Reversible Nucleolar Stress and Cell Growth Arrest Triggered by Acidic pH

ABSTRACT

The tumor microenvironment is often characterized by hypoxia and extracellular acidosis, which modulate various tumor cell phenotypes. Ribosome biogenesis is a highly energy‐demanding process that is essential for protein synthesis and cell proliferation and is sensitive to cellular stress, resulting in a nucleolar stress response. However, whether extracellular acidosis impairs ribosome biogenesis and induces nucleolar stress remains unclear. In this study, we demonstrated that an acidic pH downregulates ribosome biogenesis in cancer cells. RNA sequencing revealed the downregulation of genes related to ribosome biogenesis and cell cycle progression under acidic conditions. Consistently, acidic pH reduced the pre‐rRNA levels and induced nucleolar stress, as evidenced by NPM1 translocation from the nucleolus to the nucleoplasm, which led to G1 phase arrest and growth inhibition. Importantly, these effects were reversed upon restoration of neutral pH, with recovery of pre‐rRNA expression, NPM1 localization, and cell proliferation. Further, an acidic pH shifted the intracellular redox balance toward an oxidized state. Treatment with the reductant dithiothreitol partially reversed NPM1 translocation, suggesting that oxidative stress contributes, at least partially, to the nucleolar stress response. Overall, our findings reveal a previously unrecognized link between extracellular acidosis and impaired ribosome biogenesis, leading to nucleolar stress and reversible growth arrest. This acidosis‐driven stress response represents a therapeutic vulnerability in solid tumors, offering a novel strategy to overcome treatment resistance associated with the acidic tumor microenvironment.

Acidic pH reduced pre‐rRNA levels and triggered nucleolar stress, as evidenced by NPM1 translocation from the nucleolus to the nucleoplasm, leading to G1‐phase arrest and growth inhibition. These effects were reversible upon restoration of neutral pH, and the partial rescue by reductant treatment suggested involvement of oxidative stress. This acidosis‐induced stress response represents a potential therapeutic vulnerability in solid tumors.

1. Introduction

Solid tumors often develop a tumor microenvironment (TME) characterized by hypoxia, acidic pH (~ 6.8), and limited nutrient availability (Boedtkjer and Pedersen 2020; Hanahan 2022; Hosonuma and Yoshimura 2023). The TME strongly influences tumor cell behavior and contributes to therapeutic resistance, highlighting the importance of understanding cellular adaptation to such stresses. The Warburg effect is a key contributor to tumor acidosis, wherein tumor cells heavily rely on aerobic glycolysis for ATP production (Vander Heiden et al. 2009). This metabolic reprogramming leads to lactate accumulation and acidification of the extracellular milieu. Although acidic pH is known to modulate various tumor cell phenotypes, such as migration, invasion, and proliferation (Cortés Franco et al. 2023; de Bem Prunes et al. 2022; Estrella et al. 2013; Ward et al. 2020), the precise molecular mechanisms through which acidosis affects essential cellular processes remain unclear.

Ribosome biogenesis (RiBi) is essential to sustain cellular growth and protein synthesis (Jiao et al. 2023; Lafontaine 2015; Pecoraro et al. 2021). The nucleolus is a distinct nuclear compartment serving as the primary site of RiBi. RiBi involves three major steps: ribosomal RNA (rRNA) transcription, precursor rRNA (pre‐rRNA) processing, and ribosomal subunit assembly. RNA polymerase I (Pol I) transcribes 47S pre‐rRNA within the fibrillar center of the nucleolus. Subsequent processing and maturation generate 18S, 5.8S, and 28S rRNAs, which combine with ribosomal proteins to form functional ribosomes. As RiBi is metabolically demanding, it is tightly regulated and extremely sensitive to various cellular stressors, including DNA damage, nutrient deprivation, and oxidative stress (Kruhlak et al. 2007; Lindström et al. 2022; Pan et al. 2022; Yang et al. 2016). Perturbation of RiBi triggers a conserved nucleolar stress response characterized by nucleolar proteins such as nucleophosmin‐1 (NPM1) being released into the nucleoplasm (Yang et al. 2018). This response can stabilize the tumor suppressor p53, resulting in cell cycle arrest and cell fate modulation (Hannan et al. 2022; Kurki et al. 2004; Rubbi 2003). Although various agents and genotoxic insults trigger nucleolar stress (Yang et al. 2016), whether tumor acidosis affects RiBi remains unclear. Here, using RNA sequencing (RNA‐seq) and functional assays, we demonstrated that acidic pH downregulates the genes involved in RiBi and cell cycle progression, induces nucleolar stress as evidenced by NPM1 translocation, and promotes reversible G1 arrest. Overall, extracellular acidosis induces nucleolar stress and affects key cellular programs, with potential implications for therapeutic targeting in solid tumors.

