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Cited by 299Metabolism, Diabetes, and CancerCancer, Hypoxia, and MetabolismPI3K/AKT/mTOR signaling in cancer

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3β-MCL-1 Axis

Open access
Elgendy, Mohamed, Cirò, Marco, Hosseini, Amir, Weiszmann, Jakob, Mazzarella, Luca, Ferrari, Elisa, Cazzoli, Riccardo, Curigliano, Giuseppe +11 more·2019·Cancer Cell
DOI 10.1016/j.ccell.2019.03.007
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Abstract

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3β (GSK3β) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3β axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3β.

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Written from the paper’s full text.

TL;DR

In mice on 24-h feeding/fasting cycles, metformin given during fasting-induced hypoglycemia synergistically suppressed tumor growth, whereas metformin alone or during feeding did not. The effect required PP2A-mediated GSK3β activation, metformin-induced CIP2A inhibition, low-glucose-induced B56δ upregulation, and subsequent MCL-1 decline.

Key findings

  • In vivo, metformin (200 mg/kg, n = 5/group) impaired tumor growth only when administered during fasting-induced hypoglycemia (Met/Fast); metformin alone had no significant tumor-restraining effect, and Met/Fast was distinct from all other groups.
  • In vitro, glucose deprivation (2.5 mM) but not serum or amino acid deprivation sensitized HCT116 and HeLa cells to metformin (5 mM); the Chou-Talalay combination index was <1, indicating synergy, and the effect was observed in several cancer cell lines and patient-derived melanoma cells.
  • The combination caused GSK3β dephosphorylation/hyperactivation, MCL-1 decline, and cell death; AMPK depletion or constitutively active AMPK did not significantly modulate the synergy, indicating AMPK independence.
  • PP2A mediated the effect: PP2A depletion prevented GSK3β dephosphorylation and MCL-1 decline and increased resistance; metformin reduced CIP2A, low glucose increased B56δ, and the combination enriched a PP2A-B56δ complex with GSK3β. Overexpression of CIP2A or MCL-1, or depletion of GSK3β or B56δ, conferred resistance in vitro and/or in vivo; PPZ/thioridazine synergized with metformin.

Methods

  • In vivo: CD1 nude mice bearing HCT116 or patient-derived melanoma xenografts were assigned to Vehicle, Met, Fed/Fast, Met/Fast, or Met/Fed groups; 24-h feeding/fasting cycles; metformin 200 mg/kg by oral gavage every 48 h during fasting or feeding; tumor volume/weight measured (n = 5 per group).
  • In vitro: HCT116, HeLa, other cancer cell lines, and patient-derived melanoma cells were cultured in nutrient-rich (10 mM glucose) or glucose-deprived (2.5 mM glucose), serum-deprived, or amino-acid-deprived conditions with metformin (5 mM or dose-response); assays included immunoblotting, cell death (trypan blue/PI), proliferation (CellTiter-Glo), qRT-PCR, immunoprecipitation, clonogenic survival, lactate/O2 consumption, and AMP/ATP.
  • Statistics: Student’s t test; Chou-Talalay combination index via Compusyn; multiple linear regression for tumor growth versus intra-tumor glucose.

Limitations

  • Stated: whether the fasting/metformin protocol is equally well tolerated in patients remains to be investigated because of differences in fat and body mass between mice and humans; other GSK3β targets (e.g., c-myc) and other pathways were not ruled out; the physiological relevance of drastic glucose deprivation is unclear.
  • Stated: intra-tumor glucose reduction did not significantly correlate with tumor weight between Met/Fast and Fast (p = 0.895) or Met/Fed (p = 0.923), so the effect was not simply further glucose reduction.
  • Evident from the text: the study is preclinical, using immunodeficient mouse xenografts and cell lines with n = 5 per in vivo group, and reports no patient outcome data.

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

Integrity signals

Registry

Crossref · 10.1016/j.ccell.2019.03.007

Cites

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Statistics

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Claims the paper makes

0 of 46 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Activation of GSK3 b Mediates the Synergistic Cytotoxicity of Low Glucose/Metformin Combination · p. 6

    The synergy between metformin and glucose deprivation depended on both metformin dose and glucose level.

