High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression

In a continuation of our previous findings of a variation in basal Wnt/β-Catenin activity in canine mammary tumor cell lines [5] we searched for the mRNA expression of members of the EGF receptor family of proteins and the associated signal-transduction pathways. In the present paper, the three cell lines with high ligand-independent basal Wnt activity (CMT1, CMT-U27 and CMT9) were, through cluster analysis of the gene expression data, shown as a clear grouping, but no clear association for absent or low Wnt-ligand induced basal Wnt/β-Catenin activity in the other cell lines (Fig. 1). High basal Wnt activity is clearly grouped together with an up-regulated expression of LEF1, ID1, ID2, EGFR (only in CMT1), HER2/3, HSP90 and RAC1 mRNA and a down regulation of SLUG, NCOA3, MUC1, CYCLIN D1, BCL2, cMET and PTEN mRNA. In these highly activated Wnt cell lines, PTEN expression is completely lost. PTEN deficiency occurs in 5 to 14 % of primary human breast cancers and PTEN is also lost in human T47D and MCF7 cell lines [13]. The cluster analysis confirmed our previous finding that in the cell lines with active Wnt signaling, LEF1 is overexpressed and acts as a contributing factor of the high canonical Wnt pathway [5]. New is our finding of a strong association of the expression of the EGF pathway both at the receptor level (HER expression) as in the silencing of PTEN with high ligand independent Wnt signaling. Previously, it has been shown that binding of Wnt proteins to the Frizzled receptor may activate the EGFR. EGFR can be further activated by homo or hetero-dimerization with HER2, HSP90 as its chaperone, or HER3. These activated complexes functions as an oncogenic unit to drive the proliferation of breast cancer cells [14, 24]. In human and mouse mammary glands, the activated Wnt/β-Catenin is related to enhanced HER2 expression [25], something also found in 30 % of canine malignant mammary tumors [26]. Combining the up regulation of both HER2 and HER3 in our canine mammary cell lines and the fact that the HER2/HER3 complex is the strongest activator for downstream signaling pathways makes this an interesting area for further research [15, 27, 28].

We concluded that the association between high Wnt signaling (high TOP/FOP ratios), high LEF1 levels, loss of PTEN and high HER2/3 mRNA levels all point to the activation of the PI3K/AKT/mTor pathway [29‐32]. We therefore investigated the effects of PI3K/mTor inhibitors on cell viability, Wnt signaling, expression levels of the differently expressed genes from the clustering analysis and the cellular content of β-Catenin, and HER2/3 protein as an experimental model for HER2+/PTEN- breast cancer.

Cell lines with high Wnt activity and a cell line lacking basal Wnt activity (CIPm) were incubated with Everolimus as a specific mTor inhibitor and BEZ235 as a dual inhibitor of PI3K and mTOR. Both BEZ235 and Everolimus equally inhibited the viability of the highly activated Wnt cell lines and have only a slightly inhibitory effect on the CIPm cells (Fig. 2).

This inhibitory effect on viability was clearly dose-dependent and present in both the high basal Wnt cell line CMT-U27 and the CIPm cell line (Fig. 3 and Table 2). Comparable concentrations of BEZ235 were also used in human studies with different cell lines [33, 34]. In human MCF7 cells, Everolimus (Rad001) was tested in a range from 0-1000 nM and only the 1000 nM concentration showed a 50 % reduction in cell number [35]. Incubation of these cells with BEZ235 and Everolimus resulted in unexpected further significant enhancement of Wnt activity of CMT1, CMT-U27 and CMT9, with a more than 2-fold increase in the TOP/FOP ratios (Fig. 4).