2. Results

2.1. Acidic pH Downregulates Gene Expression Profiles Related to Ribosome Biogenesis and Cell Cycle Progression

Although the effects of acidic pH on cellular functions have been previously investigated, they remain to be completely elucidated. We initially performed RNA‐seq to determine the effect of acidic pH (6.8) on the gene expression profiles of HN30 pharyngeal cancer cells. Hierarchical cluster analysis revealed that many genes were affected at both 24 and 48 h after acidic treatment (Figure 1A,B, Supporting Information Figure S1A). Gene ontology (GO) analysis showed that the genes significantly downregulated by acidic pH were involved in biological processes related to RiBi and cell cycle‐related events (Figure 1C,D, Supplementary Information Figure S1B–D). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that acidic pH affected gene sets related to the cell cycle, DNA replication, and RiBi pathways (Figure 1E, Supplementary Information Figure S1E). Several genes related to the cell cycle and RiBi were found to be downregulated in the Reactome pathway analysis (Figure 1F, Supplementary Information Figure S1F). RNA‐seq results suggested that acidic pH was associated with downregulation of cell cycle progression and RiBi.

Figure 1.

Acidic pH‐induced downregulation of genes related to ribosome biogenesis and the cell cycle. (A) Hierarchical cluster analysis of differentially expressed genes between HN30 cells treated with neutral (pH 7.4) and acidic (pH 6.8) pH for 24 and 48 h using RNA sequencing (RNA‐seq) data (n = 2 per group). Red and green colors on the heat map represent upregulation and downregulation, respectively, of the indicated gene. (B) Volcano plot of differentially expressed genes between HN30 cells treated with neutral and acidic pH for 24 h using RNA‐seq data (n = 2 per group). Genes with at least twice as high or low expression as that at neutral pH (i.e., a log2‐fold change greater than 1 or less than −1) and an adjusted p‐value of 0.05 or less were considered differentially expressed and represented as red and green points, respectively. (C and D) Gene ontology analysis using the gene set significantly downregulated by acidic pH for 24 h in RNA‐seq results from cells treated with neutral and acidic pH for 24 h. (E and F) Kyoto Encyclopedia of Genes and Genomes (E) and Reactome (F) pathway analyses using the gene set significantly downregulated by acidic pH for 24 h in RNA‐seq results from cells treated with neutral and acidic pH for 24 h.

