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Cited by 440Sirtuins and Resveratrol in MedicineCancer, Hypoxia, and MetabolismCancer, Lipids, and Metabolism

SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

Open access
Bell, E L, Emerling, B M, Ricoult, S J H, Guarente, L·2011·Oncogene
DOI 10.1038/onc.2011.37PMC PMC3134877
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Abstract

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α.

AI snapshot

Written from the paper’s full text.

TL;DR

SirT3 acts as a tumor suppressor by suppressing mitochondrial complex III ROS, thereby limiting HIF-1α stabilization and transcriptional activity. Loss of SirT3 increases proliferation, hypoxic HIF-1α activation, and xenograft tumor growth, while SirT3 overexpression inhibits HIF-1α and tumorigenesis.

Key findings

  • SirT3 knockdown in 143B and HCT116 cancer cells increased proliferation and mildly increased normoxic HRE-luciferase activity; SirT3−/− MEFs also showed increased population doublings versus wild-type controls.
  • In hypoxia (1% O2), SirT3 knockdown strikingly increased HIF-1α protein and HRE-luciferase activity: control cells showed a 13-fold increase, whereas SirT3 knockdown cells showed a 70-fold increase. Endogenous HIF-1α targets VEGF-A, PGK-1, and PDK-1 were also further increased by SirT3 loss in hypoxia, but not in normoxia.
  • SirT3 overexpression prevented HIF-1α protein stabilization in 1% O2 and attenuated HRE-luc and PGK1 responses; DMOG restored HIF-1α stabilization, implicating proline hydroxylation.
  • HCT116 SirT3 knockdown xenografts grew faster and had greater final mass and increased VEGF-A mRNA; NAC treatment abolished these increases. Inducible SirT3 overexpression in HCT116/143B decreased proliferation, and doxycycline-induced SirT3 after tumor initiation produced smaller tumors and decreased tumorigenesis rate/final mass.

Methods

  • In vitro studies used primary MEFs from E13.5 SirT3+/− × SirT3+/− crosses and established human cancer lines 143B and HCT116 with SirT3 knockout, shRNA knockdown, or stable/inducible overexpression; hypoxia was 1% O2.
  • Main assays included HRE-luciferase reporter normalized to Renilla, quantitative real-time PCR for HIF-1α targets, western blots for HIF-1α, ROS measurement by dihydroethidium flow cytometry, and treatments with NAC, stigmatellin, antimycin A, or DMOG.
  • Xenografts used 5 × 10^6 HCT116 cells injected into either flank of 7-week-old Nu/Nu mice; tumor volume was calculated as V = L × W² × 0.52, with NAC in drinking water or doxycycline-containing food in induction experiments.

Limitations

  • The paper does not state a dedicated limitations section.
  • Evident from the text: the mechanism linking SirT3 to complex III ROS is inferred from antioxidant/stigmatellin experiments; the authors note it remains to be determined whether complex III subunits are direct SirT3 targets or whether their acetylation alters ROS generation.
  • Evident from the text: CITED2 levels differed between 143B and HCT116 SirT3 knockdown cells, so the authors state HIF effects cannot be fully explained by that mechanism; constitutive SirT3 overexpression was selected against in HCT116, requiring an inducible system; and the authors note their NAC-treated control tumors formed, unlike a prior study, possibly due to different NAC pretreatment timing.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/onc.2011.37

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 21 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    SirT3 knockdown in 143B and HCT116 cells does not alter protein levels of MnSOD, catalase, or SOD1.

    “Knocking down SirT3 in both the 143B- and HCT116-established cancer cell lines did not alter protein levels of MnSOD or the cytosolic antioxidants catalase and SOD1 (Supplementary Figure S2).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
  • Findingown data

    Tumors formed by SirT3 knockdown HCT116 cells have increased mRNA levels of VEGF-A.

    “The tumors formed by the injected cells maintained the SirT3 knockdown level of expression and also had increased mRNA levels of the pro-angiogenic HIF-1α target VEGF-A (Figure 6e), indicating that HIF-1α activity is upregulated in these tumors.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
    • Rests on an assumption: Xenograft tumor growth in Nu/Nu mice is a valid model for human tumorigenesis.
  • Findingown data

    NAC treatment prevents the increase in ROS in 143B and HCT116 cells with stable SirT3 knockdown.