BEZ235-induced inhibitions of cell proliferation have been reported previously in MCF-7/shPTEN cells and in 21 other tested human cancer cell lines [13]. The only difference is that in these cell lines, BEZ235 had a stronger effect than Everolimus on cell proliferation [36]. In human breast cancer, loss of PTEN is associated with activation of the PI3K/AKT Pathway [12], which may result in increased nuclear stabilization of β-Catenin in mammary stem cells using human breast tissue xenografts [37]. This is in contrast to the absence of any inhibitory effect in the TOPFlash reporter assays of several breast and prostate cancer cell lines treated with another inhibitor of the PI3K pathway (Wortmannin) and suggests that the PI3K pathway has no relation with Wnt-mediated transcriptional activity [30]. In our cell lines as well, neither Wortmannin nor the introduction of a PTEN-expressing plasmid inhibited Wnt signaling, indicating that PI3K inhibition alone does not influence the active Wnt signaling and that mTor inhibition is needed (data not shown). CIPm, which lacks basal Wnt activity, did not respond to both inhibitors with increased reporter activity.

We conclude that inhibition of PI3K and/or mTor in the highly activated Wnt cell lines inhibits cell viability, but further stimulates Wnt activity. Because the mechanism behind this remained unclear, we tested the effect of these inhibitors on the expression of genes from the cluster analysis.

Inhibition of the PI3K/mTor pathway with BEZ235 (a dual PI3K mTor inhibitor) or with Everolimus (a single mTor inhibitor) in cell lines with high ligand-independent canonical Wnt activity resulted in comparable upregulation of a number of candidate genes (Fig. 5). A moderate twofold increase was detected in the expression of β-Catenin, Axin2, HER2 and cMet mRNA. Only the expression of EGFR and MUC1 mRNA was higher, especially in the CMT-U27 and CMT9 cell lines. β-Catenin and Axin2 are markers for an activated Wnt/β-Catenin signaling in mammary tissues [38]. Protein levels of β-Catenin were not significantly different after treatment with both inhibitors (Fig. 6). The stable total protein expression of β-Catenin may reflect predominantly binding to E-cadherin, which is comparable high in both cell lines [5, 7] and may not be related to the cytoplasmic or nuclear ß-Catenin content. No significant effect of both inhibitors was seen on HER2/3 protein content although HER3 expression was only found in the cell line with high basal Wnt activity (Fig. 6). Apart from the expression of HER2 at the cell surface it can also form a complex with AP-1/Stat3/PR in the nucleus. This nuclear HER2 can act as a transcription factor and modulate genes involved in breast cancer proliferation [9]. In contrast with the absence of HER3 in the CIPm cell line, the CMT-U27 cells have a HER3 band visible at 180kD, which correspond to a glycosylated form. This glycosylated HER3 protein is also found in human MDA-MB-445 and MDA-MB-453 cells [39]. In MDA-MB-453 and other mammary tumor cell lines, there is strong evidence for the role of HER3 in cancer. HER3 plays a role in EMT and activated HER3 can also increase the expression of the cMET proto-oncogene independently from EGFR and HER2 [16], which may be in line with the 2-3 fold increased expression shown in Fig. 5. EGFR and HER2 expressions are upregulated after treatment with BEZ235 in a human breast cancer cell line with a lack of PTEN (MDA-MB-468). In contrast with this BCL2 is not upregulated in all tested canine cell lines and HER3 is only, although significantly, 1.5 times upregulated in some cases [40]. The most prominent change was the 7-fold increase in MUC1 expression. At the same time, in all tested cell lines, basal MUC1 protein concentrations showed no difference, even in the presence of considerable amounts of the cytoplasmic domain MUC1-CD (data not shown). Still, overexpression of MUC1 plays a role in tumor formation and progression in > 90 % of the breast cancers [41]. It has been reported that the Wnt activity is directly affected through the binding of the Wnt effector β-Catenin on MUC1-CD [42] through binding of several proteins, including cMET, cSRC and EGFR/HER. Consistent with this is the enhanced EGFR and oncoprotein MUC1 mRNA concentrations in our experiment together with the upregulation of cMET. Enhanced cMET expression has been linked to poor prognosis in human breast cancer, since it influences extravasation/angiogenesis, enhances resistance to endocrine therapy and trastuzumab treatment and may influence the regulation of β-Catenin/TCF transcription [29, 43]. The basal expression of cMET in the cell lines with elevated Wnt activity is low, but stimulated when the PI3K/mTOR pathway is blocked; blocking this pathway also affects the EGFR and HER2 expression. Overexpression of MUC1 is associated with the induction of anchorage-independent growth and tumorigenicity in human carcinomas. MUC1 overexpression is also directly associated with stabilization of β-Catenin either via MUC1 binding to E-Cadherin, or by inhibition of the adenomatous polyposis coli (APC)/GSK3β/ MUC1-CD complex, whereas phosphorylation of MUC1-CD by EGFR and cSRC increases β-Catenin [42, 44‐46].