2.2. Acidic pH Induces Nucleolar Stress Accompanied by Reduced pre‐rRNA Levels

RiBi is crucial for maintaining cellular homeostasis (Jiao et al. 2023; Lafontaine 2015; Pecoraro et al. 2021). Nucleolar stress occurs when RiBi is impaired, marked by NPM1 translocation from the nucleolus to nucleoplasm (Yang et al. 2018). Indeed, the levels of pre‐rRNA significantly decreased, and NPM1 translocation was observed when HN30 cells were treated with actinomycin D, a Pol I inhibitor (Figure 2A,B). As Pol I transcription decreased under acidic conditions, as indicated by RNA‐seq analysis, we examined the impact of acidic pH on pre‐rRNA levels and NPM1 localization. As expected, acidic treatment significantly reduced the pre‐rRNA levels compared with those at neutral pH (Figure 2C). The pre‐rRNA levels, measured using a primer set targeting a different sequence, also decreased under acidic conditions (Supplementary Information Figure S2). Immunofluorescence analysis revealed that NPM1 was localized in the nucleolus at neutral pH; however, the nucleolar NPM1 signal was weaker in acidic pH‐treated cells than in those at neutral pH (Figure 2D), suggesting NPM1 translocation. As NPM1 signals in the nucleolus and nucleoplasm decreased and increased, respectively, upon NPM1 translocation, the ratio of the NPM1 signal in the nucleolus to that in the nucleoplasm should be lower than that in the normal state. We measured this ratio to evaluate the degree of NPM1 translocation quantitatively (Figure 2E). Neutral pH did not change this ratio during observation. However, we found a significant reduction in the ratio after 12 h of acidic treatment. This reduced ratio was observed at 24 h and 48 h. Therefore, acidic pH triggers nucleolar stress and suppresses pre‐rRNA synthesis.

Figure 2.

Acidic pH‐induced nucleolar stress accompanied by reduced levels of pre‐rRNA. (A) Changes in pre‐rRNA levels after actinomycin D (Act D) treatment as determined using qPCR. HN30 cells were treated with or without 8 nM actinomycin D for 2 h. Total mRNA was extracted 2 h after treatment. Means ± S.D. (n = 3 independent experiments). (B) Representative anti‐NPM1 immunofluorescence images of control and actinomycin d‐treated HN30 cells. Scale bar: 10 μm. (C) Pre‐rRNA levels at the indicated times after neutral (N) or acidic (A) treatment quantified using qPCR. HN30 cells were treated under neutral or acidic conditions for the indicated times. Total mRNA was extracted each time after treatment. Relative levels were normalized with those obtained with neutral pH at the indicated time. Means ± S.D. (n = 3 independent experiments). (D) Representative anti‐NPM1 immunofluorescence images of HN30 cells each time after neutral or acidic treatment. Zero hour represents the time immediately before the medium was changed. Scale bar: 10 μm. (E) Ratio of the NPM1 signal in nucleoli and nucleoplasm after neutral (N) and acidic (A) treatment. Each ratio is represented as a box and whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Each plot shows the NPM1 ratio in each cell. Each group contains at least 169 cells. Zero hour represents time immediately before the medium was changed. Three independent experiments were performed. **p < 0.01, two‐tailed Student's t‐test (A, C) or Kruskal‐Wallis test followed by Mann‐Whitney U test and false discovery rate correction (E).

2.3. Acidic pH Induces Cell Cycle Arrest and Activates the p53 Signaling Pathway

We quantified cell numbers under neutral and acidic conditions to evaluate the impact of acidic pH on cell growth. Acidic pH significantly decreased the rate of cell proliferation compared to that at neutral pH (Figure 3A). Flow cytometric analysis revealed that the fraction of cells in the G1 phase significantly increased, whereas other fractions decreased under acidic conditions compared with those under neutral conditions (Figure 3B–E). It has been reported that activation of the nucleolar surveillance pathway results in p53 accumulation through the interaction between MDM2 and ribosomal proteins (Hannan et al. 2022; Kurki et al. 2004; Rubbi 2003; Yang et al. 2016). We examined the effects of acidic treatment on p53 signaling. CDKN1A is a p53‐target gene (Engeland 2022). Acidic treatment increased the CDKN1A mRNA levels over time, whereas this level did not change under neutral conditions (Figure 3F). p53 and p21 protein levels were upregulated under acidic pH compared to those under neutral conditions (Figure 3G). Furthermore, to investigate the mechanism underlying p53 accumulation under acidic conditions, we analyzed interactions between MDM2 and nucleolar proteins. Co‐immunoprecipitation failed to detect stable complexes between MDM2 and either NPM1 or ribosomal protein L11 (RPL11); however, the MDM2‐p53 interaction was detected and did not change in acidic pH (Supplementary Information Figure S3A,B). In contrast, proximity ligation assays revealed close spatial association in HN30 cells, consistent with transient or context‐dependent interactions (Figure 3H,I). The MDM2‐NPM1 interaction showed a tendency to increase under acidic pH (Figure 3H), whereas the MDM2‐RPL11 interaction was significantly enhanced under acidic conditions compared with neutral pH (Figure 3I). Notably, knockdown of RPL11 reduced p53 levels (Figure 3J). Although ARF has been shown to contribute to p53 stabilization by inhibiting MDM2 (Kung and Weber 2022), ARF protein was barely detectable in the HN30 cells used in this study (Supplementary Information Figure S3C), and no interaction between ARF and MDM2 was observed (data not shown). Collectively, these results suggest that acidic pH‐induced p53 accumulation is likely mediated by interactions between MDM2 and nucleolar proteins, including NPM1 and RPL11.