    “The synergistic effect between metformin treatment and glucose deprivation depended on both metformin concentration and glucose levels (Figures 2C-2E, S2D, and S2E)”
    Quote in the paperSubstantial claim
  • Findingown data
    § A GSK3 b -Dependent Decline in MCL-1 Levels Mediates the Synergistic Cytotoxicity of the Low Glucose/ Metformin Combination · p. 8

    The rescue of cell death correlated with the level of MCL-1 overexpression.

    “The rescue of cell death reproducibly correlated with the level of MCL-1 overexpression (Figures S4C-S4H).”
    Quote in the paperSubstantial claim
  • Findingown data
    § Hypoglycemia-Metformin Combination Effectively Restrains Tumor Growth · p. 6

    In mice on 24-hour feeding/fasting cycles, tumor growth was strongly impaired in the group given metformin during fasting compared with all other groups.

    “tumor growth was dramatically impaired in the group receiving metformin while fasting (Met/Fast) as compared with all other groups (Figures 1E and 1F)”
    • Also stated in: § SUMMARY · p. 2 · plain
      “metformin impaired tumor growth only when administered during fasting-induced hypoglycemia”
    Quote in the paperSubstantial claim
  • Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    The reduction in GSK3β phosphorylation by metformin plus 2-deoxyglucose correlated with their synergistic cytotoxicity even in glucose-rich conditions.

    “which correlated with the synergistic cytotoxicity of this combination even in cells cultured in glucose-rich conditions (Figure S3K).”
    Quote in the paperSubstantial claim
  • Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    Metformin plus 2-deoxyglucose caused a similar dramatic reduction in GSK3β phosphorylation.

    “the combination of metformin and 2-deoxyglucose, a glucose analog that inhibits glycolysis, resulted in a similar dramatic reduction in GSK3 b phosphorylation (Figure S3J)”
    Quote in the paperSubstantial claim
  • May combine two claims. 37
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 26

  • May combine two claims.

    Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    GSK3β-depleted cells treated with low glucose plus metformin proliferated almost normally and did not show the cell death seen in control cells.

    “GSK3 b -depleted cells treated with low glucose plus metformin proliferated almost normally and did not show cell death observed in control cells (Figures 3E-3G and S3L)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    GSK3β dephosphorylation by the combination depended on metformin concentration and negatively correlated with glucose concentration.

    “GSK3 b de-phosphorylation by the combination depended on the concentration of metformin and negatively correlated with that of glucose (Figures 3C3D).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § Hypoglycemia-Metformin Combination Effectively Restrains Tumor Growth · p. 6

    In mice carrying HCT116 or patient-derived melanoma xenografts, metformin alone did not significantly restrain tumor growth.

    “Metformin alone did not exert any significant tumor-restraining effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    AMPK depletion in HCT116 or constitutively active AMPK in HeLa did not significantly change the synergistic cytotoxicity.

    “depletion of AMPK in HCT116 (Figures S3C-S3E) or expression of a constitutively active form of AMPK in HeLa cells (Figures S3F-S3H) failed to modulate the synergistic cytotoxicity observed in both cell lines to a significant extent.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    GSK3β dephosphorylation by the combination was consistently seen in all cells, unlike the differential effects on AMPK and ERK phosphorylation.

    “Notably, GSK3 b dephosphorylation by the low glucose/metformin combination was consistently observed in all cells, in contrast to the differential effect on AMPK and extracellular signal-regulated kinase phosphorylation (Figure 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    Only the combination dephosphorylated GSK3β; metformin or low glucose alone slightly increased GSK3β phosphorylation.

    “GSK3 b was dephosphorylated only by the combination, while either metformin or low glucose alone-if anything-slightly increased the level of GSK3 b phosphorylation (Figures 3B and S3I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Hypoglycemia-Metformin Combination Effectively Restrains Tumor Growth · p. 4

    Fasting cycles caused a large drop in blood glucose and a 1-2 g body weight loss, both returning near normal during feeding.

