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Cited by 31Cancer-related Molecular PathwaysImmune Response and InflammationImmunotherapy and Immune Responses

Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells

Open access
Menendez, Daniel, Lowe, Julie M., Snipe, Joyce, Resnick, Michael A.·2016·Oncotarget
DOI 10.18632/oncotarget.11210PMC PMC5308678
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Abstract

// Daniel Menendez 1 , Julie M. Lowe 1, 2 , Joyce Snipe 1 , Michael A. Resnick 1 1 Genome Integrity & Structural Biology Laboratory, Inflammation Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA 2 Immunity, Inflammation Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA Correspondence to: Daniel Menendez, email: menendez@niehs.nih.gov Michael A. Resnick, email: resnick@niehs.nih.gov Keywords: apoptosis, poly (I:C), innate immunity, mutant p53, cancer therapy Received: May 30, 2016 Accepted: July 19, 2016 Published: August 11, 2016 ABSTRACT Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes ( TLRs ) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

AI snapshot

Written from the paper’s full text.

TL;DR

Cancer-associated p53 mutants differentially regulate endogenous TLR gene expression in p53-null human cancer cells, and some mutants enhance TLR3 signaling, cytokine/chemokine induction, and poly(I:C)-triggered apoptosis. Reactivation of loss-of-function mutant p53 with RITA restores TLR3 expression and synergizes with DNA-damaging drugs to amplify TLR3-mediated immune and apoptotic responses.

Key findings

  • Among 25 p53 mutants tested in p53-null HCT116 and SaOS2 cells, WT p53 induced 7 of 10 TLR genes in SaOS2 and 4 in HCT116; some mutants retained WT-like activity, while others showed null, limited, or change-of-spectrum transactivation. The loss-of-function mutants R175H, G245D, G245S, R273C, and G279E did not induce any TLR genes in either cell line.
  • For TLR3, mutants P151H, H178Y, G279R, and R337H bound the TLR3 promoter and increased TLR3 mRNA/protein similarly to WT p53, whereas A138V did not. With poly(I:C), WT p53, P151H, and R337H significantly induced IL-8, IFN-β, and IL-6 mRNA and IL-6 secretion, while A138V and G245S showed only slight induction.
  • Poly(I:C) increased apoptosis in P151H- and R337H-expressing cells but not in A138V- or G245S-expressing cells; DXR co-treatment further enhanced poly(I:C)-induced apoptosis.
  • In RAJI cells carrying loss-of-function p53 alleles R213Q and Y234H, RITA increased TLR3, p21, and PUMA expression, whereas PRIMA-1 increased p21 and PUMA but not TLR3. RITA plus DXR or 5FU increased TLR3 expression and poly(I:C)-induced cytokine responses; pifithrin-α reduced TLR3, p21, and PUMA to baseline. Poly(I:C) plus RITA+DXR reached approximately 80% apoptosis at 24 h.

Methods

  • Human cancer cell lines included HCT116 p53−/− and p53+/+, SaOS2, RAJI, SKBR3, SW620, and MDA-MB-231. p53-null cells were transiently transfected with WT p53 or 25 p53 mutants, including 21 cancer-associated mutants.
  • Assays included qPCR for TLRs and p53 targets, ChIP for p53 occupancy at TLR3 and p21 promoters, luciferase reporter assays, western blotting, IL-6 ELISA, Annexin V/PI apoptosis detection, and CellTiter proliferation assays.
  • Drug treatments included DXR, 5FU, RITA, PRIMA-1MET, poly(I:C), pifithrin-α, and Z-VAD-fmk; data were analyzed by two-way ANOVA with p<0.001 considered significant.

Limitations

  • The paper states that rodents lack direct p53 responsiveness of the mouse TLR gene family, precluding their use for developing human therapies based on p53 modulation of TLR responses, and that mechanisms of action for many p53-reactivating small molecules remain to be clarified.
  • Evident from the text: most findings rely on transient transfection in human cancer cell lines, with a limited set of p53 mutants and cell lines, and no in vivo tumor or patient data are presented.
  • The paper notes that manipulation of TLR signaling in cancer must be done judiciously and that choices of TLR targets would be tumor-specific and depend on p53 WT or mutant status.

