In the late stages of cancer, tumors acquire the ability to spread throughout the body and invade distant tissues in a process called metastasis. Studies have shown that metastasis is responsible for 90% of all cancer-related deaths, making this an important field of study. In breast cancers, 30% of patients overexpress the HER2 oncoprotein, causing a more invasive and metastatic disease. Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called EMT (epithelial-to-mesenchymal transition), where epithelial cells transition into a migratory phenotype through cell-cell junction breakdown. SLK is a Ste20-like kinase that has been linked to many processes, including cell migration and signaling downstream of the HER2 receptor complex. Here we show that the cellular migration and invasion of TGFβ-treated normal mammary epithelial cells is significantly impaired in the absence of SLK. Additionally, immunofluorescence analyses demonstrate that SLK knockdown conditions decrease a cell’s ability to progress through EMT due to the visible staining of epithelial markers. We find that SLK-depleted cultures express significantly lower levels of Snail1,and fibronectin mRNA levels following TGF-β treatment. Surprisingly, our data demonstrates that SLK kinase activity is not activated downstream of TGF-β stimulation, and that a kinase-dead SLK rescues Snail1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced epithelial-to-mesenchymal transition in a kinase activity-independent manner.

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover. SLK activity is regulated by the LIM domain-binding proteins Ldb1/2. In addition to playing role in tumor initiation and progression, these proteins have been demonstrated to interact with LMO4. Therefore, this project assessed the ability of LMO4 to interact and regulate SLK activity. Results show that LMO4 can directly bind to SLK and activate its kinase activity. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding. Cre deletion of LMO4 inhibits cell migration and SLK activation, and impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Src/Yes/Fyn-deficient cells (SYF) express very low levels of LMO4 and do not recruit SLK to the leading edge. Src-family kinase inhibition impairs SLK recruitment to the leading edge, suggesting that both expression of LMO4 and the recruitment of SLK to the leading edge require c-Src activity. In conclusion, cell migration and activation of SLK requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. This study also investigated whether LMO4 deletion through MMTV-Cre-driven excision would impair mammary tumorigenesis in a PyMT mouse model of breast cancer. No difference in Overall Survival was observed between animals with and without LMO4 expression. Western blot analysis and IHC showed that tumors expressed LMO4 protein in animals genotyped as Cre-positive. This result suggests that expression of LMO4 is required for tumor initiation in the PyMT model of murine mammary carcinoma. This project has established a novel cytosolic role for the transcriptional co-activator LMO4 and validated it’s involvement in the regulation of SLK and cell migration. This pathway may provide a novel therapeutic strategy as LMO4 appears to be critical to the initiation and progression of breast cancer.