UC Berkeley

The transforming growth factor-ß (TGF-ß) superfamily of cytokines regulates many cellular processes such as cell growth, cell survival, differentiation, and extracellular matrix deposition. TGF-ß is an important regulator of tissue homeostasis and is implicated in the development of human cancers and other diseases. During cancer development, TGF-ß acts as a tumor suppressor in the early stages of tumorigenesis and as a promoter of tumor invasiveness and metastasis in the later stages of cancer. TGF-ß exerts many of its diverse effects via the recruitment of the Smad proteins to activate transcription of TGF-ß target genes. Activities of the Smad proteins in the nucleus and cytoplasm have been shown to be regulated through interaction with positive and negative cellular regulators. Among the Smad negative regulators is the proto-oncogene of the Ski family SnoN, which was initially identified as a nuclear protein able to transform chicken and quail embryonic fibroblasts when overexpressed. Both pro- and anti-oncogenic activities of SnoN have been reported, but its function in normal epithelial cells has not been defined The overall purpose of the studies described in my dissertation was to elucidate the novel roles of SnoN in epithelial cell development, morphogenesis, differentiation, and tumorigenesis, using the mouse mammary gland, the non-malignant human mammary epithelial cells cultured on laminin-rich extracellular matrix (lrECM), and tissue tumor microarrays from human patients as the model systems. The work described here therefore lends insight into novel aspects of SnoN function and regulation in development and disease.

SnoN expression pattern has not been well characterized and examined in normal tissues. In Chapter 3 of my dissertation, I describe the expression levels of SnoN in the mammary gland at different stages of development. SnoN is expressed at relatively low levels during puberty, but is transiently upregulated at late gestation before being downregulated during lactation and early involution. Chapter 3 describes the roles of SnoN in mammary gland development and breast cancer using our generated transgenic mice expressing a SnoN fragment under the control of the mouse mammary tumor virus promoter (MMTV). In this model system, elevated levels of SnoN increased side-branching and lobular-alveolar proliferation in virgin glands, while accelerating involution in post-lactation glands. The increased proliferation stimulated by SnoN was insufficient to induce mammary tumorigenesis. Only when cooperating with the polyoma middle T antigen (PyVmT) did SnoN accelerate the formation of aggressive multifocal adenocarcinomas and increase the formation of pulmonary metastases. Our studies define functions of SnoN in mammary epithelial cell proliferation and survival and they provide the first in vivo evidence of a pro-oncogenic role for SnoN in mammalian tumorigenesis.

The roles of SnoN in epithelial differentiation and function have not been elucidated and completely understood. In Chapter 4 of my dissertation, I show that SnoN plays a very important role in maintaining mammary alveolar development, acinar structural morphogenesis, and functional differentiation of the secretory alveolar cells, using the SnoN knockout (SnoN-/-) mouse model and MCF-10A cells lacking SnoN. The impairment in alveolar development and acinar differentiation was due to a diminished prolactin-mediated STAT5 (Signal Transducer and Activator of Transcription) signaling pathway, caused by decreased STAT5 phosphorylation and total levels. While the full mechanism of SnoN and STAT5 positive regulation is still unclear, I was able to show that SnoN physcially interacts with the STAT5 protein to maintain its total levels. I also provide preliminary data showing that the high levels of SnoN observed during mammary epithelial differentiation could be regulated by both TGF-ß/Smads and prolactin/STAT5 signaling pathways. This study is the first to define novel functions of SnoN in maintaining normal mammary epithelial morphogenesis, differentiation, and function in vivo and in 3D cultures. It is also the first study to show that SnoN could regulate a different signaling pathway than TGF-ß and that SnoN expression could be regulated by a novel transcription factor different than the Smads.

Evidence suggests that SnoN has both pro-oncogenic and anti-oncogenic functions by modulating both TGF-ß and p53 pathways. It is still unclear at which stages of human cancer does SnoN antagonize TGF-ß signaling to promote oncogenesis or activate p53 to induce premature senescence as a tumor suppressor mechanism. In chapter 5 of my dissertation, I examine the localization and expression levels of SnoN in tumor microarrays from patients with esophageal, ovarian, breast and pancreatic cancers. I also stain these tumors with p53 to analyze whether its inactivation in tumor tissues correlate with high levels of SnoN at different stages of tumor progression. My result suggests that SnoN levels are not overall elevated or overexpressed in the tumor samples compared to normal matched samples. However, SnoN levels were elevated in the infiltrating inflammatory and stromal cells in the advanced stages of adenocarcinomas, suggesting that SnoN might play a role in the tumor microenvironment. Finally, there was no significant correlation between high levels of SnoN and inactivation of p53 in all stages of adenocarcinomas.