Discovery of novel MKL1 target genes important for cancer progression and metastasis : a focus on WNT1 inducible signaling pathway protein 1 (WISP1/CCN4)

Tenascin-C (TNC) is involved in tumor initiation and metastasis, and high TNC expression has been associated with poor prognosis in cancers such as glioma, breast, colon and lung carcinoma (Orend and Chiquet-Ehrismann, 2006; Oskarsson, 2013). Previous studies have shown that the transcriptional regulator megakaryoblastic leukemia-1 (MKL1) induces TNC expression in both normal and transformed mammary epithelial cells, and that this induction requires the potential DNA-binding SAP domain of MKL1 (Asparuhova et al., 2011). Therefore, we postulated that SAP-dependent MKL1 action might be responsible for the tumor-specific induction of TNC. By transcript profiling analyses, we identified genes that are co-regulated with TNC in HC11 mouse mammary epithelial cells. We found that the expression of this gene set is associated with high-proliferative poor-outcome classes in human breast cancer and with reduced survival rate for breast cancer patients independent of tumor grade. Many of the newly discovered SAP-dependent/SRF-independent MKL1 target genes are strongly implicated in cell proliferation, cell motility and cancer. Indeed, downregulation of these transcripts by overexpression of MKL1 lacking the SAP domain inhibited cell growth as well as cell migration. Interestingly, many of the SAP-dependent MKL1 target genes, including WNT1 inducible signaling pathway protein 1 (WISP1/CCN4) that we studied further, were mechanoresponsive (Gurbuz et al., 2014). WISP1 is a secreted, matricellular protein assigned to the CCN family, and aberrant WISP1 expression is observed in various pathologies including fibrosis and cancer (Berschneider et al., 2011). However, relatively little is known about the mechanistic details of its function. In our studies, we found that endogenous WISP1 expression correlates with the metastatic potential of isogenic mouse breast cancer cell lines. Furthermore, we observed that WISP1 mRNA levels within the mouse mammary gland tissue significantly increased upon irradiation, a treatment known to induce modifications in the tumor microenvironment leading to increased metastasis (Ruegg et al., 2011). Finally, we produced recombinant WISP1 protein and confirmed that WISP1 is N-glycosylated and that the secreted form of the protein undergoes additional post-translational modifications that increase its size and possibly add functional diversity to the protein. Using our purified recombinant protein we generated specific anti-WISP1 antibodies. In the future, these antibodies can be used to detect WISP1 in various tumor tissues.