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IUBMB Life, 62(10): 757–763, October 2010 Research Communication Amino Acid Starvation Sensitizes Cancer Cells to Proteasome Inhibition Sarit Mizrachy-Schwartz, Noam Cohen, Shoshana Klein, Nataly Kravchenko-Balasha Alexander Levitzki Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Summary We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines. Ó 2010 IUBMB IUBMB Life, 62(10): 757–763, 2010 Keywords Deregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of cancer, and increased activity/ expression of the ubiquitin-proteasome pathway has been monitored in many cancers [reviewed in (1, 3)]. Pharmacological inhibition of the proteasome has therapeutic utility in some cancers and the proteasome inhibitor, Bortezomib (Velcade), is approved for the treatment of multiple myeloma (MM) (4, 5). Proteasome inhibitors affect the regulation of the NF-jB pathway, induce endoplasmic reticulum stress, stabilize the proapoptotic p53 and Bax proteins and have many other effects [Reviewed in (1, 3–5)]. Here, we show that some cancer cells can be sensitized to proteasome inhibition by concomitant starvation for essential amino acids. We speculate that protein catabolism contributes to the pool of free amino acids available for protein synthesis during cancer development, leading to a crucial role of the proteasome in cancer cell survival under conditions of nutrient depletion. proteasome; protein synthesis; amino acid; starvation; bortezomib; cancer. Abbreviations CGM, complete growth medium; Cys, cysteine; Leu, leucine; Met, methionine; MM, multiple myeloma. INTRODUCTION Protein synthesis and degradation (protein homeostasis) must be balanced for normal cellular function. The major intracellular pathway controlling protein degradation is the ubiquitin proteasome system [reviewed in (1, 2)]. Received 5 July 2010; accepted 5 August 2010 Address correspondence to: Alexander Levitzki, Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel. Tel.: 972-2-6585404. Fax: 972-2-6512958. E-mail: [email protected] ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.377 EXPERIMENTAL PROCEDURES Cell Culture Tissue culture reagents were purchased from Biological Industries Bet-Haemek, Israel. DMEM without L-cysteine/Lmethionine (L-Cys/L-Met) was from Gibco. MG-132 was from Calbiochem and Bortezomib was from LC laboratories. Cholera toxin, EGF, hydrocortisone, and insulin were from Sigma. For routine growth, A431 (human epithelial carcinoma) and T-24 (bladder cancer) cell lines were maintained in DMEM supplemented with 10% fetal calf serum (FCS). The breast cancer cell line, MCF7, and the MM cell line, CAG, were grown in RPMI medium supplemented with 10% FCS. The nontumorigenic epithelial cell line, MCF10A, was grown in DMEM supplemented with 5% horse serum, 10 lg/mL insulin, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin, and 20 ng/mL EGF. 758 MIZRACHY-SCHWARTZ ET AL. To detach MCF10A cells, they were trypsinised with 0.5 mL 0.05% trypsin, 0.53 mM EDTA and incubated at 37 8C for 15 minutes. To neutralize trypsin, 7 mL of complete growth medium (CGM) were added and the cells were centrifuged at 750 3 g for 5 minutes. Then the cells were resuspended in CGM. For the experiments, A431, T-24 and CAG cells were seeded in DMEM supplemented with 10% FCS. MCF10A and MCF7 cells were seeded in DMEM supplemented with 5% FCS, 10 lg/mL insulin, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin, and 20 ng/mL EGF. All media were supplemented with 100 U/mL penicillin, 100 lg/mL streptomycin and all the cells were grown in a humidified atmosphere containing 5% CO2 at 378C. Cell Survival Assay Cells were seeded in 96-well plates. The next day, cells were treated as indicated. The fraction of surviving cells was measured after the indicated times using the methylene blue assay (6). The concentrations yielding 50% inhibition (IC50) were