Dasatinib inhibition of cSRC prevents the migration and metastasis of canine mammary cancer cells with enhanced Wnt and HER signalling

Human epidermal growth factor 2 (HER2) overexpression leads to aggressive mammary tumour growth. Although the prognosis of HER2 + tumours in humans is greatly improved using biologicals, therapy resistance, which may be caused by increased phosphatidyl-3-kinase (PI3K), rous sarcoma proto-oncogene (cSRC) or wingless-type MMTV integration site family (Wnt) activity, is a major concern. A recent analysis of 12 canine mammary cell lines showed an association between HER2/3 overexpression and phosphatase and tensin homologue (PTEN) deletion with elevated Wnt-signalling. Wnt-activity appeared to be insensitive to phosphatidyl-3-kinase (PI3K) inhibitors but sensitive to Src-I1. We hypothesized that Wnt activation, was caused by HER2/3-activated cSRC activation. The role of HER2/3 on Wnt signalling was investigated by silencing HER2/3 expression using specific small interfering RNA (siRNAs). Next, the effect of an epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor on Wnt activity and migration was investigated and compared to other tyrosine kinase inhibitors (TKIs) of related signalling pathways. Finally, two TKIs, a cSRC and a PI3K inhibitor, were investigated in a zebrafish xenograft model. Silencing of HER1-3 did not inhibit the intrinsic high Wnt activity, unravel the mechanism of cSRC activation and cSRC inhibition to restore sensitivity to HER-inhibitors in HER2/3-positive breast cancer. are

Human epidermal growth factor 2 (HER2) overexpression leads to aggressive mammary tumour growth. Although the prognosis of HER2 + tumours in humans is greatly improved using biologicals, therapy resistance, which may be caused by increased phosphatidyl-3-kinase (PI3K), rous sarcoma proto-oncogene (cSRC) or wingless-type MMTV integration site family (Wnt) activity, is a major concern. A recent analysis of 12 canine mammary cell lines showed an association between HER2/3 overexpression and phosphatase and tensin homologue (PTEN) deletion with elevated Wnt-signalling. Wnt-activity appeared to be insensitive to phosphatidyl-3-kinase (PI3K) inhibitors but sensitive to Src-I1. We hypothesized that Wnt activation, was caused by HER2/3-activated cSRC activation. The role of HER2/3 on Wnt signalling was investigated by silencing HER2/3 expression using specific small interfering RNA (siRNAs). Next, the effect of an epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor on Wnt activity and migration was investigated and compared to other tyrosine kinase inhibitors (TKIs) of related signalling pathways. Finally, two TKIs, a cSRC and a PI3K inhibitor, were investigated in a zebrafish xenograft model.

