higher than in control cells as
   early as 3h following the incubation of cells with Wnt3a ([111]S1 Fig).
   This activation was highest 6h post-treatment and started to decrease
   at 12h ([112]S1 Fig), indicating that the transcriptional activity of
   β-catenin is optimal 6 hours following the initial stimulus. Although
   the transfection of HCC38 cells was not optimal (not shown), luciferase
   activity was higher in HCC38 cells than in MDA-MB-468 cells ([113]S1
   Fig), indicating that Wnt3a activates the Wnt pathway more strongly in
   HCC38 cells than in MDA-MB-468 cells. This may be because the Wnt3a
   receptors, LRP5 and LRP6, are more strongly expressed in HCC38 cells
   than in MDA-MB-468 cells ([114]Fig 1).

   Altogether, these results indicate that the canonical Wnt pathway is
   active in TNBC cell lines, and that Wnt3a further stimulates it.

Genes activated by Wnt3a in HCC38 and MDA-MB-468 triple-negative breast
cancer cell lines

   The expression of Wnt target genes appears to be cell/tissue context
   dependent [[115]31,[116]32]. We therefore performed expression
   profiling with Gene st 1.1 Affymetrix chips in HCC38 and MDA-MB-468
   cell lines stimulated with recombinant Wnt3a to identify a
   comprehensive list of Wnt target genes in breast cancer cells. A total
   of 19,738 transcripts were included on the array. The incubation of
   cells with Wnt3a for 6h, 12h, or 24h up-regulated (fold change > 1.3)
   the expression of 59, 64, and 22 genes, respectively in MDA-MB-468
   cells ([117]Fig 2A, [118]S1 Dataset) and of 241, 385 and 362 genes,
   respectively in HCC38 cells ([119]Fig 2B, [120]S1 Dataset).

Fig 2. Wnt3a-dependent gene activation in MDA-MB-468 and HCC38 cells.

   [121]Fig 2
   [122]Open in a new tab

   Venn diagrams indicate the number of genes that were up-regulated in
   MDA-MB-468 (A) or HCC38 cells (B) following Wnt3a stimulation for 6, 12
   or 24 hours. Genes found in common in both cell lines are listed in the
   table and known Wnt target genes are shown in bold (C).

   The difference in the number of up-regulated mRNAs between the two cell
   lines is consistent with the data obtained with the reporter assay
   showing that Wnt3a activates the Wnt pathway to a greater extent in
   HCC38 cells than in MDA-MB-468 cells ([123]S1 Fig). In HCC38 cells,
   more genes were up-regulated at 12h and 24h after stimulation with
   Wnt3a than at 6h ([124]Fig 2B, [125]S1 Dataset). These genes may be
   “direct” or “secondary” Wnt target genes. However, one can postulate
   that the gene list found at the earliest time point (6h) is likely more
   enriched in “direct” Wnt target genes than those at later time points
   (12h, 24h). Therefore, we next focused on the genes up-regulated at 6h.
   Half of the target genes (30 out of 59) identified in MDA-MB-468 cells
   incubated for 6h with Wnt3a were also up-regulated in HCC38 cells after
   the same duration of treatment ([126]Fig 2C, [127]S2 Dataset). Among
   the 30 common genes, 23 were previously reported as Wnt target genes
   ([128]Fig 2C, [129]Table 1).

Table 1. Literature search for the 36 Wnt target genes (30 “up” and “6 down”)
identified in both TNBC cell lines stimulated with Wnt3a for 6h.

   Gene Symbol (official symbol) ID Wnt target gene Role in Wnt/β-catenin
   signaling pathway References