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Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction

View Article: PubMed Central - PubMed

ABSTRACT

Cancer cells depend on glutamine to sustain their increased proliferation and manage oxidative stress, yet glutamine is often depleted at tumor sites due to excessive cellular consumption and poor vascularization. We have previously reported that p53 protein, while a well-known tumor suppressor, can contribute to cancer cell survival and adaptation to low glutamine conditions. However, the TP53 gene is frequently mutated in tumors, and the role of mutant p53 (mutp53) in response to metabolic stress remains unclear. Here, we demonstrate that tumor-associated mutp53 promotes cancer cell survival upon glutamine deprivation both in vitro and in vivo. Interestingly, cancer cells expressing mutp53 proteins are more resistant to glutamine deprivation than cells with wild type p53 (wtp53). Depletion of endogenous mutp53 protein in human lymphoma cells leads to cell sensitivity to glutamine withdrawal, while expression of mutp53 in p53 cells results in resistance to glutamine deprivation. Furthermore, we found that mutp53 proteins hyper-transactivate p53 target gene CDKN1A upon glutamine deprivation, thus triggering cell cycle arrest and promoting cell survival. Together, our results reveal an unidentified mechanism by which mutp53 confers oncogenic functions by promoting cancer cell adaptation to metabolic stresses.

No MeSH data available.


Related in: MedlinePlus

Mutp53 induces expression of p53 target genes upon glutamine deprivation(a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53+/+ cells and HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
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Figure 4: Mutp53 induces expression of p53 target genes upon glutamine deprivation(a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53+/+ cells and HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).

Mentions: It has been reported that cell survival promoted by wtp53 upon metabolic stress is often mediated by the induction of cell cycle arrest and metabolic reprogramming genes including CDKN1A, GADD45A, GLS2 and TIGAR11,16,17. Therefore, we asked whether mutp53 contributes to the induction of these genes upon glutamine deprivation. Consistent with previous investigations, we found p53 was phosphorylated on Serine 15, a critical site for p53 activation, upon glutamine deprivation in EB3 cells with wild type p53 (Figure 4a)13. Moreover, mutp53 in CA46 cells was also phosphorylated upon glutamine deprivation (Figure 4a). Consistent with the phosphorylation, we found that p53-targeted pro-survival genes were significantly induced in CA46 cells upon glutamine deprivation (Figure 4b). Interestingly, the induction of these genes was more robust in CA46 cells harboring mutp53 than EB3 cells with wtp53 (Figure 4b). Importantly, we found that knockdown of p53 in both wtp53 and mutp53 cells dramatically inhibited the induction of the survival genes upon glutamine deprivation, demonstrating that, similar to wtp53, mutp53 contributes to the upregulation of the p53 target genes (Figure 4c and 4d). To further confirm that mutp53 promotes the induction of these p53-target genes, we evaluated the effect of ectopically expressed mutp53 on gene expression upon glutamine deprivation. Consistent with previous investigations, expression of CDKN1A, GLS2, GADD45A and TIGAR are significantly higher in wtp53 expressing cells compared with p53 deleted cells (Figure 4e and 4f). Strikingly, we found that expression of R248Q or R273H mutp53 in HCT116 cells robustly induced CDKN1A, GLS2, GADD45A and TIGAR expression upon glutamine starvation compared with p53 deleted cells or wtp53 expressing cells (Figure 4f). Conversely, no significant changes were found in the expression of pro-apoptotic gene, BAX (Figure 4f). Together, these results suggest that mutp53 not only retains, but also exaggerates the transactivation activity of the wtp53 protein toward pro-survival genes to promote cell survival in response to glutamine deprivation.


Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction
Mutp53 induces expression of p53 target genes upon glutamine deprivation(a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53+/+ cells and HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
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Figure 4: Mutp53 induces expression of p53 target genes upon glutamine deprivation(a) EB3 and CA46 cells were cultured in complete or Gln free medium for 24 hrs. Cells were lysed for Western blot using antibodies as indicated. (b) EB3 and CA46 cells were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to 18S was determined using qRT- PCR and normalized to the complete medium. (c) Cells were transduced with lenti-viral particles followed by puromycin selection to generate stable knockdown of wtp53 in EB3 cells and mutp53 in CA46. p53 protein levels were determined by Western blot. (d) EB3 and CA46 cells infected with virus containing control vector or shRNA against p53 were cultured in Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT- PCR and normalized to the complete control medium. (e) HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in Gln free medium for three days. p53 activation and total p53 expression was determined by Western blot analysis using anti-phospho-p53 (Ser15) and anti-p53 antibody. (f) HCT116 p53+/+ cells and HCT116 p53−/− cells expressing R248Q, R273H, or empty vector were cultured in complete or Gln free medium overnight. mRNA expression of p53 target genes relative to actin was determined using qRT-PCR and normalized to the complete medium. Data represent mean ± S.D. of duplicates from two independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
Mentions: It has been reported that cell survival promoted by wtp53 upon metabolic stress is often mediated by the induction of cell cycle arrest and metabolic reprogramming genes including CDKN1A, GADD45A, GLS2 and TIGAR11,16,17. Therefore, we asked whether mutp53 contributes to the induction of these genes upon glutamine deprivation. Consistent with previous investigations, we found p53 was phosphorylated on Serine 15, a critical site for p53 activation, upon glutamine deprivation in EB3 cells with wild type p53 (Figure 4a)13. Moreover, mutp53 in CA46 cells was also phosphorylated upon glutamine deprivation (Figure 4a). Consistent with the phosphorylation, we found that p53-targeted pro-survival genes were significantly induced in CA46 cells upon glutamine deprivation (Figure 4b). Interestingly, the induction of these genes was more robust in CA46 cells harboring mutp53 than EB3 cells with wtp53 (Figure 4b). Importantly, we found that knockdown of p53 in both wtp53 and mutp53 cells dramatically inhibited the induction of the survival genes upon glutamine deprivation, demonstrating that, similar to wtp53, mutp53 contributes to the upregulation of the p53 target genes (Figure 4c and 4d). To further confirm that mutp53 promotes the induction of these p53-target genes, we evaluated the effect of ectopically expressed mutp53 on gene expression upon glutamine deprivation. Consistent with previous investigations, expression of CDKN1A, GLS2, GADD45A and TIGAR are significantly higher in wtp53 expressing cells compared with p53 deleted cells (Figure 4e and 4f). Strikingly, we found that expression of R248Q or R273H mutp53 in HCT116 cells robustly induced CDKN1A, GLS2, GADD45A and TIGAR expression upon glutamine starvation compared with p53 deleted cells or wtp53 expressing cells (Figure 4f). Conversely, no significant changes were found in the expression of pro-apoptotic gene, BAX (Figure 4f). Together, these results suggest that mutp53 not only retains, but also exaggerates the transactivation activity of the wtp53 protein toward pro-survival genes to promote cell survival in response to glutamine deprivation.

View Article: PubMed Central - PubMed

ABSTRACT

Cancer cells depend on glutamine to sustain their increased proliferation and manage oxidative stress, yet glutamine is often depleted at tumor sites due to excessive cellular consumption and poor vascularization. We have previously reported that p53 protein, while a well-known tumor suppressor, can contribute to cancer cell survival and adaptation to low glutamine conditions. However, the TP53 gene is frequently mutated in tumors, and the role of mutant p53 (mutp53) in response to metabolic stress remains unclear. Here, we demonstrate that tumor-associated mutp53 promotes cancer cell survival upon glutamine deprivation both in vitro and in vivo. Interestingly, cancer cells expressing mutp53 proteins are more resistant to glutamine deprivation than cells with wild type p53 (wtp53). Depletion of endogenous mutp53 protein in human lymphoma cells leads to cell sensitivity to glutamine withdrawal, while expression of mutp53 in p53 cells results in resistance to glutamine deprivation. Furthermore, we found that mutp53 proteins hyper-transactivate p53 target gene CDKN1A upon glutamine deprivation, thus triggering cell cycle arrest and promoting cell survival. Together, our results reveal an unidentified mechanism by which mutp53 confers oncogenic functions by promoting cancer cell adaptation to metabolic stresses.

No MeSH data available.


Related in: MedlinePlus