    “fasting cycles resulted in a strong drop in blood glucose and a reduction (1-2 g) in body weight, both of which returned to almost normal levels during the following cycle of feeding (Figures 1B and S1H).”
    Quote in the paperNot a substantial claim

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

    Findingown data
    § A GSK3 b -Dependent Decline in MCL-1 Levels Mediates the Synergistic Cytotoxicity of the Low Glucose/ Metformin Combination · p. 8

    Metformin alone and more significantly with low glucose also modulates c-myc levels.

    “metformin alone and, more significantly, in combination with low glucose, also modulates the levels of c-myc (Figure S4I)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § A GSK3 b -Dependent Decline in MCL-1 Levels Mediates the Synergistic Cytotoxicity of the Low Glucose/ Metformin Combination · p. 8

    The decline in MCL-1 correlated with GSK3β activation and depended on metformin and glucose concentrations.

    “The decline in MCL-1 levels correlated with GSK3 b activation (Figures 4A and 4B) and depended on both metformin and glucose concentrations (Figures 4C and 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Activation of GSK3 b Mediates the Synergistic Cytotoxicity of Low Glucose/Metformin Combination · p. 6

    Glucose deprivation, but not serum or amino acid deprivation, sensitized HCT116 and HeLa cells to metformin.

    “deprivation of glucose, but not serum or A.A., sensitized cells to metformin (Figures 2A, 2B, S2A, and S2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    Depletion of CIP2A was sufficient to recapitulate metformin's effects: CIP2A depletion with low glucose triggered a decline in phosphorylated GSK3β and MCL-1 and evoked cell death comparable to metformin/low glucose.

    “depletion of CIP2A was sufficient to recapitulate the effects of metformin as the combination of CIP2A depletion with low glucose triggered a decline in phosphorylated GSK3 b and MCL-1 levels and evoked cell death comparable with that observed with the metformin/low glucose treatment (Figures 6F and 6G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Formation of an Active PP2A Holoenzyme Containing B56 d Mediates Cytotoxicity of the Combination of Low Glucose with Metformin · p. 10

    Overexpression of B56δ and subsequent enrichment of a B56δ-containing PP2A holoenzyme synergized with metformin, but not SAHA or brefeldin A, to induce GSK3β dephosphorylation, MCL-1 downregulation, and cell death.

    “Overexpression of B56 d and subsequent enrichment of a B56 d -containing PP2A holoenzyme (Figure S7B) synergized with metformin, but not SAHA or brefeldin A, to induce GSK3 b dephosphorylation, MCL-1 downregulation (Figure 7F), and cell death (Figures 7G, S7C, and S7D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION · p. 13

    Metformin did not cause additional weight loss or any other signs of toxicity when combined with fasting in the Met/Fast group.

    “and metformin did not cause additional weight loss or any other signs of toxicity when combined with fasting in the Met/Fast group, which showed the most dramatic reduction in tumor growth.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § PP2A Mediates the Synergistic Cytotoxicity of Low Glucose/Metformin Combination · p. 8

    PPZ sensitized cells to metformin, but not other cytotoxic agents.

    “PPZ sensitized cells to metformin, but not other cytotoxic agents (Figures S5C-S5F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION · p. 13

    In vitro, glucose deprivation phenocopied the effect of fasting and dramatically sensitized a panel of tumor cells to metformin.

    “In vitro , glucose deprivation phenocopied the effect of fasting and dramatically sensitized a panel of tumor cells to metformin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    The combination of metformin with low glucose simultaneously reduced CIP2A and increased B56δ levels.

    “the combination of metformin with low glucose simultaneously reduced CIP2A and increased B56 d levels (Figures 6A and S6A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    Metformin mainly promoted CIP2A proteasomal degradation, as it failed to diminish CIP2A levels in the presence of proteasome inhibitors.

    “metformin mainly promoted CIP2A proteasomal degradation as it failed to diminish CIP2A levels in the presence of proteasome inhibitors (Figures S6E-S6G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Tumors Depleted of GSK3 b or Overexpressing MCL-1 and/or CIP2A Are Resistant to Metformin Administered during Fasting · p. 13

    Tumors ectopically overexpressing CIP2A were resistant to metformin/hypoglycemia combination compared with control tumors, which were sensitive to metformin (but not to cisplatin) administered during hypoglycemia.

