Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes
the propensity for most cancer cells to take up glucose avidly and convert it primarily
to lactate, despite available oxygen 1,2. Notwithstanding the renewed interest in
the Warburg effect, cancer cells also depend on continued mitochondrial function for
metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP
and lactate3. Glutamine, which is highly transported into proliferating cells4,5,
is a major source for energy and nitrogen for biosynthesis, and a carbon substrate
for anabolic processes in cancer cells, but the regulation of glutamine metabolism
is not well understood1,6. Here, we report that the c-Myc (or Myc) oncogenic transcription
factor, which is known to regulate miRNAs7,8 and stimulate cell proliferation9, transcriptionally
represses miR-23a and miR-23b, resulting in greater expression of their target protein,
mitochondrial glutaminase (GLS). This leads to up-regulation of glutamine catabolism10.
GLS converts glutamine to glutamate that is further catabolized through the TCA cycle
for the production of ATP or serves as substrate for glutathione synthesis11. The
unique means by which Myc regulates GLS reported herein uncovers a previously unsuspected
link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive
oxygen species (ROS) homeostasis.
Oncogenes and tumor suppressors have now been linked to the regulation of glucose
metabolism, thereby connecting genetic alterations in cancers to their glucose metabolic
phenotype1,2. In particular, the MYC oncogene produces Myc protein that directly regulates
glucose metabolic enzymes as well as genes involved in mitochondrial biogenesis9,12.
In this regard, we sought to determine the role of the MYC in altering the mitochondrial
proteome to further understand regulation of tumor metabolism. We studied the human
P-493 B cells that bear a tetracycline-repressible MYC construct, such that tetracycline
withdrawal results in rapid induction of Myc and mitochondrial biogenesis, followed
by cell proliferation12,13. By comparing the mitochondrial proteome from tetracycline-treated
and untreated cells with high Myc expression, we found 8 mitochondrial proteins that
are distinctly differentially expressed in response to Myc (Figures 1a, 1b and Tables
S1). We note that mitochondrial glutaminase or GLS (MW∼58kDa) was increased ∼10-fold
in response to Myc. As such, we determined the response of glutaminase to Myc induction
in a time-course study using anti-GLS antibody10 (Figure 1c) and found that GLS levels
diminish with decreased Myc expression and recover upon Myc re-induction. However,
the level of the mitochondrial protein TFAM remained virtually unaltered. GLS levels
also correlate with Myc levels in another human B cell line (CB33) and one (CB33-Myc)
with constitutive Myc expression14. Since human prostate cancer is linked to Myc expression15,
we sought to determine whether reduction of Myc expression by siRNA in the human PC3
prostate cancer cell line was also associated with reduction of GLS expression (Figure
1d). Similar to the human lymphoid cells, the PC3 cells also displayed a correlation
between Myc and GLS levels.
We then sought to determine whether the dramatic alteration of GLS levels in response
to Myc is functionally linked to Myc-induced cell proliferation. While there are two
major known tissue-specific GLS isoforms, GLS1 and GLS216,17, our data showed that
only GLS1 is predominantly expressed in P493-6 or PC3 cells (Figure S1). We first
determined whether gain of GLS1 function through overexpression in PC3 cells would
rescue the diminished growth rate associated with siRNA-mediated reduction of Myc
(Figure S2) and found that ectopic GLS1 expression alone is insufficient to stimulate
growth. In light of the observation that no single gene could substitute for Myc 18,19
and that Myc is a pleiotropic transcription factor9, this outcome was not particularly
surprising. As such, we reduced the expression of GLS1 (herein referred to as GLS)
by RNA interference (siGLS) and found that P-493-6 cell proliferation is markedly
attenuated by siGLS but not by control siRNA (Figure 2a). Likewise, proliferation
of the human PC3 prostate cancer cell line was diminished by siGLS (Figure 2a), indicating
that GLS is necessary for cell proliferation.
Because glutamine is converted by GLS to glutamate for further catabolism by the TCA
cycle and previous studies indicate that overexpression of Myc sensitizes human cells
to glutamine withdrawal induced apoptosis11, we determined the metabolic responses
of P493-6 or PC3 cells to glutamine deprivation (Figure 2b). The growth of both cell
lines was diminished significantly by glutamine withdrawal and moderately with glucose
withdrawal. Glutamine withdrawal also resulted in a decrease in ATP levels (Figure
2c) associated with a diminished cellular oxygen consumption rate (Figures S3a and
S3b). Reduction of GLS by RNAi also reduced ATP levels (Figure 2d). Because glutamine
is a precursor for glutathione20, glutathione levels were measured by flow cytometry
and were found diminished with glutamine withdrawal or RNAi mediated reduction of
GLS (Figure S4 and Table S2) that is also associated with an increase in ROS levels
(Figure S3c) and cell death in the P493-6 cells (Figures 2e and S5). It is notable
shortly after the MYC proto-oncogene was discovered, that the MC29 retrovirus which
bears the v-myc oncogene was found to enhance glutamine catabolism and mitochondrial
respiration in transplantable avian liver tumor cells21. Thus, our findings functionally
link historical observations with Myc, glutaminase and glutamine metabolism.
Since GLS catabolizes glutamine for ATP and glutathione synthesis, its reduction affects
proliferation and cell death presumably through depletion of ATP and augmentation
of ROS, respectively. Hence, we sought to rescue the P493-6 cells with the TCA cycle
metabolite oxaloacetate (OAA) and the oxygen radical scavenger N-acetylcysteine (NAC)11.
