Download Amino acid starvation sensitizes cancer cells to proteasome inhibition

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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.
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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
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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
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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
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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).
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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).
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