    “Similarly, tumors ectopically overexpressing CIP2A were resistant to metformin/hypoglycemia combination compared with control tumors, which were sensitive to metformin (but not to cisplatin) administrated during hypoglycemia (Figures S8E and S8F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Tumors Depleted of GSK3 b or Overexpressing MCL-1 and/or CIP2A Are Resistant to Metformin Administered during Fasting · p. 13

    Tumors derived from those cells as well as patient-derived xenografts expressing high levels of CIP2A/MCL-1 were refractory to metformin/hypoglycemia combination in vivo, in contrast to tumor cells and tumors with low CIP2A and/or MCL-1 levels.

    “and tumors derived from those cells as well as patient-derived xenografts expressing high levels of CIP2A/MCL-1 were refractory to metformin/hypoglycemia combination in vivo in contrast to tumor cells and tumors with low CIP2A and/or MCL-1 levels (Figures S8J and S8K).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § PP2A Mediates the Synergistic Cytotoxicity of Low Glucose/Metformin Combination · p. 8

    PP2A-depleted cells were resistant to metformin-PPZ and did not show the reduction in phosphorylated GSK3β and MCL-1 seen in control cells.

    “cells depleted of PP2A were resistant to the combination (Figure S5G) and did not show the reduction of phosphorylated GSK3 b and MCL-1 levels observed in control cells treated with the combination (Figure S5H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Tumors Depleted of GSK3 b or Overexpressing MCL-1 and/or CIP2A Are Resistant to Metformin Administered during Fasting · p. 13

    Tumors derived from GSK3β-depleted or MCL-1-overexpressing cells grew similarly in both conditions and the metformin/hypoglycemia combination failed to exert a growth inhibitory effect, unlike control tumors.

    “Unlike control tumors, tumors derived from GSK3 b -depleted or MCL-1-overexpressing cells grew similarly in both conditions and metformin/hypoglycemia combination failed to exert a growth inhibitory effect (Figures 8D and 8E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Formation of an Active PP2A Holoenzyme Containing B56 d Mediates Cytotoxicity of the Combination of Low Glucose with Metformin · p. 10

    PPZ and thioridazine, which are synergistic in combination with metformin, enriched for the PP2A holoenzyme containing the B56δ subunit and increased recruitment of GSK3β.

    “Consistently, PPZ and thioridazine-which are synergistic in combination with metformin-enriched for the PP2A holoenzyme containing the B56 d subunit and increased recruitment of GSK3 b (Figures S5B and S7E).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given
    § Xenograft Tumor Models · p. 22

    In nude mice, fasting causes a strain-specific drop in glucose levels that is larger than in other commonly used mouse strains, such as C57B6.

    “Note that nude mice show a strain-specific decline in glucose levels upon fasting, of a higher degree as compared to other commonly used mouse strains (such as C57B6: data not shown).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § PP2A Mediates the Synergistic Cytotoxicity of Low Glucose/Metformin Combination · p. 8

    PP2A has been shown to regulate GSK3β activity by removing phosphorylation at serine 9 and other regulatory residues.

    “PP2A has been shown to regulate GSK3 b activity by removing phosphorylation at serine 9 and other regulatory residues (Bennecib et al., 2000; Mitra et al., 2012).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Bennecib et al., 2000 source

      Not checked in the free run.

    • cites Mitra et al., 2012 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Figure 2. Glucose Starvation Sensitizes Cancer Cells to Metformin · p. 6

    Phosphorylation of GSK3β at serine 9 inhibits its activity and is commonly used as a marker of inactive GSK3β.

    “Phosphorylation of GSK3 b at serine 9 inhibits its activity, and it is thus commonly used as a marker for the inactive GSK3 b .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cohen and Frame, 2001

      Not checked in the free run.

