WT1 role in mammary gland and breast cancer biology

Abstract

The Wilms' Tumour Suppressor gene 1, WT1, encodes for a complex protein which is essential in mammals throughout life. Its roles vary with the developmental stages: in the embryo, it regulates the epithelial-mesenchymal balance required for a correct organogenesis and acts as a tumour suppressor; in the adult, it is involved in the maintenance of tissue homeostasis and has been controversially considered as an oncogene.

Breast cancer is one of the adult tumours in which WT1 oncogenic function was first hypothesised. This malignancy is the most common in women, with more than one million cases being diagnosed worldwide every year, and represents the leading cause of cancer related deaths.

Because of its major health burden, this disease has been extensively studied and special attention has also been paid to normal mammary gland biology: several works have shown that breast cancer can be divided into many molecular subtypes, which may reflect the cell of origin of the tumour; moreover, many genes involved in the normal development of the mammary gland have been proven to also play a role in breast tumorigenesis.

WT1 expression has been previously reported in both healthy mammary glands and breast cancer samples, however, its function in this context is not well understood and the evidence gathered so far is extremely contradictory. This thesis aimed to investigate the exact role played by WT1 in both mammary gland and breast cancer biology, using a combination of in vivo and in vitro techniques.

Following flow cytometry isolation, Wt1 mRNA expression was detected in the myoepithelial and stem cell subpopulations of the healthy gland. To investigate the effects of WT1 loss, Wt1 conditional mice were crossed with two different mammary specific Cre lines: the knockout animals developed, bred and lactated normally,

however, they showed a significant increase of ductular branches during pregnancy, suggesting that WT1 may be involved in the regulation of branching morphogenesis. In order to study WT1 role in mammary tumours, the gene was knocked out in a breast cancer mouse model and knocked down in several breast cancer cell lines, using both constitutive and inducible lentivirus-based systems. WT1 loss did not seem to affect cell proliferation, but resulted in a significant increase in cell migration in vitro and in the upregulation of mesenchymal markers.

Furthermore, bioinformatics analysis showed that the WT1-positive tumours mainly belong to the luminal/ER-positive subtypes and express lower levels of mesenchymal markers than the WT1-negative tumours.

As a whole, the findings of this thesis characterise WT1 expression in the healthy mammary gland and provide the first evidence of its possible function in this organ; moreover, this work seems to rule out an oncogenic role for WT1 in breast cancer, while suggesting that it could be an upstream regulator of cell migration. Additional experiments are required to confirm this result in vivo and verify whether it could lead to any clinical application.