Dear Editor,
As a mitochondrial deacetylase, SIRT3 deacetylates many enzymes involved in central
metabolism and maintains mitochondrial proteostasis (Verdin et al., 2010; Papa and
Germain, 2014). Substrates of SIRT3 include components of the respiratory complexes,
proteins involved in fatty acid oxidation and TCA cycle (Yu et al., 2012). SIRT3 activates
MnSOD to maintain reactive oxygen species (ROS) homeostasis and a loss of SIRT3 contributes
to the age-associated diseases (McDonnell et al., 2015; Qiu et al., 2010). SIRT3 plays
dual roles functioning as a tumor suppressor or a promoter in tumorigenesis and progression
(Alhazzazi et al., 2011). On one hand, SIRT3 regulates the cellular ROS level and
maintains genomic stability, and mediates metabolic reprogramming to prevent tumorigenesis
(Finley and Haigis, 2012). As a result, the low expression of SIRT3 has been found
in breast cancer, glioblastoma, colon cancer, osteosarcoma, prostate, and ovarian
cancers (Kim et al., 2010; Finley and Haigis, 2012). On the other hand, SIRT3 is a
prosurvival factor that modulates p53 activities and is upregulated in oral cancer,
the node-positive breast cancer, and bladder cancer (Ashraf et al., 2006; Alhazzazi
et al., 2011). These results suggest that SIRT3 possesses the tumor-type dependent
function and its precise role needs to be elucidated in the context of a specific
cancer. Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype
of renal cancer (Cohen and McGovern, 2005). The aims of the present study were to
examine the expression of SIRT3 in ccRCC and to characterize effects of SIRT3 on tumorigenesis
and progression using 293T human embryonic kidney cells as the model system that has
cancer stem cell-like features (Debeb et al., 2010).
Equal amounts of proteins extracted from 18 paired ccRCC lesions and associated pericarcinous
tissue samples were analyzed by Western blotting and the representative Western blot
images of eight paired samples were shown in Fig. 1A, indicating that the expression
levels of SIRT3 were lower in ccRCC than those in normal tissues. The gray scale analysis
of the Western blot data for all eighteen paired samples showed that the SIRT3 expression
was statistically down-regulated in ccRCC tissues (Fig. 1B), suggesting that the low
expression of SIRT3 is important for ccRCC progression.
Figure 1
Downregulation of SIRT3 in ccRCC compared to associated pericarcinous tissues and
characterization of SIRT3 overexpression cells. (A) Representative Western blot images
of the expression levels of SIRT3 of eight paired samples, N (pericarcinous tissue),
C (ccRCC tissue). (B) The gray scale analysis of SIRT3 presented in (A). (C) Growth
curve of SIRT3-OE and the control cells. (D) Graphical representation of ROS levels
of SIRT3-OE cells compared to the control cells. and (E) Survival rate of SIRT3-OE
cells and the control cells treated with different concentration of H2O2 for 12 h.
Data were analyzed using student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
*P < 0.05 is considered statistically significant. Error bars represent ±SEM
To understand the role of SIRT3 in tumorigenesis and progression of ccRCC, stable
cells overexpressing SIRT3 were established in 293T cells. The overexpression of SIRT3
in 293T cells (SIRT3-OE) was examined by Western blotting (Fig. S1), confirming that
the expression level of SIRT3 in SIRT3-OE cells was four fold higher than that in
control cells. The SIRT3 overexpression in 293T led to a decrease in proliferation
rates (Fig. 1C). The ROS level in SIRT3-OE cells is two and half fold higher than
that in the control cells as detected using the CellROX® Deep Red kit (Fig. 1D). To
determine the susceptibility of SIRT3-OE cells to oxidative stress, cells were treated
with various concentrations of hydrogen peroxide for 12 h. The cell viability was
measured using CCK-8 assay. The effects of hydrogen peroxide were represented as the
percentage of viable cells after 12 h treatment (Fig. 1E). When cells were treated
with 400 µmol/L H2O2 for 12 h, the percentages of viable cells were 20% and 90% for
the control and SIRT3-OE cells, respectively (Fig. 1E). This declares that SIRT3-OE
cells are more resistant to H2O2 treatment.