    “The 143B and HCT116 cells that have SirT3 stably knocked down were treated with N-acetyl-cysteine (NAC), and the increase in ROS was prevented (Supplementary Figure S1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
  • Findingown data

    NAC treatment decreases the normoxic increase in HIF-1α transcriptional activity in 143B and HCT116 SirT3 knockdown cells.

    “NAC treatment also decreased the normoxic increase of HIF-1α transcriptional activity in 143B and HCT116 SirT3 knockdown cells (Figures 5b and c).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
    • Rests on an assumption: The effects of NAC in these experiments are due to its antioxidant activity (reduction of ROS) rather than other effects.
    • Rests on an assumption: The HRE-luc reporter construct reflects endogenous HIF-1α transcriptional activity.
  • Findingown data

    SirT3 knockdown in 143B and HCT116 cancer cell lines increases proliferation.

    “We knocked down SirT3 in the human osteosarcoma cell line 143B (Figure 1c) and the human colon carcinoma cell line HCT116 with two different short-hairpin (sh)RNAs (Figure 1d), and found that both knockdown cell lines displayed increased proliferation (Figures 1c and d).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
  • May combine two claims. 8
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingown data

    NAC-treated scramble shRNA cells formed tumors, inconsistent with a previous study.

    “Interestingly, our NAC treated scramble shRNA expressing cells did form tumors, which is not consistent with the aforementioned study.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gao et al., 2007 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Overexpression of SirT3 decreases basal ROS levels and the increased ROS levels mediated by antimycin A.

    “Further, overexpression of SirT3 decreased basal ROS levels, as well as the increased ROS levels mediated by antimycin A (Figure 4d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Induction of SirT3 decreases the rate of tumorigenesis and final tumor mass in HCT116 xenografts.

    “The rate of tumorigenesis was decreased, as well as the final tumor mass, with the cells that stably induced SirT3 expression (Figures 7c and d).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Xenograft tumor growth in Nu/Nu mice is a valid model for human tumorigenesis.
    • Rests on an assumption: The doxycycline-inducible system specifically induces SirT3 without off-target effects.

  • The quote only partly states this claim.

    Findingown data

    SirT3 knockdown in 143B and HCT116 cells mildly increases HRE-luciferase activity under normoxic conditions.

    “Both the 143B and HCT116 SirT3 knockdown cell lines showed a mild increase in luciferase activity when transfected with HRE-luc (Figures 1e and f).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
    • Rests on an assumption: The HRE-luc reporter construct reflects endogenous HIF-1α transcriptional activity.

  • May combine two claims.

    Findingown data

    In the presence of NAC, tumors from SirT3 shRNA cells do not display an increase in growth rate, final mass, or VEGF-A mRNA.

    “In the presence of NAC, the tumors derived from the SirT3 shRNA cells did not display an increase in the rate of tumor growth (Figure 6c), final mass (Figure 6d) or VEGF-A mRNA (Figure 6e).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.
    • Rests on an assumption: The effects of NAC in these experiments are due to its antioxidant activity (reduction of ROS) rather than other effects.
    • Rests on an assumption: Xenograft tumor growth in Nu/Nu mice is a valid model for human tumorigenesis.

  • May combine two claims.

    Premisecitation

    SirT3−/− Myc/Ras MEFs exhibit increased glycolysis, decreased oxidative phosphorylation, and increased ROS.

    “Kim and colleagues also demonstrated that SirT3−/− Myc/Ras MEFs exhibit increased glycolysis, decreased oxidative phosphorylation and increased ROS.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kim et al., 2010 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    SirT3 deficiency increases ROS to activate HIF-1α and facilitate tumorigenesis.

    “This data implies that SirT3 deficiency increases ROS to activate HIF-1α and facilitate tumorigenesis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The effects of NAC in these experiments are due to its antioxidant activity (reduction of ROS) rather than other effects.

  • Classified as probably not a substantial claim.

    Premisecitation

    SirT3 deficiency alone is sufficient for transformation of primary MEFs with Myc and/or RAS to form tumors in xenograft models.

    “Specifically, SirT3 deficiency alone is sufficient for the transformation of primary MEFs with Myc and/or RAS to from tumors in xenograft models.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kim et al., 2010 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The discrepancy with the previous study may be explained by pretreatment timing of NAC.