K E Y W O R D S
cSRC, dasatinib, HER2, mammary cancer, migration, Wnt

| INTRODUCTION
The life-time risk of mammary cancer is high not only in humans but also in dogs. 1 Most of these carcinomas in both species are oestrogen receptor (ER) and progesterone receptor (PR)-positive and respond to endocrine therapy in humans. [2][3][4][5] However, not all canine mammary tumours are well characterized, and immunohistochemical results may vary. 4,6 Approximately, 15% to 20% of human breast cancers are characterized by a combination of human epidermal growth factor 2 (HER2) overexpression and a lack of steroid hormone receptors, leading to more aggressive tumour growth and a worse prognosis. 7,8 Despite improved treatment options using therapeutic monoclonal antibodies against HER2, the acquisition of drug resistance remains a major problem and could lead to tumour recurrence. 9 For triple negative carcinomas, no targeted therapy is available. 10 HER2 overexpression in human breast cancer indicates a risk of recurrence independent of whether the axillary node is positive or negative for micrometastases. 11 Overexpression of HER2 is often observed (50%) in ductal carcinomas. 12 A clear role for HER2 overexpression in the progression from ductal carcinoma in situ (DCIS) to invasive carcinoma has not been shown, 13 although dimerization of epidermal growth factor receptor (EGFR) and HER2 induces motility. 14 In human breast cancer, approximately, 50% of clinical samples have an elevated level of nuclear or cytoplasmic ß-catenin, implicating active wingless-type MMTV integration site family (Wnt) signalling. 15 In contrast to colon carcinomas, where mutations in adenomatous polyposis coli (APC) or ß-catenin are the cause of activated Wnt signalling, these mutations are rarely found in breast cancer. 16 Nevertheless, in human breast cancer, Wnt/β-catenin signalling has a clear contribution to tumour progression, metastasis and a poor clinical outcome in all molecular subtypes of invasive breast cancer. 17 In addition, HER2-mediated breast cancer progression requires ß-catenin signalling 18 ; therefore, inhibition of Wnt signalling may be relevant in HER2-positive tumours. HER2 also acts among other pathways through the activation of rous sarcoma proto-oncogene (cSRC) kinases. 19 Because we have shown that Wnt activity is sensitive to cSrc inhibition, 20 we investigated the hypothesis that the enhanced expression of HER2/3 causes high Wnt activity through cSrc activation in some canine mammary tumour (CMT) cell lines.
Ductal carcinomas in dogs are comparable to the most common forms of human breast cancer. 21 One-third of malignant mammary carcinomas in dogs may form life-threatening distant metastases. 22 Tumour cells with cancer stem cell properties, such as a phenotypical epithelial-mesenchymal transition (EMT), can migrate and form metastases. In this regrowth and metastasis, elevated activity of the Wnt pathway plays an important role. 23 In a panel of 12 different canine mammary cell lines, we identified three cell lines in which the basal Wnt activity was highly elevated in a ligand-independent manner, as indicated by high T-cell-specific transcription factor (TCF) reporter activity. 24 This Wnt activity was not caused by enhanced expression of Wnt ligands, whereas overexpression of lymphoid enhancer-binding factor 1 (LEF1) messenger RNA (mRNA) could only partially explain this elevated signalling. 24 A rounded cell morphology characterized the cell lines, suggesting EMT.
In general, EMT, which is characterized by alterations in cell shape and loss of attachment to other cells and the extracellular matrix (ECM), is caused by loss of E-cadherin expression. 23 However, the cell lines with high Wnt activity and rounded cell morphology had relatively high E-cadherin expression, 24 excluding the likelihood that loss of E-cadherin caused high canonical Wnt activity 25 and elevated β-catenin levels. [26][27][28] Further analysis by quantitative polymerase chain reaction (qPCR) of selected genes showed that the cell lines with high Wnt signalling overexpressed HER2,3 mRNA with LEF1, heat shock protein 90 (HSP90) and ras-like protein (RAC1), among others, and silenced phosphatase and tensin homologue (PTEN) mRNA expression. 20 The loss of PTEN expression in combination with an increased expression level of HER3, which promotes HER2-driven mammary tumour proliferation, 29 could indicate a highly activated PI3K/mammalian target of rapamycin (mTOR) pathway. However, inhibition of this pathway with the mTOR inhibitor everolimus or the dual PI3K/mTOR inhibitor BEZ325 further upregulated the already high basal activated Wnt activity. 24 Wnt activity could only be inhibited by Src-I1, a tyrosine kinase inhibitor that targets cSRC, among others. 20 Recently, SRC has been shown to promote resistance to trastuzumab in HER2-positive breast cancer. Upregulation of cSRC stabilizes HER2 and vice versa. 30 However, to the best of our knowledge, no literature is available on HER2/3 activation of Wnt signalling through cSRC activation.
HER signalling occurs predominantly by activating the PI3K/AKT/mTOR pathway, 31 but HER dimers may also transduce signals through cSRC/focal adhesion kinase (FAK) complexes. 32 These complexes influence the Wnt pathway by interacting with β-catenin.
Therefore, it is likely that a connection exists between Wnt, cSRC and HER signalling. Considering that cSRC could be activated by EGFR and HER2/3, this likely makes cSRC a key player in the activation of highly activated basal Wnt cells. 33 The central role of cSRC signalling was further investigated in vitro by cell migration and invasion assays and in an in vivo zebrafish embryo xenograft model. This model has been validated extensively, showing that short-term experiments in zebrafish embryos correlate well with long-term rodent xenograft models of breast cancer cell lines with variable grades of malignancy. 34 In general, dye-labelled cells are injected into the yolk sac, followed by invasion into the blood stream and the formation of distant metastases.
However, cells may also be inserted directly into the blood stream through injection into the duct of Cuvier to investigate metastatic properties of breast cancer cell lines. 35 This model has also been validated in our laboratory for human insulinoma cell line metastasis and compared to xenografts in mice. 36 The same model was used for investigating the effects of mTOR and cSRC inhibition on the metastatic properties of cells with high Wnt signalling in the current study.
For cSRC inhibition, dasatinib was used because this selective cSRC inhibitor is FDA-approved.
The aim of this study was to investigate the role of HER signalling in enhanced canonical Wnt signalling and to investigate tyrosine kinase inhibitors (TKIs) related to HER signalling as inhibitors of Wnt activity and associated cell migration and metastasis.