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

Integrity signals

Registry

Crossref · 10.18632/oncotarget.11210

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

28 of 46 cited sources checked · 13 supported · 2 flagged · accuracy not yet measured

  • Findingcitation

    TLR4 was only expressed in SaOS2 cells.

    “Consistent with our previous findings, TLR8 mRNA was not detected in either cell type while TLR4 was only expressed in SaOS2 cells [25].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] Resnick, 2012, The human TLR innate immune gene family is differentially influenced by DNA stress and p53 status in cancer cells, Cancer research source

      The Results section on p53 modulation of TLR genes says that all TLRs except TLR8 are expressed in U2OS cells, which includes TLR4. It also reports increased TLR4 mRNA in SaOS2 cells, so expression was not limited to SaOS2. (LLM, conf=0.91)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    The RAJI cell line harbors the loss-of-function mutant alleles R213Q and Y234H.

    “The cell line RAJI, which harbors the loss-of-function mutant alleles R213Q and Y234H [34–36] for p53 targets including TLR3 and p21 (Supplementary Figure S4A), was treated with the reactivating drugs RITA [36] and PRIMA-1 [37] as well as with the anticancer drugs doxorubicin (DXR) and 5-fluorouracil (5FU) to induce p53.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [34] Tock, 1993, Alternative splicing of the p53 tumor suppressor gene in the Molt-4 T-lymphoblastic leukemia cell line, Cancer Lett source
    • cites [35] Crook, 1991, p53 is frequently mutated in Burkitt's lymphoma cell lines, EMBO J source

      Table I (p. 2880) identifies Raji’s p53 mutation as R213Q, but does not list Y234H. The functional-analysis passage reports that Raji mutant p53 lost suppressor activity in one assay but retained it in another, so the unqualified description of both alleles as loss-of-function is not supported. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [36] Selivanova, 2010, Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA, Cell cycle source

      Table 1, in the section on RITA’s growth suppression, identifies Raji as carrying mutant p53 but lists its mutations as “Arg213, thr234,” not the claimed R213Q and Y234H. The source therefore supports that Raji has p53 mutations at those positions, but gives different amino-acid identities. (LLM, conf=0.84)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    TLR3 can directly trigger apoptosis in human cancer cells in response to poly(I:C).

    “TLR3 can directly trigger apoptosis in human cancer cells in response to the double-strand RNA synthetic agonist polyinosinic-polycytidylic acid (poly(I:C)) [28, 29], which is a mimic for viral double-stranded RNA that triggers an innate immune response following viral infection.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [28] Riccioli, 2008, Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC-alpha-dependent mechanism, Carcinogenesis source
    • cites [29] Kai, 2010, TLR3 induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis, Cancer Sci source
  • Findingown data

    RITA and PRIMA-1 increased the expression of the p53 targets p21 and PUMA in RAJI cells.

    “As shown in Figure 5A, RITA and PRIMA-1 increased the expression of the p53 targets p21 and PUMA, based on qPCR measurements at 24 after their addition to cell cultures, similar to previous reports for other p53 mutants [36, 37].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [36] Selivanova, 2010, Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA, Cell cycle source

      The section “Restoration of the transcriptional function of mutant p53 in cells” (page not specified) reports qPCR induction of p21 in SKBR-3 cells and PUMA in A431 cells after RITA treatment, supporting the comparison with other p53 mutants. It does not report that PRIMA-1 induced either target; its PRIMA-1 comparison concerns cell-killing potency. The RAJI result is presented as the citing authors’ own finding, not as a result of this source. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [37] Selivanova, 2002, Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound, Nature medicine source
  • Premisecitation

    Wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses.

