Mutant p53K120R expression enables a partial capacity to modulate metabolism

The TP53 tumor suppressor gene is one of the most studied gene in virtue of its ability to prevent cancer development by regulating apoptosis, cell cycle arrest, DNA repair, autophagy and senescence. Furthermore, the modulation of metabolism by P53 is fundamental for tumor suppressor activity. Studies in mouse models showed that mice carrying TP53 mutations affecting the acetylation in the DNA binding domain still retain the ability to transactivate genes involved in metabolism. Noteworthy, mice expressing the triple 3KR or the single K117R mutant do not show early on-set tumor development in contrast to TP53 −/− mice. Interestingly, the mouse K117R mutation corresponds to the human tumor-derived K120R modification, which abrogates P53-dependent activation of apoptosis without affecting growth arrest. In this study, we investigated the property of the human P53 K120R mutant in the regulation of metabolism by analyzing the transcriptional specificity in yeast- and mammalian-based reporter assays, the metabolic phenotype associated to its expression in colon cancer HCT116 TP53−/− cells and the induction of P53 targets and proteins involved in the antioxidant response. These properties were analyzed in comparison to wild type P53 protein, the human triple mutant corresponding to mouse 3KR and the cancer hot-spot R273H mutant. We confirm the selective functionality of P53 K120R mutant, which shows a transcriptional activity on cell cycle arrest but not on apoptotic targets. Interestingly, this mutant shows a partial transactivation activity on p53 response element belonging to the metabolic target TIGAR. Moreover, we observe a significant uncoupling between oxygen consumption and ATP production associated with higher lipid peroxidation level in all P53 mutants carrying cells with respect to wild type P53 expressing cells. Noteworthy, in the absence of a pro-oxidative challenge, cells expressing K120R mutant retain a partial capacity to modulate glucose metabolism, limiting lipid peroxidation with respect to the other P53 mutants carrying cells. Lastly, especially in presence of human 3KR mutant, a high expression of proteins involved in the antioxidant response is found. However, this response does not avoid the increased lipid peroxidation, confirming that only wild type P53 is able to completely counteract the oxidative stress and relative damages.


Percentage of living cells following H2O2 treatment in HCT116 TP53-/cells expressing p53 WT (WT), p53 3KR (3KR), p53 K120R (K120R) and p53 R273H (R273H) proteins.
HCT116 TP53-/cells were transiently transfected with the empty vector (empty) or plasmid expressing p53 WT , p53 3KR , p53 K120R and p53 R273H . After 16 hours of transfection, cells were treated with 100 M H2O2 for further 6 hours. At the end of treatment, cells were trypsinized and counted; living cells were evaluated with trypan blue exclusion using a TC20 apparatus (Bio-Rad). As shown in the histogram, the percentage of living cells was comparable among samples. The same number of cells (1x10 5 ) was used to determine metabolic end points. Data were obtained from at least five independent experiments and reported as mean ± SD.
A) Representative western blots showing the level of P53 and beta-actin (-act) in HCT116 TP53-/cells transiently transfected with the empty vector (EV), p53 WT , p53 3KR , p53 K120R , p53 R273H and p53 2KR expression plasmids. B) Histogram representing the amount of p53 protein detected in HCT116 TP53-/transfected cells and normalized for -actin. Data were obtained after chemiluminescence analysis of western blots from at least four independent experiments and are reported as mean ± SD.
The expression level of different P53 protein in HCT116 TP53-/cells following transfection was comparable. A plasmid carrying the double P53 mutation K120R+K164R, was also constructed and expressed in HCT116 TP53-/cells. The expression level of the human p53 3KR (K120R+K164R+ Q165R) carrying the Q165R amino acid substitution, did not significantly affect the formation of the P53 protein.
Samples were treated with 1 M Rotenone or 10 M Antimycin A, specific inhibitors of Complex I and Complex III, respectively, to verify that OCR and ATP synthesis are dependent on OxPhos machinery. Oxygen consumption rate and ATP synthesis were analyzed as described in the Material and Methods section in the main text. A) OCR induced by pyruvate + malate (P/M). B) ATP synthesis induced by P/M. C) OCR induced by succinate. D) ATP synthesis induced by succinate. Each panel is representative of at least four independent experiments; data are reported as mean ± SD. The symbol **** indicates significant differences for p<0.0001 between untreated and rotenone-or antimycin A-treated samples.
Western blots showing the level of GLUT1 and -actin (-act) in untreated HCT116 TP53-/cells transiently expressing p53 WT , p53 3KR , p53 K120R and p53 R273H . The expression level of GLUT1 normalized for -actin is reported as Glut1/-act ratio after chemiluminescence analysis of the membrane by UVITEC (Cambridge, UK). With respect to cells transfected with the empty vector (EV), all samples showed a slightly lower level of GLUT1, as expected for an inhibitory effect of P53 on GLUT1 expression. However, this analysis did not reveal differences among cells expressing wild type or mutant P53 protein.
A) Glutaminase (GLS) activity. B) Glutamate dehydrogenase (GDH) activity. C) 3-hydroxyacyl-CoA dehydrogenase activity. Activities reported in panels A and B are markers of aminoacidic metabolism; the activity reported in panel C is a marker of fatty acids beta-oxidation. Each panel is representative of at least four independent experiments; data are reported as mean ± SD. The symbols *, **, and **** indicate significant differences for p<0.05, 0.01, and 0.0001, respectively, between empty and the other samples; the symbol #, ##, and #### indicates significant differences for p<0.05, 0.001, and 0.0001, respectively, between p53 K120R and the indicated samples.