| Canine mammary cell lines and culture
In our studies, a panel of 12 different canine mammary cell lines were used as described previously. [37][38][39] For detailed studies, the well-

| Protein isolation and Western blot
CMT-U27 cells were seeded at a density of 600 000 in 6-well

| Xenograft
To investigate the effect of inhibition of cSRC or PI3K signalling on the metastasis of canine mammary cells, a xenograft model in zebrafish was used that has been validated previously 35,45 and has been used in our laboratory to investigate the metastasis of canine insulinoma cells. 36  Abbreviations: CHK1/2, cell cycle checkpoint kinase 1/2; CMT, canine mammary tumour cell line; IGF-1, Rinsulin-like growth factor 1 (receptor); JAK2, Janus kinase 2; LCK, lymphocyte specific protein-tyrosine kinase; PP2, inhibitor for cSRC-family kinases. IC-50 values for proliferation inhibition of CMT-U27 and CIPm cells analysed by MTT assays using various TKIs. For dose response curves, see Figure S1.   Figure 1(B)). The lack of the effect was not caused by stable protein expression, because Western blot analysis after 72 hours also showed decreased expression levels in the protein bands ( Figure 1C). Additional analysis of various Wnt target genes even showed limited increases in the mRNA expression levels of axis inhibitor 2 (AXIN2), LEF1, low-density-lipoprotein related protein (LGR5) and SRC after 72 hours ( Figure S2 siRNA qPCR).  (Table 2). No differences in cell viability were observed between CMT-U27 and CIPm cells in the 0.2% DMSO control compared with medium. In general, CMT-U27 cells were slightly more sensitive to inhibition than CIPm cells. The inhibitors PP2 and Src-I1 also showed strong inhibition of CMT-U27 cell migration in a scratch assay together with inhibitors of the PI3K pathway (Figure 3 A, C). None of the investigated inhibitors affected the migration of the control CIPm cells (Figure 3 B, D). The  When CMT RED cells were injected into the yolk sac in the presence of everolimus, the number of metastases increased ( Figure 6).

| The effect on metastasis in vivo
More clumps of tumour cells were observed when CMT RED cells were injected in the duct of Cuvier, extravasation of the CMT RED cells was observed and everolimus slightly increased the amount of metastases.

| DISCUSSION
In the present study, we investigated our hypothesis that overexpression of HER2/3 is related to the ligand-independent high basal Wnt activity of canine mammary cancer cells through the activation of cSRC (Figure 7).  65 or interacting with β-catenin. 66 Increased Wnt/β-catenin activity leads to elevated SLUG (SNAIL) expression that promotes EMT. 67 As expected, inhibition of Wnt activity resulted in the inhibition of the invasive and migratory ability of CMT-U27 cells. However, PI3K/mTOR inhibition also resulted in decreased migratory rates, indicating that both pathways were related to inhibition of the migratory ability of these cells. To investigate whether this also had consequences for the metastatic properties, a zebrafish xenograft experiment was performed.
For these experiments, we used dasatinib as a cSRC inhibitor and everolimus as a PI3K/mTOR inhibitor because these compounds have also been used in human clinical trials. 30,68 Cell viability and incidence of metastases were strongly reduced after pre-treatment of CMT-U27 cells with dasatinib followed by injection in the yolk sac or in the duct of Cuvier of zebrafish larvae. When cells were pre-treated with 10 μM everolimus and then injected into the yolk or duct of Cuvier larvae, survival was good, but in contrast with dasatinib, it resulted in an increased incidence of metastasis. The fact that no decrease in metastasis but rather a limited increase was observed was consistent with our findings that everolimus further upregulated the already high basal Wnt activity. 20 It is concluded that the highly activated Wnt signalling in the CMT-U27 canine mammary cell line, which is similar to human HER2-overexpressing luminal cell lines, is not related to HER2/3 signalling. Additionally, no relation was observed between HER proteins and cSRC activation. Inhibition of PI3K/mTOR activity due to HER signalling and loss of PTEN expression was associated with enhanced metastasis in a zebrafish xenograft model.
The cSRC inhibitor dasatinib decreased Wnt signalling, migration and metastasis. The fact that the cSRC inhibitor dasatinib was effective in decreasing metastasis of our HER2 + and PTEN − CMT-U27 cells makes it worthwhile to further evaluate this drug for the treatment of patients with this type of breast cancer because PTEN loss has been associated with a worse outcome in HER2-amplified human breast cancer patients. 69 Because human breast cancer patients have already been treated with dasatinib in a phase II study, 70,71 it may be of interest to relate the clinical outcome to data on HER and Wnt signalling.