Both OAA and NAC partially rescued the decreased proliferation and death of P493-6
cells deprived of GLS (Figures 2e and S6). Likewise, OAA and NAC both partially rescued
glutamine-deprived P493-6 cells (Figures S5 and S6). These findings support the notion
that glutamine catabolism through GLS is critical for cell proliferation induced by
Myc and protection against ROS generated by enhanced mitochondrial function in response
to Myc11,20.
Given that GLS is critical for cell proliferation and is induced by Myc, we determined
the mechanism by which Myc regulates GLS. Because Myc is a transcription factor9,
we hypothesized that Myc transactivates GLS directly as a target gene. Despite the
presence of a canonical Myc binding site (5′-CACGTG-3′) in the GLS gene intron 1,
GLS mRNA levels do not respond to alterations in Myc levels in the P493-6 cells, suggesting
that GLS is regulated at the post-transcriptional level (Figure 3a). As such, we hypothesized
that GLS could be regulated by miRNAs that are in turn directly regulated by Myc.
The TargetScan algorithm predicts that miR-23a and miR-23b could target the GLS 3′UTR
seed sequence. Intriguingly, our earlier studies uncovered that both miR-23a and miR-23b
are suppressed by Myc in P493-6 cells7, and both miR-23s are decreased in human prostate
cancers22, which are associated with elevated Myc expression15.
To verify that miR-23a and miR-23b (herein referred to as miR-23) are suppressed by
Myc and could be diminished by antisense miR-23 locked nucleic acid (LNA) oligomers,
northern analysis was performed and miR-23 was suppressed by Myc and profoundly diminished
by antisense miR-23 LNAs (Figure 3b). Using quantitative real-time PCR, we document
(Figure S7) that miR-23 levels increased with diminished Myc expression and then decreased
upon Myc re-induction in a manner that is compatible with the GLS protein levels seen
in Figure 1c. We also found an inverse relationship between Myc and the levels of
miR-23a and miR-23b in the CB33 human lymphoid cells and PC3 prostate cancer cell
line (Figure S8). Furthermore, Myc directly bound the transcriptional unit, C9orf3,
encompassing miR-23b, as demonstrated for other Myc miRNA targets7 by chromatin immunoprecipitation
(Figure 3c). Because the transcriptional unit involving miR-23a has not been mapped,
we did not study miR-23a in this context. These observations document that Myc represses
miR-23a and miR23b, which appear to be directly regulated by Myc.
We determined next whether miR-23 targets and inhibits the expression of GLS through
the 3′UTR. In this regard, we cloned the 3′UTR sequence of GLS including the predicted
binding site for miR-23 to the pGL3 luciferase reporter vector and transfected MCF-7
cells, which is known to express miR-2323. The GLS 3′UTR inhibited luciferase activity
in a fashion that was blocked by co-transfection with the antisense miR-23 LNAs, but
not with control LNAs (Figure 3d). We next mutated the predicted binding site by a
site-directed mutagenesis strategy8 and observed that mutant 3′UTR did not inhibit
luciferase activity as the wild-type sequence did. Using these reporters in PC3 cells,
we observed that siRNA-mediated diminished Myc expression resulted in decreased luciferase
activity with wild-type but not with the mutant 3′UTR reporter (Figure S9). Most importantly,
diminished GLS protein level, which follows decreased Myc expression (Figure 1c),
was rescued by antisense miR-23 LNAs (Figure 3e). The antisense miR-23 LNAs also partially
rescued the diminished GLS level associated with RNAi-mediated reduction of Myc expression
in the PC3 cells (Figure 3e).
We also examined events upstream and downstream of GLS24 and found that the glutamine
transporter SLC7A5 is induced by Myc in P493-6 cells at the transcriptional level
(5-fold by nuclear run-on, unpublished data) with a >7-fold induction of its mRNA
level. The glutamine transporter ASCT2 is induced by Myc at the mRNA level by 2-fold,
whereas glutamate dehydrogenase mRNA levels appear unaltered (unpublished data). Furthermore,
we found that elevated levels of Myc protein in human prostate cancer correspond to
levels of GLS, which was not increased in the accompanying normal tissue from the
same patients (Figure S10). Intriguingly, miR-23a and miR-23b are significantly decreased
in human prostate cancer as compared with normal prostate tissue22. It is notable
that loss of GLS function by antisense suppression significantly inhibited the tumorigenesis
of Ehrlich ascites tumor cells in vivo25. Our findings here uncover a pathway by which
Myc suppression of miR-23, which targets GLS, enhances glutamine catabolism through
increased mitochondrial glutaminase expression. Taken together, these observations
provide a regulatory mechanism involving Myc and miRNAs for elevated expression of
glutaminase and glutamine metabolism in human cancers.
Methods Summary
Human cell lines were cultured under standard conditions. Isolation of mitochondria,
enrichment for mitochondrial proteins, and proteomic analysis were performed as described26-29.
RNA interference experiments and luciferase reporter analysis of miRNA activity were
as reported8,30. Flow cytometric analyses of reactive oxygen species, cell death and
glutathione level were performed as described11,30. Human samples were acquired with
the approval of the Johns Hopkins University School of Medicine Institutional Review
Board.
Supplementary Material
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