Supplementary Materials & Methods
Glutaminase and glutamic dehydrogenase activity assay Glutaminase activity was assayed spectrophotometrically at 340 nm following NAD + reduction. The assay mix contained: Tris-HCl (pH 8), 50 mM glutamine, 5 mM NAD + , and 5 IU glutamic dehydrogenase (1). Glutamic dehydrogenase was assayed spectrophotometrically at 340 nm following NADH oxidation. The assay solution contained: Tris-HCl (pH 7.4), 20 mM α-ketoglutarate, 0.15 mM NADH, and 1 mM ADP (1). In both cases, 0.1 mM rotenone was added to inhibit the NADH oxidation from Complex I. 50 μg of total protein were used for both assays and data were normalized on the sample protein content.

Fatty acid metabolism evaluation
The activity of 3-hydroxyacyl-CoA dehydrogenase, used as a marker of fatty acids beta-oxidation metabolism, was assayed spectrophotometrically at 340 nm following the oxidation of NADH in the presence of acetoacetyl-CoA. The reaction mix contained: 100 mM sodium phosphate (pH 6.0), 0.2 mM NADH and 0.1 mM acetoacetyl-CoA (2). Oxidative stress status in HCT116 TP53-/cells expressing p53 WT , p53 K120R , p53 3KR and p53 R273H proteins.
A) Evaluation of ROS production in p53 WT , p53 3KR , p53 K120R , and p53 R273H expressing cells; the symbols *, *** and **** indicate significant differences for p=0.0245, p=0.0001 and p<0.0001, respectively, between empty and the other samples. B) Intracellular level of GSH. The symbols ** and **** indicate significant differences for p≤0.0082 and p<0.0001, respectively, between empty and the other samples. Data are representative of at least four independent experiments and are reported as mean±SD. C) ROS/GSH values in the same transfected cells; the symbols **** indicate significant differences for p<0.0001 between empty and the other samples; the symbols ### and #### indicate significant differences for p < 0.0001 (p53 K120R vs and p53 WT and p53 R273H ; p=0.002, p53 K120R vs p53 3KR

ROS and GSH evaluation
To evaluate the reactive oxygen species (ROS) level, cells were washed and re-suspended in PBS and stained for 10 minutes at 37° C with 20,70-dichlorodihydro-fluorescein diacetate (H2DCFDA) at a concentration of 5 μM (Thermo Fisher Scientific, Waltham, MA, USA). H2DCFDA is a non-fluorescent dye, which is cleaved inside cells to 20,70-dichlorofluorescein (H2DCF). In the presence of oxidants, H2DFC is converted in turn to the fluorescent compound DCF. Samples were measured on a FacsCalibur flow cytometer (Becton Dickinson, San José, CA). The analysis was confined to viable cells only, after gating based on forward-and side-scatter characteristics. Ten thousand cells per sample were analyzed (Ravera et al., 2020, PMID: 33020573). Glutathione (GSH) was measured by flow cytometry after staining cells with monobromobimane (MBB Thermo Fisher Scientific, Italy) (Cuccarolo et al., 2012, PMID: 22578062). When excited at 405 nm, MBB becomes fluorescent after conjugation with intracellular thiols. This reaction is far more competitive for free GSH than for protein sulfhydryls. The decrease in the intracellular GSH concentration is reflected by a decrease in the MBB fluorescence peak.