Nelfinavir, a new anti-cancer drug with pleiotropic effects and many paths to autophagy

Abstract

The development of cancer drugs is slow and costly. One approach to accelerate the availability of new drugs is to reposition drugs approved for other indications as anti-cancer agents. HIV protease inhibitors (HIV PIs) are useful in treating HIV infection and cause toxicities in humans that are similar to those observed when the kinase Akt, a target for cancer therapy, is inhibited. To test whether HIV PIs inhibited Akt and cancer cell proliferation, we screened 6 HIV PIs and found that three, ritonavir, saquinavir and nelfinavir, inhibit the growth of over 60 cancer cell lines derived from 9 different tumor types; Nelfinavir is the most potent. Nelfinavir causes caspase-dependent apoptosis and non-apoptotic death, as well as endoplasmic reticulum (ER) stress and autophagy. Nelfinavir blocks growth factor receptor activation and decreases growth factor-induced and endogenous Akt signaling. In vivo, nelfinavir inhibits tumor growth and upregulates markers of ER stress, autophagy and apoptosis. Nelfinavir is currently being tested in cancer patients in Phase I clinical trials where biomarkers will be assessed. Current studies are focused on measuring autophagy in clinical specimens and identifying combination strategies that will exploit the induction of autophagy and increase the effectiveness of nelfinavir.

Pharmacological eEF 2K activation promotes cell death and inhibits cancer progression

“…EEF2K deficiency did not confer a promiscuous resistance to cell death as demonstrated by unaltered loss of viability in the presence of other compounds such as tunicamycin (TM) or the apoptosisinducing drug staurosporine (Appendix Fig S4). Other pathways including those related to the ISR are activated by NFR and could affect cell viability [7,10,17,35]. Cells deficient in key signaling components of the UPR pathway including PERK, ATF4, IRE1, and XBP1 and cells unable to phosphorylate eIF2a were tested for NFR sensitivity.…”

“…To quantify the role of eEF2K in NFR-mediated translation inhibition, we measured global translation rates using 35 S-labeled A B C D Figure 3. NFR-mediated eEF2 phosphorylation is AMPK and mTOR independent.…”

“…Nuclei and mitochondria were cleared by centrifugation at 13,000 rpm for 10 min at 4°C; 250 ll post-mitochondrial supernatant (PMS) was mixed with an equal volume of polysomal buffer (25 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 25 mM NaCl, 0.05% Triton X-100, 0.14 M sucrose, 500 lg/ml heparin), and 250 ll was removed to measure incorporation of 35 S-methionine and cysteine into total protein (nascent and completed). Polysomes were pelleted by centrifugation of the remaining supernatant at 55,000 g for 1 h at 4°C in a Beckman TLA120 rotor, and 200 ll post-ribosomal supernatant (PRS) was removed to measure the incorporation of 35 S-methionine and cysteine into completed proteins. Hundred microliters of PMS and PRS samples was collected on a glass fiber filter (GF/C, Whatman).…”

“…Ribosome half-transit time measurements were assessed as in [37,39] with the following modifications; 200,000 cells seeded for 24 h in 6-well plates were treated for 6 h with 20 lM NFR; after 5.5 h of treatment, medium was replaced with labeling medium (DMEM, with 4,500 mg/l glucose and sodium bicarbonate, and without L-methionine, L-cystine, and L-glutamine [Sigma-Aldrich], supplemented with 10% FCS, 1% nonessential amino acids (Gibco Life Technologies TM ), and 2 mM L-glutamine (AMIMED, Bioconcept)) for 30 min before addition of 1 lCi/well/ml of L- 35 Smethionine/cysteine. For cycloheximide treatment, CHX was added at 1 lg/ml to the labeling medium for 30 min.…”

“…We therefore tested eEF2K contribution to elongation by examining NFR regulation of the ribosome half-transit time [34]. We compared the kinetics of 35 S-methionine incorporation into total protein isolated from the post-mitochondrial supernatant (PMS) with the kinetics of radioactive amino acid incorporation measured in released polypeptides harvested from the post-ribosomal supernatant (PRS). The time separating the PMS and PRS lines indicates the ribosome halftransit time (Fig 4D).…”