    “We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses.”
    • Also stated in: § INTRODUCTION · plain
      “Recently, our lab and others identified TLR human gene family members as p53 targets and responsive to chromosomal stress.”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [23] Kai, 2008, p53 regulates Toll-like receptor 3 expression and function in human epithelial cell lines, Molecular and cellular biology source

      The Results and Discussion show that p53 regulates TLR3 expression in human epithelial cells and affects downstream cytokine responses. The Discussion mentions only preliminary evidence for TLR7 and TLR8 regulation and does not establish regulation of most TLR genes. No page numbers were supplied for these passages. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [24] Resnick, 2011, The Toll-like receptor gene family is integrated into human DNA damage and p53 networks, PLoS genetics source
    • cites [25] Resnick, 2012, The human TLR innate immune gene family is differentially influenced by DNA stress and p53 status in cancer cells, Cancer research source
    • cites [26] Resnick, 2015, p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways, Oncotarget source

      The Introduction says p53 induction alters expression of TLR family genes in human primary leukocytes and cancer cell lines, and the Results describe enhanced cytokine responses to TLR ligands. The copy does not establish the claim’s more specific assertion that p53 regulates *most* TLR genes. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 24
  • May combine two claims. 23

  • Classified as probably not a substantial claim.

    Premisecitation

    Many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets.

    “Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors.”
    • Also stated in: § INTRODUCTION · plain
      “Many of these can result in a change-of-spectrum transactivation of various p53 targets, thereby altering subsequent cellular responses such as DNA repair, genome stability and programmed cell death that facilitates tumorigenesis [4, 6, 8].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [4] Inga, 2003, Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity, Proceedings of the National Academy of Sciences of the United States of America source
    • cites [6] Resnick, 2006, The biological impact of the human master regulator p53 can be altered by mutations that change the spectrum and expression of its target genes, Molecular and cellular biology source
    • cites [8] Fronza, 2007, Transcriptional functionality of germ line p53 mutants influences cancer phenotype, Clinical cancer research source

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

    Premisecitation

    TLRs are activated by a wide array of endogenous molecules released from self-cells following tissue damage (DAMPs) such as hyaluronic acid and molecules derived from necrotic and apoptotic cells including nucleic acids.

    “In addition, TLRs are activated by a wide array of endogenous molecules released from self-cells following tissue damage (known as damage-associated molecular pattern, DAMP) such as hyaluronic acid and molecules derived from necrotic and apoptotic cells including nucleic acids [42].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [42] Nunez, 2010, Sterile inflammation: sensing and reacting to damage, Nature reviews Immunology source

      The section on TLR recognition of endogenous DAMPs reports activation by molecules from necrotic cells or the extracellular matrix, explicitly including endogenous nucleic acids and hyaluronan. It also describes TLR sensing of RNA and DNA from necrotic cells. The cited passages do not establish the claim's additional attribution to molecules derived from apoptotic cells. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The senescence-associated phenotype identified with secretion of pro-tumorigenic cytokines and chemokines can be restrained or qualitatively modified by p53 in several cell types in mouse.

    “This senescence-associated phenotype identified with secretion of pro-tumorigenic cytokines and chemokines can be restrained or qualitatively modified by p53 in several cell types in mouse [39, 41].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [39] Coussens, 2012, Accessories to the crime: functions of cells recruited to the tumor microenvironment, Cancer cell source
    • cites [41] Lowe, 2013, Non-cell-autonomous tumor suppression by p53, Cell source

      The Results and Discussion report that p53 qualitatively changes cytokine and chemokine secretion in murine hepatic stellate cells, promoting an anti-tumorigenic microenvironment. The Discussion also mentions an earlier report of p53 suppressing secretion in senescent fibroblasts, but this paper does not establish the claimed effect in several mouse cell types. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Rodents lack direct p53 responsiveness of the mouse TLR gene family, which precludes their use in developing human therapies based on p53 modulation of TLR responses.