Next, proteomic analysis was carried out on SIRT3-OE and control cells in biological
replicates. Equal amounts of proteins from SIRT3-OE and the control cells were in-solution
digested and labeled with TMT reagents. The generated tryptic peptides were fractionated
using off-line HPLC and each fraction was further analyzed by nano-LC-MS/MS. Differentially
expressed proteins were identified and quantified using the TMT-based quantification.
We identified 7536 proteins in two biological replicates and the false-positive rate
was estimated to be less than 1%. Based on the average reporter ion ratios (>1.5 or
<0.67), 188 proteins were found to be differentially expressed between SIRT3-OE and
control cells, in which 93 proteins were down-regulated and 95 were up-regulated (Tables
S1 and S2). To understand the biological relevance of the differentially expressed
proteins, the Gene Ontology (GO) was used to cluster differentially expressed proteins
according to their associated biological processes. The annotations of gene lists
are summarized via a pie plot based on the functional classification from Panther
as shown in Fig. 2A. One hundred and eighty eight differentially expressed proteins
participated in a variety of cellular processes including metabolic process, cellular
process, and cellular component organization process. The primary metabolic process
shows the dominant difference between SIRT3-OE and the control cells. About 25% of
the down-regulated proteins are classified as mitochondrial proteins, indicating that
SIRT3 overexpression has a great impact on the mitochondrial protein expressions.
Five subunits of respiratory complex IV were down-regulated in SIRT3-OE cells including
COX7C, COX6A1, COX7A2, COA7, and COA5 (Fig. 2B), suggesting that the SIRT3 overexpression
disrupted the integrity of the respiratory complexes. We also identified that three
proteins in the fatty acid β-oxidation pathway were downregulated in SIRT3-OE cells
including enoyl-CoA hydratase, very long-chain specific acyl-CoA dehydrogenase and
hydroxyacyl-coenzyme A dehydrogenase. More importantly, proteomic analysis showed
that nine subunits of mitochondrial ribosomes were downregulated in SIRT3-OE cells
(Fig. 2B). All these results indicated that SIRT3-overexpression disrupted mitochondrial
proteostasis and contributed to mitochondrial dysfunction.
Figure 2
Proteomic, qPCR and Western blot analysis of differentially expressed proteins between
SIRT3-OE and control cells. (A) GO analysis of the differentially expressed proteins
in SIRT3-OE cells compared to the control cells with PANTHER (http://www.pantherdb.org).
(B) Graphical representation of TMT ratios for proteins in SIRT3-OE cells compared
to control cells. (C) qPCR analysis of mRNA expressions of selected mitochondrial
ribosomal genes and other selected genes from SIRT3-OE cells and control cells. (D)
Western blot analysis of selected proteins from SIRT3-OE and control cells. (E) Cell
cycle analysis of SIRT3-OE cells and the control cells. (F) Western blotting images
showing expression level of SIRT3 in HSP60-KN cells compared to the control cells,
WCL (whole cell lysates), Mito (mitochondria); and (G) mRNA expression level of SIRT3
in HSP60-KN cells and control cells. Data were analyzed using student’s t-test. *P
< 0.05, **P < 0.01 and ***P < 0.001. Error bars represent ±SEM
qPCR analysis was conducted and showed that the mRNA expression levels of these nine
mitochondrial ribosomal genes were lower in SIRT3-OE cells than those in the control
cells (Fig. 2C). qPCR analysis also confirmed the downregulation of other genes including
SDHB, COA7, CDK1, and CDK4 (Fig. 2C). Additionally, Western blotting was employed
to examine expressions of the selected proteins and showed that the expression levels
of EEF1A2, CDK1, and CDK4 were down-regulated whereas those of vimentin and angiomotin
were upregulated in SIRT3-OE cells (Fig. 2D), consistent with the proteomic results
displayed in Tables S1 and S2. The above results also showed that SIRT3-OE cells grew
slower than the control cells (Fig. 1C), indicating that cell cycle progression varies
between those two cells. Indeed, the cell cycle analysis of SIRT3-OE and control cells
showed that SIRT3-OE cells had the higher G1-phase accumulation (Fig. 2E), in consistent
with the down-regulation of CDK1 and CDK4 in SIRT3-OE cells.
