Flotillin-1 palmitoylation is essential for its stability and subsequent tumor promoting capabilities.

Flotillin-1 contributes to invasion and metastasis in triple negative breast cancer (TNBC). Palmitoylation, the process of conjugating palmitoyl-CoA to proteins, plays an essential role in protein stability and trafficking. Flotillin-1 is modified by palmitoylation, however, the role of its palmitoylation in the context of metastasis has not been explored. Using palmitoylation defective flotillin-1 constructs, we have demonstrated that flotillin-1 palmitoylation contributes to its stability and metastatic capabilities in vivo. Further investigation led to the identification of zDHHC5 as the main palmitoyl acyl transferase responsible for palmitoylating flotillin-1, which also contributed to its stability by preventing its poly-ubiquitylation. To assess the ability to target flotillin-1 palmitoylation therapeutically, we designed a competitive peptide, which displayed efficacy in blocking flotillin-1 palmitoylation in vitro without altering palmitoylation of other zDHHC5 substrates, highlighting its specificity. Additionally, multiple TNBC tumor models expressing a doxycycline inducible flotillin-1 palmitoylation inhibiting peptide construct displayed attenuated tumor growth and lung metastasis. The current study has demonstrated the palmitoylation of flotillin-1, a known metastasis inducing protein, to be essential for its protein stability. The demonstrated methods in blocking its palmitoylation through the delivery of a competitive peptide provide proof-of-concept data for further development as a potential targeted therapeutic in TNBC. To evaluate the effects of flotillin-1 palmitoylation, MDA-MB-231 cells were engineered to express flotillin-1 WT or palmitoylation defective (C34A). WT flotillin-1 cells were labelled with a GFP reporter and C34A cells with a dTomato reporter. GFP-WT and dTomato-C34A cells were implanted bilaterally into mice in their #4 mammary fat and allowed to form tumors for 8 weeks. Tumors were digested into single cell supsensions and flow sorted based on GFP or dTomato reporters. Gene expression was then compared between GFP-flotillin-1-WT and dTomato-flotillin-1-C34A MDA-MB-231 cells from xenograft tumors.