Leaving aside the question of how EGFR/HER2/HER3 signaling is involved in high basal Wnt activity, it is clear that the upregulation of the MUC1 and EGFR/HER2 expression have a relationship with the increased Wnt activity that occurs after inhibition of the PI3K/mTor pathway. The presence of glycosylated HER3 protein in the highly activated Wnt cell line is new, and because our results suggest that activation of cSRC could play a role [15, 46], we next studied the influence of cSRC inhibitors on the Wnt activity and HER3 levels.

Activation of EGFR initiates the activation of PI3K/AKT/mTor and MAPK as well as various other signaling pathways, including the PLC-Υ1, JAK/STAT, and cSRC pathway [15, 47]. Because the EGFR can form a complex with cSRC, FAK (focal adhesion kinase), integrins and β-Catenin resulting in growth, viability, survival and migration [46], we tested the effect of the cSRC inhibitor 1 (Src-I1) and a FAK inhibitor [48, 49]. Treatment with the FAK inhibitor resulted in a small non-significant decreases in the TOP/FOP ratio with no effect on cell proliferation (data not shown), however Src-I1 had a clear effect on cell viability and was found to be an inhibitor of the Wnt activity via inhibition of cSRC signaling [50, 51]. The same inhibition on the Wnt pathway was found in the human cell lines NIH 3 T3 with the cSRC inhibitor PP1 [52]. The used concentration of Src-I1 reduced cell proliferation for some 20 % and acted as a weaker inhibitor in comparison with BEZ235 and Everolimus (Fig. 2). Unlike BEZ235 and Everolimus, the cSRC inhibitor Src-I1 reduced the Wnt/β-Catenin signaling by almost half in canine mammary tumor cell lines with high canonical Wnt activity, but not in the CIPm cells that lack basal Wnt activity (Fig. 4). Inhibiting mouse cells with cSRC inhibitors PP2 and Genistein gave comparable results on LEF/TCF sensitive transcription and on the level of activated phosphorylated cSRC. This indicates that cSRC is a positive regulator of the Wnt/β-Catenin signaling [50]. To investigate the Wnt-regulation by both the PI3K pathway and the cSRC pathway, we tested the same target genes used in the clustering analyses (Fig. 1).

The cSRC inhibitor Src-l1 decreased the cell viability (Fig. 2), decreased Wnt signaling (Fig. 4) without affecting MUC1, EGFR and HER2/HER2 expression levels (Figs. 5 and 6). The inhibition of Wnt activity as measured by a decrease in TOP/FOP ratios was also clearly dose-dependent (data not shown) in the activated Wnt cells. Marginal increases were found in SLUG and HER3 mRNA concentrations (Fig. 5c) after cSRC inhibition. Remarkably inhibition with Src-I1 showed a significant decrease in HER3 protein concentrations whereas the HER3 mRNA expression was slightly elevated indicating that cSRC inhibition is associated with a higher turnover or a decreased glycosylation of HER3 at the protein level. At the moment information on the regulation of HER3 expression is scarce. Several microRNAs regulate the expression levels and also epigenetic effects have been shown to influence HER3 expression in breast cancer cells [16].