SIRT3 is known to play a crucial role in maintenance of mitochondrial proteostasis.
Proteomic analysis showed that multiple subunits of respiratory complex IV were down-regulated
in SIRT3-OE cells, which compromised the integrity and assembly of respiratory complexes,
leading to over-production of ROS (Fig. 1D). Excessive ROS can negatively regulate
cell growth and may activate the Nrf2/Keap1 pathway that protects SIRT3-OE cells from
oxidative stress (Fig. 1E). An alternative explanation for the high resistance to
oxidative stress exhibited in SIRT3-OE cells is that deacetylation of antioxidant
proteins by SIRT3 enhances their enzymatic activities, which needs to be confirmed
in the future study. Three key proteins in fatty acid β-oxidation pathway were also
down-regulated in SIRT3-OE cells (Table S2). Growth and proliferation of tumor cells
require fatty acids for synthesis of membranes and signaling molecules. Disruption
of fatty acid oxidation pathway causes a decrease in both acetyl-CoA production that
is essential for the de novo lipid synthesis and NADH and FADH2 generation that are
important for ATP and citrate production (Carracedo et al., 2013). Moreover, SIRT3-meidated
disruption of fatty acid β-oxidation can lead to the accumulation of fatty acids to
induce lipotoxicity. Cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase
4 (CDK4) were found to be downregulated in SIRT3-OE cells as confirmed by qPCR and
Western blotting. CDK4 is associated with D-type cyclins to promote cell-cycle entry
and progression through G1 by inactivating the retinoblastoma protein Rb (Sherr and
Roberts, 1999). SIRT3-induced CDK4 downregulation causes the prolonged G1 cell cycle
arrest that contributes to the slower growth of SIRT3-OE cells as compared to the
control cells.
To further identify factors that regulate SIRT3 stability, we isolated the SIRT3 complexes
from SIRT3-OE cells. Protein components of the SIRT3 complexes were separated on a
1D SDS-PAGE gel and changes in band intensities were identified between SIRT3-OE and
the control cells (Fig. S2). These bands were excised, digested by trypsin and analyzed
by LC-MS/MS, resulting in the identification of HSP60 as the major binding partner
of SIRT3. Similarly, immunoprecipitation of HSP60 from 293T cells was carried out
and showed that SIRT3 bound to HSP60, suggesting that SIRT3 directly interacts with
HSP60. In order to confirm that HSP60 regulates SIRT3 stability, we established the
HSP60 knockdown cells. Western blotting showed that SIRT3 was down regulated in HSP60
knockdown cells as compared to the control cells (Fig. 2F). On the other hand, qPCR
analysis revealed that the SIRT3 mRNA level was unchanged between these two cells,
suggesting that HSP60 regulated SIRT3 stability (Fig. 2G). This is consistent with
an early study showing that two murine SIRT3 isoforms interacted with HSP60 (Yang
et al., 2011). HSP60 is the major mitochondrial chaperone and is essential in maintenance
of mitochondrial proteostasis. More work is needed to examine the molecular mechanisms
of SIRT3 degradation in HSP60-knockdown cells. Nevertheless, our results propose that
HSP60 is important in the maintenance of SIRT3 protein stability.
Taken together, we demonstrate that SIRT3 overexpression disrupts mitochondrial proteostasis
that causes overproduction of ROS and the cell cycle arrest to suppress cell proliferation,
proposing that the low level expression of SIRT3 is important for tumorigenesis and
progression in ccRCC. Our data also show that HSP60 mediates the stability of SIRT3
and proteomics is a powerful approach to decipher the complex cellular processes.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary material 1 (PDF 386 kb)