    “We note that while rodents have served as excellent models systems for addressing p53 functions the lack of direct p53 responsiveness of the mouse TLR gene family precludes their use in developing human therapies based on p53 modulation of TLR responses [24].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Resnick, 2011, The Toll-like receptor gene family is integrated into human DNA damage and p53 networks, PLoS genetics source

      In Results, ‘Evolution of TLR gene family responsiveness to p53’ (page not provided), the authors report small, apparently p53-independent TLR responses in the mouse cells tested and no predicted functional p53 targets among several mouse TLR sequences. This supports the distinction from human TLR responses, but the paper does not establish that rodents cannot be used to develop human therapies; ‘precludes their use’ is a stronger conclusion than these findings support. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Anticancer drugs including 5FU, cisplatin, etoposide, and interferons could increase poly(I:C)-induced cell death in HCT116 cells that are WT p53.

    “Taura et al. [29] reported that anticancer drugs including 5FU, cisplatin, etoposide, and interferons could increase poly(I:C)-induced cell death in HCT116 cells that are WT p53.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Kai, 2010, TLR3 induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis, Cancer Sci source

  • Classified as probably not a substantial claim.

    Premisecitation

    p53 can directly influence expression of the TLR gene family and downstream immune/inflammatory responses.

    “Since p53 can directly influence expression of the TLR gene family and downstream immune/inflammatory responses [24–26]”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [24] Resnick, 2011, The Toll-like receptor gene family is integrated into human DNA damage and p53 networks, PLoS genetics source
    • cites [25] Resnick, 2012, The human TLR innate immune gene family is differentially influenced by DNA stress and p53 status in cancer cells, Cancer research source
    • cites [26] Resnick, 2015, p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways, Oncotarget source

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

    Premisecitation

    Several small molecules have been identified that inactivate mutant p53 or restore WT p53 response in mutant p53 protein including PRIMA-1, MIRA-1, Elipticin, CDB3, WR1065, NSC319726, p53R3 and CP-31398, although their mechanisms of action in many cases remain to be clarified.

    “Over the past decade, several small molecules have been identified that inactivate mutant p53 or restore WT p53 response in mutant p53 protein including PRIMA-1, MIRA-1, Elipticin, CDB3, WR1065, NSC319726, p53R3 and CP-31398, although their mechanisms of action in many cases remain to be clarified [9].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [9] Selivanova, 2015, Pharmacological reactivation of p53 as a strategy to treat cancer, J Intern Med source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    RITA was found to suppress growth of cancer cell lines carrying various p53 mutations and restored the transcriptional functions of several p53 mutant alleles including R175H, R213Q, Y234H, R248W, R273H and R280K that resulted in induction of apoptosis.

    “In particular, RITA was found to suppress growth of cancer cell lines carrying various p53 mutations and restored the transcriptional functions of several p53 mutant alleles including R175H, R213Q, Y234H, R248W, R273H and R280K that resulted in induction of apoptosis [36, 48].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [36] Selivanova, 2010, Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA, Cell cycle source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [48] Clardy, 2015, Small-Molecule Reactivation of Mutant p53 to Wild-Type-like p53 through the p53-Hsp40 Regulatory Axis, Chemistry & biology source

      Split into 2 atoms — 0 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

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

    Premisecitation

    p53 has non-cell-autonomous effects in stimulating an anti-tumorigenic microenvironment.

    “recent studies have shown non-cell-autonomous effects of p53 in stimulating an anti-tumorigenic microenvironment [10].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [10] Lohrum, 2008, Characterization of genome-wide p53-binding sites upon stress response, Nucleic acids research source

      The Introduction describes p53-mediated tumor suppression through transcriptional regulation, and the Results mention genes associated with cell–cell signaling. Neither passage addresses p53 stimulating an anti-tumorigenic microenvironment through non-cell-autonomous effects. Searches for the claim’s phrasing and for microenvironment-related terms found no passage establishing that point. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Nutlin, an activator of WT p53 functions, can greatly enhance the TLR5 downstream mediated responses to its natural ligand flagellin.

