ISM1 in mammary gland morphogenesis and malignant transformation

Abstract

Isthmin 1 (ISM1) is a secreted protein that was first discovered in the isthmus of the brain of Xenopus. It is conserved in many animals, including zebrafish, mice and humans, which suggests that it plays an important role in development. ISM1 is enriched in the lung, brain, bone marrow, ovary, mammary gland (MG) and other organs for the ontogeny and homeostasis. It may contribute to extracellular matrix organization, cell interaction and angiogenesis. However, the mechanisms and functions of ISM1 remain largely unexplored.

Ism1 transgenic (ISM1 Tg) mice were used to investigate the functions of ISM1 during MG morphogenesis and tumorigenesis in this project. In the MGs of female ISM1 Tg mice, ISM1 was found to be expressed by basal epithelial cells. The epithelial ductal trees of ISM1 Tg mice showed increased luminal cells and defective branch morphogenesis. The expressions of Girdin, Itgb4, Olfml2A, Lypd6, Ank3 in ISM1 Tg MGs were significantly altered.

ISM1 Tg mice were crossed with the PyMT mice, a luminal breast cancer mouse model. PyMT;ISM1 mice exhibited increased hyperplastic lesions as early as postnatal day 12. PyMT;ISM1 mice had increased total tumor number and weight at later stages, accompanied by increased pulmonary metastasis burdens. PyMT;ISM1 conditional knockout mice, which expressed fewer ISM1, had decreased tumor burdens. The expressions of Esr1, Pgr, Her2, Gata3, Csn1s1, Pymt, Girdin, Itgb4 and Tbx3 in PyMT;ISM1 tumors were significantly altered. The pro-tumorigenic role of ISM1 in breast cancer is associated with its ability to promote the generation and maintenance of tumor-initiating cells (TICs). PyMT;ISM1 tumors were found to have more TICs than PyMT tumors through fluorescence-activated cell sorting (FACS) analysis. Sorted PyMT;ISM1 TICs could be maintained as luminal/tumor cells, while sorted PyMT TICs became basal cells after a long-term culture. Sorted PyMT;ISM1 TICs generated tumor spheroids of increased number and size compared with PyMT TICs. The number and size of tumor spheroids could be increased by the medium containing a higher level of ISM1. Inhibition of the fibroblast growth factor (FGF) pathway affected spheroid formation considerably, and inhibition of the epidermal growth factor (EGF) and Src pathways led to spheroid death. In addition, enhanced ISM1 expressions were found in clinical samples of breast cancers with poor prognosis.

Collectively, these results indicate that ISM1 is important for MG morphogenesis and plays a pro-tumorigenic role in breast cancer.