c-Src protein expression is increased in human breast cancer. An immunohistochemical and biochemical analysis.

In human breast cancer, c-Src activity is elevated compared to normal breast tissue. It is not yet known whether this increase in c-Src activity is accompanied by an increase in c-Src protein expression. In this study, c-Src activity and protein expression were determined in a series of human breast cancers and in normal breast tissue, using immune complex kinase assays and immunoblotting. As the heterogeneity of breast cancer is not taken into account in these biochemical experiments, immunohistochemistry was also used to distinguish between normal and malignant cells. In human breast cancers, the c-Src activity is increased 4- to 30-fold, compared with normal breast tissue. This enhanced activity is accompanied by an increase in c-Src protein expression, as shown by both immunoblotting and immunohistochemistry. Immunohistochemistry indicates that the majority of c-Src appears to be concentrated around the nucleus in malignant cells, whereas in normal cells, it is distributed more evenly in the cytoplasm. These data confirm that c-Src activity is increased in human breast cancer. In addition, this study provides strong immunohistochemical evidence that the c-Src protein is also overexpressed, enabling a distinction to be made between normal and malignant cells.

Protein tyrosine phosphatase activity as a diagnostic parameter in breast cancer.

Cellular phosphotyrosine levels are regulated by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). It is supposed that this balance is disturbed in tumour cells, making the increased or altered activity of PTKs and PTPs likely hallmarks of tumour tissues. Indeed it could be shown that the PTK activity was increased in breast cancer in correlation with prognosis (Hennipman et al., Cancer Res. 49, 516-522, 1989). In the present report we measured the PTP activities in breast cancer and normal breast tissues. An increase of approximately three- to four-fold was measured in the cytosolic tumour fractions compared to normal, whereas the solubilized membrane fraction PTP activity showed an increase in tumours of approximately 1.5-fold. Remarkably, the membrane PTP activity correlated with the presence of tumour positive axillary lymph nodes (p = 0.004), whereas the cytosolic PTP activity correlated with the mitotic index, a higher PTP activity occurring when the mitotic index was higher than 10 (p = 0.0004). These results indicate the membrane PTP activity may be considered as an index of metastatic potential, whereas cytosolic PTP activity may be a measure of the growth capacity of the tumour. The increase of PTP activity in breast cancers was confirmed by enzyme-histochemical studies. In frozen sections of tumours a strong to moderate activity was found in both tumour cells and interstitial cells. In the interstitium membrane activity was most pronounced, whereas in the tumour cells diffuse staining of the cytoplasm together with a clear membrane staining was demonstrated. Immunoblotting with anti-phosphotyrosine antibodies also reveals differences between the tumours and normal tissues, confirming the disturbance of the balance between protein tyrosyl phosphorylation and dephosphorylation in the tumour cells.

Functional interaction between the epidermal growth factor receptor and c-Src kinase activity.

To study the relationship between the tyrosine kinase c-Src and the epidermal growth factor receptor (EGF-R), we used the breast cancer cell line ZR75-1, which was transfected with the EGF-R. The EGF-R transfected cell line expressed 60 times more EGF-R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF-R over-expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c-Src. However, the cell line over-expressing the EGF-R showed a twofold enhancement of c-Src kinase activity after EGF stimulation. The activation of c-Src kinase by EGF was confirmed in other EGF-R expressing cell types.

Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product.

Tyrosine phosphorylation is an important regulatory mechanism in response to the action of growth factors and oncogenes. Since many oncogenes code for tyrosine kinases, increased or altered oncogene expression may be reflected in increased tyrosine kinase activity. In a recent study (Hennipman et al., Cancer Res., 49: 516-521, 1989), we found that the tyrosine kinase activity of the cytosolic and membrane fractions of malignant human breast tissue was significantly higher compared to the benign or the normal breast tissue. Moreover, the increase in the cytosolic fractions was found to be of prognostic value. In the present study we determined the protein tyrosine kinase (PTK) activity of another 72 breast cancer specimens, and it could be shown again that the PTK activity in all 72 of these tumors was elevated compared to normal controls. We characterized these cytosolic PTKs by anion exchange chromatography using fast protein liquid chromatography, and it could be shown that at least two different forms of PTK exist. Using antibodies against a number of known oncogene products, we could determine that at least 70% of the PTK activity in the cytosol originated from the presence of the c-src oncogene product. Both of the PTK activity peaks seen in the fast protein liquid chromatography patterns could be precipitated with the anti-Src antibody. Furthermore, using the MCF-7 breast cancer cell line, it could be shown that the antibody against c-src also precipitated a part of the cytosolic PTK activity. In normal human peripheral lymphocytes, no precipitation of the cytosolic and membrane PTK activity could be achieved using the anti-Src antibody. Inasmuch as the cytosolic PTK activity parallels the malignancy in breast tumors (Hennipman et al., Cancer Res., 49: 516-521, 1989), and the majority of this activity is precipitated by anti-Src antibodies, the c-src protooncogene may play a key role in the manifestation of breast cancer.