calculated with GraphPad Prism software using nonlinear regression (Prism, GraphPad Software). Protein Determination For denatured lysates, proteins were quantitated using the bound Coomassie blue method (6). Immunoblotting Cells were washed with PBS (50 mM NaH2PO, 50 mM Na2HPO4, 0.77 M NaCl) and denatured cell lysates were prepared by scraping the cells with Laemmli sample buffer (40% glycerol, 0.2 M Tris pH 6.8, 20% b-mercaptoethanol, 12% sodium dodecyl sulfate (SDS), and bromo phenol blue) and boiling for 10 min. Aliquots of cell extracts containing equal amounts of protein were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes (Sartorius). Antibodies for western blotting were as follows: anti-b-catenin (#610153, BD Transduction Laboratories, 1:10000 dilution) and antipolyubiquitin (1:4000), kindly provided by Prof. R. Kulka. Immunoreactive bands were visualized using enhanced chemiluminescence. Densitometry of immunoblots was performed with NIH image 1.61 software. When needed, blots were stripped using Restore Plus Western blot stripping buffer (Pierce), according to the manufacturer’s instructions, or by incubating in 2% SDS, 10 mM b-mercaptoethanol, 62.5 mM Tris-HCl pH 6.8 at 55 8C for 20 min, washed with TBST, and then blocked with 5% milk in TBST and reprobed. Pulse Labeling of Proteins Proteins were labeled in the presence or absence of MG-132 or Bortezomib as described in (7). 20S Proteasomal Activity Assay The 20S Proteasome Activity Assay Kit (Chemicon) was used to measure the 20S proteasome activity in 20 lg of total protein, according to the manufacturer’s instructions. RESULTS Enhanced Proteasome Activity during Cell Transformation To explore the role of the ubiquitin proteasome pathway in carcinogenesis, we compared MCF10A cells (a nontumorigenic epithelial cell line) to MCF7 cells (metastatic adenocarcinoma of the breast). Targeting of most substrates to the 26 S proteasome requires their prior marking by a covalently linked polyubiquitin chain(s) [reviewed in (1, 2)]. We examined the polyubiquitination profiles of MCF10A and MCF7 cells, using an antibody that recognizes polyubiquitin-protein conjugates. MCF7 cells had more protein ubiquitination than MCF10A cells (Fig. 1A). Polyubiquitin can be linked through each of seven lysine residues in the ligation site, and the different linkages may represent different functions (2, 8). Furthermore, increased ubiquitination does not necessarily mean that the proteasome is more active, but could rather indicate a problem in the proteasome machinery. To evaluate directly the proteasome activity in our cell lines, we used a proteasome activity assay kit (9, 10). MCF7 cells had dramatically higher proteasome activity than MCF10A cells (Fig. 1B). However, in CGM, there was no significant difference in the sensitivity of MCF7 and MCF10A cells to the proteasome inhibitors, MG-132 (Fig. 1C) or Bortezomib (Fig. 1D). Proteasome Activity Is Important to Cell Survival during Amino Acid Depletion It has been shown that during nutrient depletion the proteasome releases amino acids that are then used for protein synthesis (7). We therefore hypothesized that the increased proteasome activity that we observed in MCF7 cells only becomes essential for survival when the cells are starved for amino acids. The experiments shown in Fig. 1 were performed in a medium highly enriched with amino acids, and therefore amino acids were not a limiting factor. Proteasome inhibition by MG-132 or Bortezomib treatment, in amino acid depleted medium, led to reduced survival of MCF7 cells when compared with MCF10A cells (Figs. 2A and 2B). Furthermore, during amino acid starvation, increased polyubiquitination was monitored in MCF7 cells (Fig. 2C), and proteasome activity was enhanced in MCF7 but not in MCF10A cells (Fig. 2D). Proteasome Activity Is Required to Sustain Protein Synthesis Under Amino Acid Depletion We next examined