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

    Premisecitation
    § A GSK3 b -Dependent Decline in MCL-1 Levels Mediates the Synergistic Cytotoxicity of the Low Glucose/ Metformin Combination · p. 8

    Akt is a major kinase that phosphorylates GSK3β.

    “Akt has been established as a major kinase that phosphorylates GSK3 b (Cohen and Frame, 2001).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Cohen and Frame, 2001

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation
    § Significance · p. 4

    Because tumor cells can switch metabolic pathways, blocking only one pathway may fail.

    “Targeting one specific metabolic pathway could thus be ineffective.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    Metformin-induced downregulation of CIP2A mediates PP2A activation and can account for largely, if not all, sensitization of cells to low glucose.

    “These results thus suggest that metformin-induced downregulation of CIP2A mediates PP2A activation and can be attributed for-largely, if not all-sensitization of cells to low glucose conditions.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given
    § DISCUSSION · p. 13

    The weight loss observed is relatively modest, especially compared with the weight loss observed in mice treated with established anti-cancer drugs.

    “The weight loss observed is relatively modest, especially compared with the weight loss observed in mice treated with established anti-cancer drugs.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    CIP2A is an endogenous PP2A inhibitor overexpressed in several cancers and contributes to malignant transformation by inhibiting PP2A.

    “cancerous inhibitor of protein phosphatase 2A (CIP2A) is an endogenous PP2A inhibitor that is overexpressed in several types of cancer and contributes to malignant transformation through inhibition of PP2A (Junttila et al., 2007).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Junttila et al., 2007 source

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

    Premisecitation
    § DISCUSSION · p. 13

    Previous studies reported synergism of the metformin/low glucose combination.

    “These results are consistent with previous studies reporting the synergism of the metformin/low glucose combination (Birsoy et al., 2014; Zhuang et al., 2014).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Birsoy et al., 2014 source

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    • cites Zhuang et al., 2014 source

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    Interpretationown data
    § DISCUSSION · p. 15

    Cell death in response to the combination is mediated by specific modulation of the PP2A-GSK3β-MCL-1 axis, wherein PP2A dephosphorylates and activates GSK3β, which diminishes MCL-1 levels, leading to cell death and reduction of tumor growth.

    “Further mechanistic investigation showed that cell death in response to the combination is mediated by specific modulation of the PP2A-GSK3 b -MCL-1 axis whereas PP2A dephosphorylates and thus activates GSK3 b , which in turn acts to diminish MCL-1 levels and ultimately leading to cell death and reduction of tumor growth.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given
    § DISCUSSION · p. 13

    Both intermittent fasting and the timing of metformin administration during the fasting/feeding cycles dictate the sensitivity of tumors to metformin.

    “Our results suggest that both IF and the timing of metformin administration during the fasting/feeding cycles dictate the sensitivity of tumors to metformin.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Intermittent fasting is a clinically feasible, safe, and effective approach to lower glucose availability.
    • Rests on an assumption: Metformin is an OXPHOS inhibitor.

  • May combine two claims.

    Interpretationreasoning
    § SUMMARY · p. 2

    Specific activation of the PP2A-GSK3β axis was the sum of metformin-induced CIP2A inhibition and low glucose-induced B56δ upregulation, leading to an active PP2A-B56δ complex with high affinity toward GSK3β.

    “Mechanistically, specific activation of the PP2A-GSK3 b axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56 d by low glucose, leading to an active PP2A-B56 d complex with high affinity toward GSK3 b .”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § DISCUSSION · p. 15

    Cancer cells with reduced OXPHOS due to either mutated mitochondrial DNA or impaired glucose utilization were unable to initiate this adaptive response, and were thus particularly sensitive to glucose deprivation to one-tenth of normal levels.

    “Cancer cells with reduced OXPHOS due to either mutated mitochondrial DNA or impaired glucose utilization were unable to initiate this adaptive response, and were thus particularly sensitive to glucose deprivation to one-tenth of normal levels (Birsoy et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Birsoy et al., 2014 source

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    Premisecitation
    § DISCUSSION · p. 15

    Simultaneous targeting of multiple metabolic pathways through inhibition of nutrient uptake triggered PP2A activation.

