Cellular targets of HAMLET, their role in tumor cell death and therapeutic potential

Sammanfattning: Protein-lipid complexes have broad and specific effects against cancers of different origins. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a complex of partially unfolded α-lactalbumin and oleic acid that kills a wide range of tumor cells but leaves healthy differentiated cells unaffected. Therapeutic efficacy of HAMLET has been demonstrated in several animal models, without apparent side effects. HAMLET has also been used, successfully, in patients with skin papillomas or bladder cancer. The overall aim of this thesis was to extend the therapeutic studies to include colon cancer and to identify molecular mechanisms of HAMLET-induced tumor cell death.Paper I shows that HAMLET can be given perorally to treat colon cancer in APCMin/+ mice- a model of human familial and sporadic colorectal cancer. Peroral administration of HAMLET reduced tumor numbers and mortality, and this effect was accompanied by a decrease in total β-catenin levels and β-catenin dependent proliferation markers. Transcriptomic analysis of surviving tumors showed suppression of oncogene activity, including the Wnt signaling pathway. Finally, prophylactic use of HAMLET in the drinking water was shown to prevent tumor formation in young APCMin/+ mice. Paper II examined the hypothesis that affinity for conserved molecular motifs may explain the apparent multitude of cellular targets for HAMLET in tumor cells. Using a proteomic screen, we identified nucleotide binding proteins as HAMLET targets, including ATPases, Ras GTPases and kinases. HAMLET acted as a pan-kinase inhibitor, in vitro and this broad effect was confirmed in protein lysates from HAMLET treated cells. The results identify HAMLET as an inhibitor of kinases and GTPases, to which tumor cells are addicted.Paper III identifies three critical features for the membrane response to HAMLET. I. Rapid membrane perturbations, and gross remodeling of tumor cell membranes. II. Formation of HAMLET-Ras membrane clusters in tumor cells. III. Absence of membrane responses in healthy primary cells, indicating tumor selectivity. Paper IV provides a molecular basis for the tumoricidal activity of the HAMLET complex. Synthetic alpha-helical peptides were selectively internalized by tumor cells in the presence of oleate, accumulated in nuclear speckles and suppressed transcription, through a direct effect on the speckle constituents SC-35, PKC and Pol II. Therapeutic efficacy was demonstrated in a bladder cancer model, where alpha-helical peptide-oleate complexes targeted cancer tissue selectively and reduced tumor load. These findings identify new, peptide-based HAMLET biosimilars with strong therapeutic potential.