whether the increased proteasome activity in MCF7 cells is essential for protein synthesis. In Cys/Met- PROTEASOME INHIBITION AND PROTEIN SYNTHESIS IN CANCER 759 Figure 1. Enhanced proteasome activity in MCF7 versus MCF10A cells. A: Lysates were analyzed by immunoblot with antipoly ubiquitin antibody. The blots, which are part of the same gel, were cut for ease of presentation. b-catenin served as a gel loading control. Polyubiquitinated protein levels were normalized to b-catenin levels. The graph shows the calculated averages and S.D. from two independent experiments. B: The 20S proteasome activity assay kit (Chemicon) was used to evaluate proteasome activity in cells in CGM. The proteasome inhibitor, Lactacystin (In.) was used as a control. (MCF10A without inhibitor 5 100%). C, D: MCF7 and MCF10A cells were treated with MG-132 (C) or Bortezomib (D) at increasing concentrations for 48 h in CGM. Cell survival was assayed using methylene blue (V 5 vehicle 5 100%). depleted medium, less 3H-leucine (3H-Leu) was incorporated into MCF7 cells in the presence of a proteasome inhibitor, MG132 or Bortezomib, indicating decreased protein synthesis (Fig. 2F). This effect was not observed in MCF10A cells (Fig. 2E). On the other hand, in CGM there was no reduction in 3H-Leu incorporation when the proteasome was inhibited (Figs. 2E and 2F). Combination of Proteasome Inhibition and Amino Acid Depletion in Various Cancer Cells We next explored whether the proteasome plays a role in the survival of additional cancer cell lines, under conditions of depletion of Cys and Met. Since Bortezomib was approved for the treatment of MM, we examined the MM cell line, CAG, and cell lines A431(epithelial carcinoma) and T-24 (bladder car- cinoma). All three lines were sensitive to Cys/Met depletion. However, the various cell lines differed in their sensitivity to Bortezomib. In CGM, CAG cells were quite sensitive to Bortezomib, and A431 cells were barely sensitive. CAG and A431 cells were more sensitive to Bortezomib when starved for amino acids, and the IC50s of the inhibitor significantly decreased under these conditions (Figs. 3A and 3B). T-24 cells were unaffected at concentrations of up to 100 nM Bortezomib, in complete or depleted medium (Fig. 3C). We examined whether the different sensitivities to proteasome inhibition among the cell lines might result from differences in proteasome activity. However, no significant differences were found in proteasome activity among the different cell lines (Fig. 3D). We next examined the effect of combining Bortezomib treatment with amino acid starvation on the protein synthesis rates 760 MIZRACHY-SCHWARTZ ET AL. Figure 2. Amino acid depletion renders MCF7 cells sensitive to proteasome inhibition. MCF7 and MCF10A cells were treated with MG-132 (A) or Bortezomib (B) at increasing concentrations for 48 h in Cys/Met depleted medium. Cell survival was assayed using methylene blue (V 5 vehicle 5 100%). C: Polyubiquitinated protein levels increased in MCF7 cells, but less so in MCF10A cells, when starved for Cys/Met. Anti-b-catenin was used to control for gel loading. D: Evaluation of proteasome activity in cell lysates (20 lg) using the proteasome activity assay kit (Chemicon) (MCF10A in CGM 5 100%). The proteasome inhibitor, Lactacystin (In.) was used as a control. E, F: MCF10A (E) and MCF7 (F) cells were seeded in 6-well plates. After 24 h, cells were washed with PBS and incubated in complete or Cys/Met depleted medium in the presence or absence of proteasome inhibitor (MG-132 or Bortezomib). After 30 min starvation, cells were labeled with 100 lCi/mL 3H-Leu for an additional 25 min. The incorporation of 3H-Leu into TCA-insoluble material was measured. (V 5 vehicle 5 1). PROTEASOME INHIBITION AND PROTEIN SYNTHESIS IN CANCER 761 Figure 3. Cancer cell lines differ in their sensitivity to proteasome inhibition. A–C: CAG cells (A) A431 cells (B) and