Figure 7.

Pin1-FOXM1-blocking peptides repress FOXM1 activity and malignant proliferation in fresh human melanoma biopsy material. (a–c) The S331/704 peptides repress CENPF expression and malignant proliferation of a human metastatic melanoma biopsy. A melanoma was isolated, divided in 300-μM sections and either directly treated with the S331/704 peptides as in Figure 6c or left untreated. After 6 days, one set was processed for CENPF (b) immunostaining and quantified as in Figure 6c. Another set was incubated with EdU on day 5, fixed 24 h later and processed for EdU incorporation (c), which was quantified similarly. **P<0.01. (d) The FOXM1-S704 D-Retro-Inverso (DRI) peptide reduces viability of a patient-derived 3D-cultured melanoid, EM5. A node-resected Stage III metastatic melanoma was sectioned and cultured in Matrigel as in a. Subsequent passaging resulted in monocultures (left panel) with >96% BRAF^V600E and CDKN2A mutations as determined by Next Generation sequencing (bottom table and Supplementary Figure k). The melanoid cultures, dubbed ErasmusMC melanoma #5, EM5, were split into in 96-well plates and exposed to 0.25 μM PLX4032, 100 μM of the S704 DRI peptide or their combination. Cell viability determined 4 days later by AqueousOne Celltiter Assay (top right panel). (e) Model of the regulation of FOXM1 by Pin1 in BRAF^V600E-driven melanoma. (1) Active BRAF^V600E induces a senescence-associated G0-based growth arrest resulting in low FOXM1 expression. (2) In melanomas where this arrest is escaped, FOXM1 becomes elevated and MEK-dependent FOXM1 phosphorylation occurs in the G2/M phase of the cell cycle. (3) MEK-mediated phosphorylation allows Pin1 binding, ensuring full FOXM1 activity and proliferation. (4) Interference with Pin1 or the Pin1-FOXM1 interaction by (a) specific blocking peptide(s) represses FOXM1 activity and proliferation, providing a potential point of intervention for melanomas expressing high levels of Pin1-FOXM1 signaling. *P<0.05; **P<0.01.