    “We recently demonstrated that Nutlin, an activator of WT p53 functions, can greatly enhance the TLR5 downstream mediated responses to its natural ligand flagellin [26].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Resnick, 2015, p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways, Oncotarget source

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

    Premisecitation

    p53 directly induces numerous genes involved in triggering the recruitment of immune cells and immune surveillance in human cells, which can involve the TLR gene family.

    “gene expression analyses has shown that p53 directly induces numerous genes involved in triggering the recruitment of immune cells and immune surveillance in human cells [11], which can involve the TLR gene family.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [11] Resnick, 2013, Diverse stresses dramatically alter genome-wide p53 binding and transactivation landscape in human cancer cells, Nucleic acids research source

      The Introduction says p53 can transactivate genes involved in innate immune responses, and the Results (Novel p53 target genes; page unavailable) identify many putative direct p53 targets in human cancer cells. Our copy does not establish that numerous targets trigger immune-cell recruitment or immune surveillance, or identify the TLR gene family as part of that finding. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    p53-dependent tumor regression in a mouse liver carcinoma model was related to induction of a senescence program through the upregulation of pro-inflammatory cytokines and activation of innate immune response, characterized by the recruitment of neutrophils, macrophages and natural killer (NK) cells.

    “Xue et al. [40] found that p53-dependent tumor regression in a mouse liver carcinoma model was related to induction of a senescence program through the upregulation of pro-inflammatory cytokines and activation of innate immune response, characterized by the recruitment of neutrophils, macrophages and natural killer (NK) cells.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [40] Lowe, 2007, Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas, Nature source

  • May combine two claims.

    Findingown data

    Transient transfection with WT or mutant p53 had no apparent influence on expression of IL-8, IFN-β and IL-6, nor did poly(I:C) on parental p53−/− cells or empty vector-transfected cells.

    “Transient transfection with WT and plasmids expressing mutant p53 had no apparent influence on the expression of the IL-8 (CXCL8), IFN-β (IFNB1) and IL-6, nor did poly(I:C) (3h) on parental p53−/− cells or those transfected with the empty vector.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    p53 mutant proteins also bound the p53 RE associated with p21, with relative differences for A138V and H178Y.

    “Binding by these mutant proteins was also observed at the p53 RE associated with p21 although there were relative differences, as for A138V and H178Y, suggesting that p53 mutants can have different specificities for p53 binding sequences.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    p53 mutants T125R, M133T, A138V, P190L, V272L, C277W and R337H exhibited limited transactivation potential as well as change-of-spectrum of TLR genes expressed.

    “while another group that included T125R, M133T, A138V, P190L, V272L, C277W and R337H exhibited a limited transactivation potential as well as change-of-spectrum of TLR genes expressed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In cells expressing WT p53 and mutants P151H and R377H, significant induction of IL-8, IFN-β and IL-6 mRNA levels was observed after poly(I:C) treatment, whereas only slight induction of cytokines resulted with A138V and G245S mutants.

    “In cells expressing WT p53 and mutants P151H and R377H a significant induction of IL-8, IFN-β and IL-6 mRNA levels was observed after poly(I:C) treatment, whereas only a slight induction of cytokines resulted with A138V and G245S mutants (Figure 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    p53 mutants P151H, H178Y, G279R and R337H, but not A138V, bound the TLR3 p53 response element located in the promoter region and enhanced TLR3 mRNA levels.

    “Similar to WT p53, the p53 mutants P151H, H178Y, G279R and R337H, but not A138V, bound the TLR3 p53 response element located in the promoter region (Figure 2B) and enhanced TLR3 mRNA levels (Figure 2A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    24h exposure to poly(I:C) significantly increased the incidence of apoptosis in P151H and R337H expressing cells, similar to WT p53, but not in A138V and G245S transfected cells.

    “As shown in Figure 4A, 24h exposure to poly(I:C) significantly increased the incidence of apoptosis in P151H and R337H expressing cells, as indicated by Annexin V staining, similar to WT p53. There was no increase for the A138V and G245S transfected cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cell line-specific expression patterns were observed for TLR9: in HCT116, expression of WT p53 and several mutants resulted in reduced TLR9 expression, while in SaOS2 there was increased expression.