    “it has been shown that simultaneous targeting of multiple metabolic pathways through inhibition of nutrient uptake triggered PP2A activation (Kim et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kim et al., 2016 source

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    Premisecitation
    § DISCUSSION · p. 15

    Inactivation of PP2A is an important step toward full-blown transformation.

    “the inactivation of PP2A has become widely accepted as an important step toward full-blown transformation.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Janssens et al., 2005 source

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    • cites Mumby, 2007 source

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

    Premisecitation
    § Upregulation of the PP2A Regulatory Subunit B56 d and Downregulation of CIP2A Mediate the Effects of the Low Glucose/Metformin Combination · p. 10

    PP2A is a trimeric complex of catalytic, scaffold, and regulatory B subunits; different B subunits produce holoenzymes with unique substrates and functions.

    “PP2A is a trimeric protein complex consisting of a catalytic subunit (PP2Ac or C), a scaffold subunit (PR65 or A), and one of several regulatory B subunits. Such variability in PP2A composition results in heterogeneity in the composition of active PP2A holoenzymes, each with unique substrates and functions (Janssens et al., 2005).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Janssens et al., 2005 source

      Not checked in the free run.

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

    Premisecitation
    § DISCUSSION · p. 15

    CIP2A interacts with the A subunit and impairs binding of B subunits to PP2A complexes.

    “It has also been proposed that CIP2A interacts with the A subunit and impairs binding of B subunits to PP2A complexes (Junttila et al., 2007; Khanna et al., 2013).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Junttila et al., 2007 source

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    • cites Khanna et al., 2013

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    Premisecitation
    § DISCUSSION · p. 15

    Genetic and/or functional inactivation of different PP2A subunits, and loss of its phosphatase activity, have been found in several types of tumors.

    “Indeed, genetic and/or functional inactivation of different PP2A subunits, and, therefore, loss of its phosphatase activity, have been found in several types of tumors (Janssens et al., 2005; Mumby, 2007).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Janssens et al., 2005 source

      Not checked in the free run.

    • cites Mumby, 2007 source

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

    Premisecitation
    § DISCUSSION · p. 15

    CIP2A is specifically overexpressed in numerous types of tumors, while barely detectable in normal cells.

    “In addition, PP2A inhibitor CIP2A is specifically overexpressed in numerous types of tumors, while is barely detectable in normal cells, making it a potential therapeutic target.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Junttila et al., 2007 source

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    Premisecitation
    § DISCUSSION · p. 15

    CIP2A overexpression correlates with poor prognosis in several cancer subtypes.

    “CIP2A overexpression has been shown to correlate with poor prognosis in several cancer subtypes (Junttila et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Junttila et al., 2007 source

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    Premisecitation
    § Patient-Derived Cells · p. 22

    The primary melanoma cell lines GaLa1949 and LuCa1970 were established from metastatic melanoma samples as described in an earlier publication.

    “Establishment of primary cells GaLa1949 and LuCa1970 from metastatic melanomas was previously described in (Elgendy et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Elgendy et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning
    § DISCUSSION · p. 15

    Downregulation of CIP2A by metformin leads to reduced trapping of B56 proteins and, when combined with low glucose-induced B56δ upregulation, ultimately enhances recruitment of B56δ to the PP2A complex.

    “Downregulation of CIP2A by metformin leads to reduced trapping of B56 proteins and, when combined with low glucose-induced B56 d upregulation, ultimately enhances recruitment of B56 d to the PP2A complex.”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § DISCUSSION · p. 17

    Other tumor-promoting roles of MCL-1 in autophagy and cellular bioenergetics are emerging.

    “other tumor-promoting roles in autophagy and cellular bioenergetics are emerging (Elgendy, 2017; Elgendy et al., 2014, 2017; Elgendy and Minucci, 2015; Perciavalle et al., 2012).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Elgendy, 2017

      Not checked in the free run.

    • cites Elgendy et al., 2014 source

      Not checked in the free run.

    • cites Elgendy et al., 2017

      Not checked in the free run.

