Abstract
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, whereas the HER kinase inhibitor afatinib showed enhanced Wnt activity. The strongest inhibition of Wnt activity and cell viability and migration was shown by cSRC inhibitors, which also showed strong inhibition of cell viability and metastasis in a zebrafish xenograft model. HER2/3 overexpression or HER2/3-induced cSRC activation is not the cause of enhanced Wnt activity. However, inhibition of cSRC resulted in a strong inhibition of Wnt activity and cell migration and metastasis. Further studies are needed to unravel the mechanism of cSRC activation and cSRC inhibition to restore sensitivity to HER-inhibitors in HER2/3-positive breast cancer.
Highlights
The life-time risk of mammary cancer is high in humans and in dogs.[1]
We investigated the underlying cause of the high basal wingless-type MMTV integration site family (Wnt) activity in the canine mammary tumour (CMT)-U27 metastatic canine mammary cell line, which was selected from a panel of canine mammary cell lines where 3 out of 12 cell lines were characterized by high ligandindependent Wnt signalling.[24]
Additional studies showed that these cell lines had human epidermal growth factor 2 (HER2)/3 overexpression, absent phosphatase and tensin homologue (PTEN) messenger RNA (mRNA), and sensitivity to Src-I1, indicating a possible relationship between HER signalling and Wnt activity.[20]
Summary
The life-time risk of mammary cancer is high in humans and in dogs.[1]. 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 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. 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
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