Figure 3.

Acidic pH‐induced cell cycle arrest and p53 accumulation. (A) Quantitative analysis of cell proliferation assay under neutral and acidic conditions. Cell numbers were counted at each time after treatment. The medium was changed to neutral or acidic pH after 24 h of seeding HN30 cells. Zero hour represents the time immediately before the medium was changed. Means ± S.D. (n = 3 independent experiments). (B–E) Representative histograms (B) and quantification of the fractions of G1 (C), S (D), and G2/M (E) phases based on DNA content using flow cytometry analyses of HN30 cells treated with neutral (black) or acidic (gray) pH for 24 and 48 h. Zero hour represents the time immediately before the medium was changed. Means ± S.D. (n = 3 independent experiments). (F) Quantitative analysis of CDKN1A mRNA levels under neutral and acidic conditions. Total mRNA was extracted at each time point after treatment. The medium was changed to neutral or acidic pH after 24 h of seeding HN30 cells. Zero hour represents the time immediately before the medium was changed. Means ± S.D. (n = 3 independent experiments). (G) Representative images of p53, p21, and β‐tubulin protein levels obtained by western blotting. HN30 cells were treated with acidic or neutral pH for 12, 24, and 48 h. Cell lysates were extracted at each time point after treatment. Three independent experiments were performed, and a representative result is shown. (H and I) Representative images (left) and quantification (right) of the proximity ligation assay (PLA). PLA signals (red) for MDM2 and NPM1 (H) or RPL11 (I) were detected in HN30 cells treated with acidic or neutral pH for 24 h. Two independent experiments were performed. Images were acquired from six fields per sample for each treatment and quantified. Scale bar: 10 µm. (J) Representative western blot images of RPL11, p53, and GAPDH (upper) and immunofluorescence staining for RPL11 and p53 (lower). HN30 cells were transfected with control and RPL11 siRNA and treated for 24 h with neutral or acidic treatment. Three independent experiments were performed, and a representative result is shown. **p < 0.01, two‐way ANOVA followed by Sidak's multiple comparisons post hoc tests (A, C–F) and Mann‐Whitney U test (H and I).

2.4. Acidic pH Inhibits Cell Growth Irrespective of p53 Expression

Given that acidic pH appeared to induce G1 arrest through activation of the p53 signaling pathway, we next knocked down p53 in HN30 cells to examine whether acidic pH–induced nucleolar stress is dependent on p53. Although siRNA against p53 decreased its expression (Supplementary Information Figure S4A), NPM1 translocation was still induced under acidic conditions, similar to that in control cells (Supplementary Information Figure S4B,C). CDKN1A mRNA levels were decreased in p53‐knockdown cells treated with neutral or acidic pH compared to those in control cells (Supplementary Information Figure S4D). However, cell proliferation was inhibited under acidic conditions even in p53‐knockdown cells (Supplementary Information Figure S4E). We confirmed the results obtained with siRNA‐treated cells using another cell line in which shRNA was used for p53 knockdown (Supplementary Information Figure S5A). As expected, acidic pH induced NPM1 translocation (Supplementary Information Figure S5B) and inhibited cell proliferation (Supplementary Information Figure S5C) in both the control and p53‐knockdown cells. Overall, acidic pH induces nucleolar stress and growth inhibition, irrespective of p53 expression.