    “This discrepancy may be explained by the fact that in that study they pretreated mice with NAC one week before injection whereas we started treating with NAC on the day of injection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    ROS are necessary for hypoxic activation of HIF-1α; a mitochondrial-targeted antioxidant prevents HIF-1α protein stabilization during hypoxia.

    “These ROS are necessary for hypoxic activation, as when a mitochondrial-targeted antioxidant is added during hypoxia HIF-1α protein is not stabilized.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    FOXO3a regulates expression of HIF-1α transcriptional inhibitor CITED2.

    “Previous reports have demonstrated that the increase in ROS in the absence of SirT3 inhibits FOXO3a activity (Sundaresan et al., 2009) and FOXO3a regulates expression of the HIF-1α transcriptional inhibitor CITED2 (Bakker et al., 2007), thus it is possible that CITED2 may have a role in our results.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bakker et al., 2007 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The HIF pathway has a role in life span of C. elegans.

    “Numerous reports have indicated that the HIF pathway has a role in life span of C. elegans (Chen et al., 2009; Zhang et al., 2009).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Chen et al., 2009 source

      Not checked in the free run.

    • cites Zhang et al., 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    SirT1 negatively regulates HIF-1α activity in hypoxia by direct deacetylation.

    “SirT1 was recently demonstrated to negatively regulate HIF-1α activity in hypoxia by direct deacetylation (Lim et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lim et al., 2010 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    SirT3 has been demonstrated to act as a tumor suppressor by negatively regulating MnSOD/ROS-mediated genomic instability.

    “Previously, SirT3 has been demonstrated to act as a tumor suppressor by negatively regulating MnSOD/ROS-mediated genomic instability (Kim et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kim et al., 2010 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The increase in ROS in absence of SirT3 inhibits FOXO3a activity.

    “Previous reports have demonstrated that the increase in ROS in the absence of SirT3 inhibits FOXO3a activity (Sundaresan et al., 2009) and FOXO3a regulates expression of the HIF-1α transcriptional inhibitor CITED2 (Bakker et al., 2007), thus it is possible that CITED2 may have a role in our results.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sundaresan et al., 2009

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SirT6 represses HIF-1α-mediated transcription by binding to chromatin on HREs and attenuating activation of glycolytic genes via deacetylating histones around promoters.

    “In addition, SirT6 has been shown to repress HIF-1α-mediated transcription by binding to chromatin on HREs and attenuating the activation of glycolytic genes via deacetylating histones around the promoters (Zhong et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Zhong et al., 2010 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Two enzymes involved in ROS detoxification, MnSOD and IDH2, are direct targets of SirT3.

    “Recently, two enzymes that have a role in the detoxification of ROS were demonstrated to be direct targets of SirT3 (Qiu et al., 2010; Someya et al., 2010; Tao et al., 2010).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Qiu et al., 2010 source

      Not checked in the free run.

    • cites Someya et al., 2010 source

      Not checked in the free run.

    • cites Tao et al., 2010 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    HIF-1α activates metabolic genes such as PGK-1 and PFK1A, which increase glycolysis for ATP production during hypoxia.

    “HIF-1α also activates metabolic genes, such as PGK-1 and PFK1A, which increase glycolysis for ATP production during hypoxia (Semenza, 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Semenza, 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    NAC administration completely inhibits tumor formation in xenograft models.

    “Previously it was reported that NAC administration completely inhibits tumor formation in xenograft models (Gao et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gao et al., 2007 source

      Not checked in the free run.

19 of 19

  • The paper relies on this without support.

    SirT3 knockdown by shRNA specifically reduces SirT3 without off-target effects.

    “We knocked down SirT3 in the human osteosarcoma cell line 143B (Figure 1c) and the human colon carcinoma cell line HCT116 with two different short-hairpin (sh)RNAs (Figure 1d), and found that both knockdown cell lines displayed increased proliferation (Figures 1c and d).”

  • The paper relies on this without support.

    The effects of NAC in these experiments are due to its antioxidant activity (reduction of ROS) rather than other effects.

    “Strikingly, addition of NAC to primary SirT3−/− MEFs abolished the increase in population doublings observed in the primary SirT3−/− MEFs (Figure 5a).”