T-24 cells (C) were treated with Bortezomib at increasing concentrations for 48 h in CGM or in Cys/Met depleted medium (Starvation). Cell survival was assayed by the methylene blue method (V 5 vehicle 5 100%). D: Evaluation of proteasome activity in 20 lg of cell lysates using the proteasome activity assay kit (Chemicon). E–G: CAG cells (E) A431 cells (F) and T-24 cells (G) were seeded in 6-well plates. After 24 h, the cells were washed with PBS and incubated in CGM or Cys/Met depleted medium with/without Bortezomib at the indicated concentrations. After 30 min the cells were labeled with 3H -Leu (100 lCi/mL) for 25 minutes more. The incorporation of 3H-Leu into TCA-insoluble material was measured (V 5 vehicle 5 1). 762 MIZRACHY-SCHWARTZ ET AL. in these three cell lines. For CAG cells, proteasome inhibition led to reduced protein synthesis rates in both complete and Cys/ Met-depleted media (Fig. 3E). For A431 cells, Bortezomib treatment led to a reduction in protein synthesis rate in Cys/ Met-depleted medium, but not in CGM (Fig. 3F). In T-24 cells, proteasome inhibition had no effect on the protein synthesis rate (Fig. 3G). These results are in correlation with the results of the survival assay. DISCUSSION Here, we investigated the effect of proteasome activity on protein synthesis in cancer cells. While enhanced proteasome activity was monitored in cancer cells (Figs. 1A and 1B), proteasome inhibition had only a mild effect on the survival of both cancerous (MCF7) and noncancerous (MCF10A) cells under complete growth conditions (Figs. 1C and 1D). However, under amino acid depletion, proteasome activity was enhanced in the cancer cells when compared with the noncancer cells (Figs. 2C and 2D). Proteasome inhibition during amino acid deprivation not only reduced the rate of protein synthesis in MCF7 cells (Fig. 2F), but also affected the survival of MCF7 cells much more strongly than that of MCF10A cells (Figs. 2A and 2B). Certain cancer cells may be more dependent on protein synthesis than nontumorigenic cells, so that when the cancer cells are starved for amino acids, their enhanced proteasome activity becomes essential for their survival. Combining amino acid depletion and proteasome inhibition had an effect on A431 cells and CAG cells, but not on T-24 cells (Fig. 3). Proteasome activity in these cells was similar (Fig. 3D), but there were differences in the effects of proteasome inhibition on their protein synthesis rates (Figs. 3E–3G) and on the survival of these cells (Figs. 3A and 3B). Several studies have shown that the induction of apoptosis by proteasome inhibitors is p53-dependent (e.g., 11–13), but others have reported p53-independent cell death (e.g., 14). There was no correlation between the p53 or pRb status of the cell lines in our study and their response to proteasome inhibition: both MCF10 and MCF7 are wild type for p53 and pRb, whereas both A431 and T-24 have p53 mutations (http://p53.free.fr/Database/Cancer_cell_lines/HB_cell_lines. html). We hypothesize that one role of the proteasome is to ensure cell survival by allowing efficient translation under conditions of amino acid starvation. Solid tumors are often starved for nutrients (15), and proteasome over-activation may be a way to overcome this. We have found that primary keratinocytes are resistant to leucine limitation (85% survival after 36 h starvation), and that amino acid starvation does not render primary keratinocytes sensitive to proteasome inhibition (data not shown). Amino acid starvation has been shown to cause autophagy in yeast and mammalian cells, and in multicellular organisms, such as Caenorhabditis elegans. Dietary restriction, proteasome activity, and autophagy have been shown to contribute to longevity (16–19). Bortezomib (Velcade) is the first proteasome inhibitor approved for newly diagnosed and relapsed MM (4, 5). Our data suggest that Bortezomib and similar