    “Cell line-specific expression patterns were also observed, as for the case of TLR9. In HCT116, expression of WT p53 and several mutants resulted in a reduced TLR9 expression while in SaOS2 there was increased expression, suggesting the involvement of cell specific co-regulatory factors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Loss-of-function mutants R175H and G245S showed no binding to the TLR3 promoter region or increased TLR3 mRNA levels.

    “The loss-of-function mutants R175H and G245S, which are unable to interact with canonical p53 binding sequences or induce TLR3 expression, were used as negative controls in these experiments and showed no binding to the TLR3 promoter region or increased TLR3 mRNA levels, as expected.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    No binding was observed at the promoter region of the negative control GAPDH.

    “No binding was observed at the promoter region of the negative control GAPDH (Supplementary Figure S1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    p53 mutants vary in their influence on immune and apoptotic signaling mediated by TLR3 in response to poly(I:C), with some mutants retaining a strong p53 mediated enhancement of ligand responsiveness.

    “Using TLR3 and the poly(I:C) agonist as a model, we have shown how p53 mutants can vary in their influence on immune and apoptotic signaling mediated by this receptor in response to a ligand, with some mutants retaining a strong p53 mediated enhancement of ligand responsiveness.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the mutants evaluated in the present study, P151H, H178Y and P190L resulted in increased TLR5 expression, whereas the M133T mutant down-regulated expression.

    “Among the mutants evaluated in the present study, P151H, H178Y and P190L resulted in increased TLR5 expression, whereas the M133T mutant down-regulated expression.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both RITA and DXR alone increased the level of apoptosis approximately 2-3 fold, and this was increased by the addition of poly(I:C).

    “As shown in Figure 6F, both RITA and DXR alone could increase the level of apoptosis approximately 2-3 fold, and this was increased by the addition of poly(I:C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both DXR and 5FU increased poly(I:C) signaling for some of the cytokines and this was generally additive with the effect of RITA.

    “Both DXR and 5FU increased poly(I:C) signaling for some of the cytokines and this was generally additive with the effect of RITA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Increases in expression are reflected in p53 protein occupancy at the TLR3 promoter and an increase in TLR3 protein.

    “The increases in expression are reflected in p53 protein occupancy at the TLR3 promoter (Figure 5C and Supplementary Figure S4) and an increase in TLR3 protein (Figure 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RITA synergizes with DNA damaging drugs to increase TLR3 expression, enhancing its immune and apoptotic responses mediated by its agonist poly(I:C).

    “It synergizes with DNA damaging drugs to increase TLR3 expression, enhancing its immune and apoptotic responses mediated by to its agonist poly(I:C).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Crosstalk between tumor cells and cells of the microenvironment is crucial for regulating tumorigenesis.

    “Crosstalk between tumor cells and cells of the microenvironment is crucial for regulating tumorigenesis [39].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Coussens, 2012, Accessories to the crime: functions of cells recruited to the tumor microenvironment, Cancer cell source

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

    Study design and conductnone given

    Statistically significant differences were identified using two way ANOVA, with p-values < 0.001 considered statistically significant.

    “Statistically significant differences were identified using two way ANOVA. p-values < 0.001 were considered statistically significant.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Sequences of all the constructs developed for this study were confirmed by DNA sequencing.

    “Sequences of all the constructs developed for this study were confirmed by DNA sequencing.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    TLRs are expressed in many cancer epithelial cells and can affect tumor growth.

    “Besides the canonical pathogen protection functions of TLRs, they are also expressed in many cancer epithelial cells and can affect tumor growth [18].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Medzhitov, 2009, Toll-like receptors and cancer, Nature reviews Cancer source

  • Classified as probably not a substantial claim.

    Premisecitation

    Poly(I:C) treatment induced TLR3 dependent apoptosis in human prostate cancer cells.

