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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.


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Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment(a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
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Figure 2: Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment(a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).

Mentions: To determine whether loss of mutp53 could sensitize cancer cells to glutamine starvation, we used shRNA to knock down mutp53 in CA46 cells. We generated stable cell lines with p53 knocked down (Figure 2a and 2b), and found that mutp53 deficiency significantly sensitized CA46 cells to glutamine deprivation compared to control (Figure 2c). Based on the annexin V/PI staining, glutamine deprivation resulted in more dramatic apoptotic cell death in mutp53 knockdown cells compared to vector control cells (Figure 2d and 2e). In addition, we found that knockdown of mutp53 promoted cell sensitivity to different glutaminase inhibitor treatment, such as L-DON, BPTES and Compound 968 (Figure 2f). These results demonstrated that mutp53 is required for cell survival under glutamine deprivation and glutaminase inhibitor treatment.


Tumor-associated mutant p53 promotes cancer cell survival upon glutamine deprivation through p21 induction
Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment(a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
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Related In: Results  -  Collection

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Figure 2: Loss of mutp53 sensitizes cancer cells to glutamine deprivation and glutaminase inhibitor treatment(a–b) p53 mRNA levels (a) relative to actin and protein levels (b) in control vector and p53 shRNA transduced CA46 cells was assessed using qRT-PCR and Western blot. (c) The indicated control vector or shRNA p53 transduced cells were cultured in complete or Gln free medium for the indicated time points. Viability was assessed by PI exclusion and normalized to cells cultured in complete medium. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (d–e) Annexin-V and PI staining of stably transfected CA46 cells cultured in Gln free medium or complete medium for 24 hrs. Representative graphs of cells in late apoptosis (annexin-V and PI positive) are shown. Data represent mean ± S.D. of three independent experiments (***P≤.001, Student’s t test). (f) The control vector transduced cells and p53 shRNA transduced cells were treated with 12.5µM L-DON for two days, 40µM Compound 968 and 50µM BPTES for four days. Cell viability was assessed by PI exclusion and normalized to control treated cells. Data represent mean ± S.D. of three independent experiments (*P<.05, **P<.01, ***P≤.001, Student’s t test).
Mentions: To determine whether loss of mutp53 could sensitize cancer cells to glutamine starvation, we used shRNA to knock down mutp53 in CA46 cells. We generated stable cell lines with p53 knocked down (Figure 2a and 2b), and found that mutp53 deficiency significantly sensitized CA46 cells to glutamine deprivation compared to control (Figure 2c). Based on the annexin V/PI staining, glutamine deprivation resulted in more dramatic apoptotic cell death in mutp53 knockdown cells compared to vector control cells (Figure 2d and 2e). In addition, we found that knockdown of mutp53 promoted cell sensitivity to different glutaminase inhibitor treatment, such as L-DON, BPTES and Compound 968 (Figure 2f). These results demonstrated that mutp53 is required for cell survival under glutamine deprivation and glutaminase inhibitor treatment.

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