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

    Premisecitation
    § DISCUSSION · p. 15

    Orally bioavailable small-molecule activators of PP2A inhibit the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models.

    “Orally bioavailable small-molecule activators of PP2A have been shown to inhibit the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models (Sangodkar et al., 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Sangodkar et al., 2017 source

      Not checked in the free run.

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

    Study design and conductown data
    § Immunoblotting · p. 23

    For immunoblots, band intensities were averaged from three independent biological replicates and reported as fold change relative to the first band with standard deviation.

    “Average intensities of the bands from three biologically-independent repeats expressed as fold change of the first band +/- SD are presented under each lane.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § DISCUSSION · p. 15

    CIP2A forms homodimers, which interact with the PP2A B56 subunits leading to CIP2A stabilization.

    “A recent study resolved the 3D structure of CIP2A (Wang et al., 2017) and showed that CIP2A forms homodimers, which interact with the PP2A B56 subunits leading to CIP2A stabilization.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Wang et al., 2017 source

      Not checked in the free run.

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

    Study design and conductnone given
    § Patient-Derived Cells · p. 22

    The patient-derived cells were grown in DMEM with 10% FBS and were periodically checked by immunohistochemistry for S100, MelanA, and HMB45 at different passages while in culture.

    “Cells were grown in DMEM+10%FBSandwereperiodicallycharacterized by immunohistochemistry staining with S100, MelanA and HMB45 antibodies at various passages during the time they are kept in culture.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § DISCUSSION · p. 15

    The B56δ-containing PP2A complex plays important tumor suppressor roles through its established function in the regulation of GSK3β dephosphorylation.

    “Particularly, the B56 d -containing PP2A complex plays important tumor suppressor roles through its established function in the regulation of GSK3 b dephosphorylation (Lambrecht et al., 2018; Haesen et al., 2016) and possibly other substrates.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Lambrecht et al., 2018 source

      Not checked in the free run.

    • cites Haesen et al., 2016 source

      Not checked in the free run.

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

    Study design and conductown data
    § Xenograft Tumor Models · p. 22

    Once tumors had formed, the mice were assigned randomly to different experimental groups.

    “After the tumors were established, mice were randomized in different groups.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given
    § Figure 4. GSK3 b -Depleted MCL-1 Degradation Mediates Synergistic Cytotoxicity of Low Glucose and Metformin · p. 10

    The results presented are representative of three biologically independent experiments.

    “Results are representative of three biologically independent experiments.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given
    § Glucose Measurement in Tumors · p. 24

    Tumor glucose was identified using purified external standards, and peak areas were normalized to internal standards and tissue fresh weight.

    “Glucose was identified by measuring purified external standards and peak areas were normalized to internal standards (Pentaerythritol and Phenyl b -D-glucopyranoside, both Sigma-Aldrich) and tissue fresh weight.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § DISCUSSION · p. 15

    GSK3β is a target of metformin.

    “GSK3 b has been shown previously to be a target of metformin (Gwak et al., 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gwak et al., 2017 source

      Not checked in the free run.

54 of 54

  • The paper relies on this without support.

    Intermittent fasting is a clinically feasible, safe, and effective approach to lower glucose availability.

    “we exploited IF as a clinically feasible, safe, andeffective approach to lower glucose availability”

  • The paper relies on this without support.

    Metformin is an OXPHOS inhibitor.

    “the OXPHOS inhibitor metformin”
2 of 2

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?

  • CIP2A drives tumour growth in xenograft and transgenic mouse models.

    “Of these, CIP2A is widely over-expressed in different human cancer types and drives tumour growth both in xenograft and transgenic mouse models18–24.”

    From Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A (2023) · cites this paper as [23]

    The section on CIP2A and metformin resistance (p. 13) reports that CIP2A-overexpressing tumors in mice resisted growth inhibition by metformin during fasting. The paper also says CIP2A contributes to malignant transformation (p. 10), but it does not show that CIP2A drives tumor growth in transgenic mice or establish that it increases baseline xenograft growth. The transgenic-model finding in the Discussion (p. 15) concerns PP2A activators, not CIP2A. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Therapeutic impact of inhibition of CIP2A protein expression has been validated by numerous studies across human cancer types.