    “Paone et al., [28] showed that poly(I:C) treatment induced TLR3 dependent apoptosis in human prostate cancer cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Riccioli, 2008, Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC-alpha-dependent mechanism, Carcinogenesis source

  • May combine two claims.

    Interpretationown data

    Functional restoration of endogenous p53 mutants can increase TLR3 expression and synergize with DNA damaging agents to modulate TLR3 signaling responses in the presence of a cognate ligand.

    “Together, these results show that functional restoration of endogenous p53 mutants can increase TLR3 expression and synergize with DNA damaging agents to modulate TLR3 signaling responses in the presence of a cognate ligand.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The different effects of RITA and PRIMA suggest different mechanisms for restoring function and its potential transcriptional targets.

    “Yet, the combination of RITA, but not PRIMA, with DXR or 5FU led to a considerable increase in TLR3, p21 and PUMA expression (Figure 5B, Supplementary Figure S4), suggesting different mechanisms for restoring function and its potential transcriptional targets.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Triplicate wells were assayed for each condition in proliferation assays.

    “Triplicate wells were assayed for each condition.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The data presented correspond to the mean ± SD of at least three experiments where each was done in triplicate.

    “The data presented correspond to the mean ± SD of at least three experiments where each was done in triplicate.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    TLR3 has been implicated in the induction of apoptosis in response to genotoxic stresses or immune challenges.

    “In addition, TLR3 has been implicated in the induction of apoptosis in response to genotoxic stresses or immune challenges.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    TLR antagonists as well as agonists of their negative regulators are now being developed for the treatment of cancer, allergies and viral infections and as vaccine adjuvants to enhance immune responses against tumors and infectious diseases.

    “TLR antagonists as well as agonists of their negative regulators are now being developed for the treatment of cancer, allergies and viral infections and as vaccine adjuvants to enhance immune responses against tumors and infectious diseases [44, 45].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Coffman, 2007, Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists, Nature medicine source
    • cites [45] Rezaei, 2012, Prophylactic and therapeutic implications of toll-like receptor ligands, Medicinal research reviews source

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

    Study design and conductown data

    Mouse Ig was used as a negative control in ChIP assays.

    “Mouse Ig (Santa Cruz Biotechnology) was used as a negative control.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The immune response stimulated via TLR3 has been proposed for development of anticancer therapies, and several poly(I:C) derived agonists of TLR3 are under clinical investigation.

    “The immune response stimulated via TLR3 has been proposed for development of anticancer therapies, and several poly(I:C) derived agonists of TLR3 are under clinical investigation [31, 46].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [31] Pavelic, 2014, The exploitation of Toll-like receptor 3 signaling in cancer therapy, Current pharmaceutical design source
    • cites [46] Toth, 2014, Toll-like receptor agonists: a patent review (2011 - 2013), Expert opinion on therapeutic patents source

  • May combine two claims.

    Interpretationreasoning

    Choices of TLR targets would be tumor specific and depend on p53 WT or mutant status.

    “Choices of TLR targets would be tumor specific and depend on p53 WT or mutant status.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Many DAMPs are generated by cancer cells in response to chemotherapeutic drugs such as DXR and cyclophosphamide.

    “Many DAMPS are generated by cancer cells in response to chemotherapeutic drugs such as DXR and cyclophosphamide [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Kroemer, 2010, Decoding Cell Death Signals in Inflammation and Immunity, Cell source

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

    Study design and conductnone given

    Amplification of GADPH promoter region was used as a negative control in ChIP assays.

    “Amplification of GADPH promoter region was used as a negative control.”
    Quote in the paperNot a substantial claim
43 of 43

  • The paper relies on this without support.

    The frequency of p53 change-of-spectrum mutants in tumors is low compared with classical p53 hotspot mutants.

    “Although p53 change-of-spectrum mutants can impact TLR expression and signaling, their frequency in tumors is low when compared with classical p53 hotspot mutants, which have lost the ability to interact with p53 binding sequences.”

  • The paper relies on this without support.

    Classical p53 hotspot mutants have lost the ability to interact with p53 binding sequences.

