Targeting Subcellular Organelles in Breast Cancer with Enzyme Induced Self-Assembly: Stimulated Response with Eyes-Absence Enzymes (EYA)

Self-assembly of short peptides induced by enzymes is an emerging pathway to overcome drug resistance in cancer. However, specific targeting requires optimal precursor design and understanding of subcellular interactions. In our study, we synthesized six short peptides by modifying the N-terminal motif with fluorenylmethyloxycarbonyl (Fmoc), diphenyalanine (FF), arginylglycylaspartic acid (RGD), naphthyl (Nap), and 4-nitrobenz-2-oxa-1,3-diazole (NBD) motifs and including C terminal activation groups of tyrosine phosphate (pTyr) and threonine phosphate (pThr). We utilized the tyrosine phosphatase activity of Eye Absent Enzyme (EYA) to induce self-assembly of short peptides in breast cancer cells. The subcellular interaction of P1-P6 on DNA, mitochondria, and the cell membrane were evaluated in MDA-MB-231 (receptor-negative), MCF-7 (receptor-positive), and MCF10 (nontumorigenic epithelial breast) cells. Being dephosphorylated by EYA, the peptide transformed from monomers to assembled, which was confirmed by UV absorption shift and released phosphate. Studying protein expression showed that EYA2 is expressed in breast cancer cells but is absent in normal epithelial breast cells. The peptide/assemblies first aggregate on the cell membrane, then enter the cells, and accumulate in nuclei. Fmoc-FF-pTyr and RGD-FF-pTyr demonstrated significant binding affinity for phosphatidylserine on the surface of apoptotic cells. Among these, Fmoc-FF-pTyr effectively induced DNA damage and triggered apoptosis in cancer cells while remaining nontoxic to normal cells at optimal concentrations (10 μM). Peptides containing Nap groups selectively accumulated in the mitochondria of MCF-7 and MDA-MB-231 cells, enhancing the effectiveness of the doxorubicin treatments. These results demonstrate the critical role of EISA precursor in modulating phosphatase induced pathways by EYA's in breast cancer and the importance of considering these properties in nanotherapeutic design.