    “therapeutic impact of inhibition of CIP2A protein expression has been validated by numerous studies across human cancer types18,20,21,23,33,34.”

    From Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A (2023) · cites this paper as [23]

    The Results (p. 10) show that CIP2A depletion, combined with low glucose, induced tumor-cell death; the in vivo Results (p. 13) link lower CIP2A levels to the tumor-restraining effect of metformin during fasting. The Discussion (p. 15) says CIP2A is overexpressed in numerous tumor types, but this paper does not establish that numerous studies have validated the therapeutic impact of inhibiting CIP2A expression across human cancer types. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • CIP2A is widely over-expressed in different human cancer types.

    “Of these, CIP2A is widely over-expressed in different human cancer types and drives tumour growth both in xenograft and transgenic mouse models18–24.”

    From Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A (2023) · cites this paper as [23]

  • CIP2A is required for malignant cellular growth in vitro and tumor formation in vivo in several cancers, and its overexpression broadly promotes cancer cell drug resistance.

    “Furthermore, CIP2A is required for malignant cellular growth in vitro and for tumor formation in vivo in a number of cancers, and its overexpression broadly promotes cancer cell drug resistance (6)(7)(8)(9)(10)(11).”

    From Discovery of a Novel CIP2A Variant (NOCIVA) with Clinical Relevance in Predicting TKI Resistance in Myeloid Leukemias (2021) · cites this paper as [7]

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.

  • Patient-derived melanoma cell line has two identifiers

    Minor

    The methods identify the patient-derived melanoma cells as LuCa1970, while the key resources table lists LuCa1973. The text does not establish whether these are different cell lines or a mismatched identifier.

1 of 1

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Authors
Elgendy, M., Cirò, M., Hosseini, A., Weiszmann, J., Mazzarella, L., Ferrari, E., Cazzoli, R., Curigliano, G., DeCensi, A., Bonanni, B., Budillon, A., Pelicci, P. G., Janssens, V., Ogris, M., Baccarini, M., Lanfrancone, L., Weckwerth, W., Foiani, M., & Minucci, S.
Affiliations
  • European Institute of Oncology (IT)
  • IFOM (IT)
  • University of Vienna (AT)
  • Ente Ospedaliero Ospedali Galliera (IT)
  • Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale" (IT)
  • University of Milan (IT)
  • KU Leuven (BE)
Corresponding author
  • Elgendy, Mohamed
  • Minucci, Saverio
ORCID
Venue
Cancer Cell
Publisher
Elsevier BV
Published
2019-05
Volume
35
Issue
5
Pages
798-815.e5
ISSN
1535-6108 (ISSN-L)
DOI
10.1016/j.ccell.2019.03.007
OpenAlex
W2941131991
Semantic Scholar
29d6222ca10f501757c49fddd7056d6bca81ff03
Cited by
299
References
49
Citations by year
2026: 15 · 2025: 33 · 2024: 43 · 2023: 59 · 2022: 53 · 2021: 53 · 2020: 29 · 2019: 14
FWCI
12.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Lirias (KU Leuven) (submittedVersion, other-oa)
  • Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
Funders
  • Associazione Italiana per la Ricerca sul Cancro
  • FP7 People: Marie-Curie Actions
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
Keywords
metformin, hypoglycemia, intermittent fasting, oxidative phosphorylation, glycolysis, protein phosphatase 2A, GSK3β, Mcl-1, CIP2A, tumor growth, cell death, glucose availability
MeSH terms
Glycogen Synthase Kinase 3 beta, Animals, Cell Survival, Fasting, Glycolysis, HeLa Cells, Humans, Hypoglycemia, Metformin, Neoplasms, Oxidative Phosphorylation, Signal Transduction, Gene Expression Regulation, Neoplastic, Xenograft Model Antitumor Assays, HCT116 Cells, Cell Line, Tumor, Cell Proliferation, Mice, Protein Phosphatase 2, Myeloid Cell Leukemia Sequence 1 Protein

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Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

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