    “Although p53 change-of-spectrum mutants can impact TLR expression and signaling, their frequency in tumors is low when compared with classical p53 hotspot mutants, which have lost the ability to interact with p53 binding sequences.”

  • The paper relies on this without support.

    In response to poly(I:C), TLR3 induces type I interferon and inflammatory cytokine/chemokines.

    “In response to poly(I:C), TLR3 induces a type I interferon (IFN) along with inflammatory cytokine/chemokines.”

  • The paper relies on this without support.

    DXR and 5FU can activate WT p53 and even some change-of-spectrum mutants.

    “Although DXR and 5FU can activate WT p53 and even some change-of-spectrum mutants (see Figure 4B), neither DXR nor 5FU treatment alone affected TLR3 expression in the RAJI cells.”

  • The paper relies on this without support.

    Most commonly found p53 mutations are loss-of-function.

    “Since most of the commonly found p53 mutations are loss-of-function, pharmacological restoration of p53 function is an obvious therapeutic strategy against human cancers.”

  • The paper relies on this without support.

    The interpretation of signals by innate immune responding cells such as macrophages or dendritic cells can stimulate tumor-specific immune responses that in some settings enhance anticancer therapy.

    “The interpretation of such signals by innate immune responding cells such as macrophages or dendritic cells can stimulate tumor-specific immune responses that in some settings enhance anticancer therapy.”
6 of 6

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.

  • Luciferase assay timing differs between caption and methods

    Minor

    The Figure 2 caption says luciferase activity was assessed 24 hours after transfection, whereas the methods say it was measured 48 hours after transfection.

  • Responding mutant is labeled R377H instead of R337H

    Minor

    The results identify R377H as a mutant that increased cytokine expression, but the mutant identified in the surrounding analysis and Figure 4 is R337H.

  • Figure 6 summary statistic conflicts with the statistics section

    Minor

    Figure 6 specifies medians and standard deviations, while the statistics section says the presented data are means and standard deviations.

  • DXR and 5FU effects on poly(I:C) signaling are described oppositely

    Minor

    The results say both drugs increased poly(I:C) signaling for some cytokines without mentioning p53 reactivation. The discussion says that, without reactivation, either drug alone has no impact on downstream signaling.

4 of 4

Cites

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Authors
Menendez, D., Lowe, J. M., Snipe, J., & Resnick, M. A.
Affiliations
  • Genome Integrity & Structural Biology Laboratory, Inflammation Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA
  • National Institutes of Health (US)
ORCID
Resnick, Michael A.
Venue
Oncotarget
Publisher
Impact Journals, LLC
Published
2016-08-11
Volume
7
Issue
38
Pages
61630-61642
ISSN
1949-2553 (ISSN-L)
DOI
10.18632/oncotarget.11210
PMC
PMC5308678
OpenAlex
W2508373487
Semantic Scholar
1bf4692497b3998a148234a06dc4f348025c1b7d
Cited by
31
References
51
Citations by year
2026: 1 · 2025: 2 · 2024: 3 · 2023: 5 · 2022: 2 · 2021: 5 · 2020: 5 · 2019: 2 · 2018: 1 · 2017: 5
FWCI
1.24
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Oncotarget (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • National Institutes of Health (ES065079, Z01 ES065079)
  • National Institute of Environmental Health Sciences (Z01ES065079)
Topics
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
mutant p53, TLR3 signaling, Toll-like receptors, poly(I:C), apoptosis, RITA, p53 reactivation, innate immunity, TLR gene expression, DNA damage-induced apoptosis, chemokines, cytokines, cancer therapy
MeSH terms
Animals, DNA Damage, Humans, Immunity, Innate, Mutation, Plasmids, Signal Transduction, Transcriptional Activation, Gene Expression Regulation, Neoplastic, Tumor Suppressor Protein p53, Apoptosis, Gene Expression Profiling, Cell Line, Tumor, Cell Proliferation, Toll-Like Receptor 3, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-24.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-24.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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