  • The paper relies on this without support.

    The HRE-luc reporter construct reflects endogenous HIF-1α transcriptional activity.

    “Using a synthetic HIF-1α reporter construct (HRE-luc) that consists of three putative HRE sites upstream of firefly luciferase, we found that MEFs displayed mildly elevated HIF-1α transcriptional activity in the absence of SirT3 (Figure 1b).”

  • The paper relies on this without support.

    Xenograft tumor growth in Nu/Nu mice is a valid model for human tumorigenesis.

    “When HCT116 cells with stable knockdown of SirT3 were injected into the flanks of Nu/Nu mice, they formed tumors with an increased rate of growth and increased final mass compared with control HCT116 cells expressing a scrambled shRNA injected in the same mouse on the opposite flank (Figures 6a–d).”

  • The paper relies on this without support.

    The doxycycline-inducible system specifically induces SirT3 without off-target effects.

    “When induced by the addition of doxycycline, both the 143B and HCT116 cells that expressed SirT3 had decreased proliferation compared with the GFP cells and the un-induced cells (Figures 7a and b).”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • SIRT3 has been reported as pro-apoptotic, and SIRT3 loss is associated with a tumor-permissive environment via inhibition of ROS production.

    “SIRT3 has been reported as pro-apoptotic ((Allison and Milner 2007)) and SIRT3 loss is associated with a tumor-permissive environment via the inhibition of ROS production ((Kim et al. 2010);(Bell et al. 2011);(Finley et al. 2011)).”

    From The Mitochondrial Acylome Emerges: Proteomics, Regulation by Sirtuins, and Metabolic and Disease Implications (2018) · cites this paper as Bell et al. 2011

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Two different references share the same article identifiers

    Minor

    References 13 and 14 list different authors, years, journals, and page ranges but assign both papers the same title, PMID, and DOI.

  • SirT3's effect on hypoxic HIF-1α stabilization is reversed in the Discussion

    Minor

    The Discussion says SirT3 stimulates greater HIF-1α protein stabilization in hypoxia, whereas the Results say SirT3-expressing cells failed to stabilize it under hypoxia.

2 of 2

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Related

Authors
Bell, E. L., Emerling, B. M., Ricoult, S. J. H., & Guarente, L.
Affiliations
  • Massachusetts Institute of Technology (US)
  • Beth Israel Deaconess Medical Center (US)
Corresponding author
Guarente, L
ORCID
Venue
Oncogene
Publisher
Springer Science and Business Media LLC
Published
2011-02-28
Volume
30
Issue
26
Pages
2986-2996
ISSN
  • 0950-9232 (ISSN-L)
  • 1476-5594
DOI
10.1038/onc.2011.37
PMC
PMC3134877
OpenAlex
W1585889626
Semantic Scholar
da64f22fc3684471cb1cca187aa23660811da2f5
Cited by
440
References
43
Citations by year
2026: 9 · 2025: 18 · 2024: 14 · 2023: 20 · 2022: 19 · 2021: 29 · 2020: 25 · 2019: 29 · 2018: 30 · 2017: 34 · 2016: 40 · 2015: 35 · 2014: 40 · 2013: 46 · 2012: 39
FWCI
21.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Oncogene (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
  • PMC
Funders
  • Glenn Foundation for Medical Research
  • National Institutes of Health
  • National Institute on Aging (R01AG015339, R01AG011119)
Topics
  • Health Sciences › Medicine › Geriatrics and Gerontology › Sirtuins and Resveratrol in Medicine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Lipids, and Metabolism
Keywords
SIRT3, sirtuins, mitochondrial sirtuins, hypoxia-inducible factor-1α, reactive oxygen species, tumor suppression, carcinogenesis, hypoxia, N-acetylcysteine, cancer metabolism, tumor xenografts
MeSH terms
Animals, Cells, Cultured, Embryo, Mammalian, Humans, Mice, Nude, Mitochondria, Neoplasms, Down-Regulation, Gene Expression Regulation, Neoplastic, Reactive Oxygen Species, Xenograft Model Antitumor Assays, RNA, Small Interfering, HCT116 Cells, Cell Proliferation, Mice, Hypoxia-Inducible Factor 1, alpha Subunit, Sirtuin 3

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