proteasome inhibitors might be more effective in treating multiple myeloma, and applicable to a broader range of cancers, if treatment were combined with amino acid depletion. Many human cancer cell lines and primary tumors have absolute requirements for Met, whereas normal cells are relatively resistant to exogenous Met restriction (19). Prolonged Met restriction is not suitable for clinical use (19), but temporary Met restriction is being tested in the clinic in association with various chemotherapeutic agents (19). We suggest that the combination of Met restriction and Bortezomib should be investigated. ACKNOWLEDGEMENTS This study was supported by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israel’s Ministry of Science and Technology (MOST). REFERENCES 1. Mani, A. and Gelmann, E. P. (2005) The ubiquitin-proteasome pathway and its role in cancer. J. Clin. Oncol. 23, 4776–4789. 2. Navon, A. and Ciechanover, A. (2009) The 26 S proteasome: from basic mechanisms to drug targeting. J. Biol. Chem. 284, 33713–33718. 3. Paul, S. (2008) Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches. Bioessays 30, 1172– 1184. 4. Adams, J. and Kauffman, M. (2004) Development of the proteasome inhibitor Velcade (Bortezomib). Cancer Invest. 22, 304–311. 5. Shah, J. J. and Orlowski, R. Z. (2009) Proteasome inhibitors in the treatment of multiple myeloma. Leukemia 23, 1964–1979. 6. Mizrachy-Schwartz, S., Kravchenko-Balasha, N., Ben-Bassat, H., Klein, S., and Levitzki, A. (2007) Optimization of energy-consuming pathways towards rapid growth in HPV-transformed cells. PLoS One 2, e628. 7. Vabulas, R. M. and Hartl, F. U. (2005) Protein synthesis upon acute nutrient restriction relies on proteasome function. Science 310, 1960– 1963. 8. Pickart, C. M. (2000) Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548. 9. Bhat, U. G., Halasi, M., and Gartel, A. L. (2009) FoxM1 is a general target for proteasome inhibitors. PLoS One 4, e6593. 10. Fortun, J., Li, J., Go, J., Fenstermaker, A., Fletcher, B. S., and Notterpek, L. (2005) Impaired proteasome activity and accumulation of ubiqutinated substrates in a hereditary neuropathy model. J. Neurochem. 92, 1531–1541. 11. An, W. G., Hwang, S. G., Trepel, J. B., and Blagosklonny, M. V. (2000) Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition. Leukemia 14, 1276–1283. 12. Dietrich, C., Bartsch, T., Schanz, F., Oesch, F., and Wieser, R. J. (1996) p53-dependent cell cycle arrest induced by N-acetyl-L-leucinylL-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts. Proc. Natl. Acad. Sci. USA 93, 10815–10819. 13. Lauricella, M., D’Anneo, A., Giuliano, M., Calvaruso, G., Emanuele, S., Vento, R., and Tesoriere, G. (2003) Induction of apoptosis in human osteosarcoma Saos-2 cells by the proteasome inhibitor MG132 and the protective effect of pRb. Cell Death Differ. 10, 930–932. PROTEASOME INHIBITION AND PROTEIN SYNTHESIS IN CANCER 14. Strauss, S. J., Higginbottom, K., Juliger, S., Maharaj, L., Allen, P., Schenkein, D., Lister, T. A., and Joel, S. P. (2007) The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines. Cancer Res. 67, 2783–2790. 15. Brech, A., Ahlquist, T., Lothe, R. A., and Stenmark, H. (2009) Autophagy in tumour suppression and promotion. Autophagy in tumour suppression and promotion. Mol. Oncol. 3, 366–375. 16. Droge, W. (2004) Autophagy and aging–importance of amino acid levels. Mech. Ageing Dev. 125, 161–168. 763 17. Fontana, L., Partridge, L., and Longo, V. D. Extending healthy life span–from yeast to humans. Science 328, 321–326. 18. Ghazi, A., Henis-Korenblit, S., and Kenyon, C. (2007) Regulation of Caenorhabditis elegans lifespan by a proteasomal E3 ligase complex. Proc. Natl. Acad. Sci. USA 104, 5947–5952. 19. Cellarier, E., Durando, X., Vasson, M. P., Farges, M. C., Demiden, A., Maurizis, J. C., Madelmont, J. C., and Chollet, P. (2003) Methionine dependency and cancer treatment. Cancer Treat Rev. 29, 489– 499.