Mortalin peptides exert antitumor activities and act as adjuvants to antibody-mediated complement-dependent cytotoxicity.

Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress­70 protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer pro­survival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complement­dependent cytotoxicity (CDC) and may therefore protect cells from antibody­based immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a C­terminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, Mot­P2 and Mot­P7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, Mot­P2 and Mot­P7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, Mot­P7, but not Mot­P2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At sub­toxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalin­binding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with Mot­P2 or Mot­P7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complement­activating antibody therapy to target mortalin for precision cancer therapy.

Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9.

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Mortalin/GRP75 binds to complement C9 and plays a role in resistance to complement-dependent cytotoxicity.

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.