2.5. Oxidative Stress Partially Contributes to Nucleolar Stress Induced by Acidic pH

Nucleolar stress can be triggered by various cellular stressors, ribotoxins, and cellular insults, including DNA damage and oxidative stress (Yang et al. 2016; Yang et al. 2018). As shown in Figures 1 and 2, acidic pH reduced pre‐rRNA levels, which could contribute to nucleolar stress induction. To determine whether DNA damage is involved in this response as another mechanism, we assessed focus formation using markers of DNA double‐strand (γH2A.X) and single‐strand (RPA32) breaks under acidic conditions. However, neither focus was clearly observed (Supplementary Information Figure S6A,B), suggesting that DNA damage was not a major contributor to acidic pH‐induced nucleolar stress.

Oxidative stress induces nucleolar oxidation, leading to S‐glutathionylation of NPM1 at cysteine 275, which promotes its translocation to the nucleoplasm (Yang et al. 2016). To investigate whether acidic pH‐induced nucleolar stress resulted from a redox mechanism, we analyzed the production of the reduced (GSH) and oxidized (GSSG) forms of glutathione. Acidic pH reduced the amount of GSH but increased the amount of GSSG, significantly reducing the ratio of GSH/GSSG compared to that at neutral pH (Figure 4A–C). Because glutathione S‐transferase Pi 1 (GSTP1) catalyzes protein S‐glutathionylation (Mazari et al. 2023), we thus knocked down GSTP1 to investigate its involvement in NPM1 translocation (Supplementary Information Figure S7A). However, NPM1 localization was not affected by GSTP1 knockdown under neutral or acidic conditions (Supplementary Information Figure S7B,C). To test whether the redox imbalance is involved in NPM1 translocation, we treated the cells with dithiothreitol (DTT), a protein disulfide reductant. DTT treatment did not alter NPM1 localization at a neutral pH (Figure 4D,E). In contrast, NPM1 translocation induced by an acidic pH was partially restored by DTT treatment (Figure 4D,E). Additionally, DTT treatment attenuated the acidic pH–induced increase in p53 levels, although a reduction in p53 levels was also observed after DTT treatment in neutral pH (Figure 4F,G). Next, we analyzed the effect of N‐acetylcysteine (NAC), an antioxidant, on cell proliferation under acidic conditions, as prolonged incubation with DTT exhibited cytotoxicity. Similar to DTT, NAC treatment also partially restored NPM1 localization and decreased p53 levels (Supplementary Information Figure S7D–F). Interestingly, cell proliferation was not restored by NAC treatment under acidic pH, despite the recovery of p53 levels (Figure 4H). These findings suggest that oxidative stress partially contributes to NPM1 translocation and p53 accumulation, but attenuation of oxidative stress alone is insufficient to rescue acidic pH–induced inhibition of cell growth.

Figure 4.

Partial contribution of oxidative stress to nucleolar stress induced by acidic pH. (A–C) Quantitative analysis of the ratio of GSH and GSSG (A), total GSH (B), and GSSG (C) amounts under neutral and acidic conditions. HN30 cells were subjected to neutral or acidic conditions for 24 h. Means ± S.D. (n = 3 independent experiments). (D) Representative anti‐NPM1 immunofluorescence images of HN30 cells at 24 h after neutral or acidic treatment with or without dithiothreitol (DTT). DTT was applied to each medium for at least an hour. Scale bar: 10 μm. (E) Ratio of NPM1 signals in the nucleoli and nucleoplasm at 24 h after neutral and acidic treatment with or without DTT. Each ratio is represented as a box and whisker plot showing the outliers, distribution intervals, interquartile range (box), and median. Each plot shows the NPM1 ratio in each cell. Each group contains at least 184 cells. Three independent experiments were performed. (F) Representative western blot images of p53 and GAPDH (upper) and immunofluorescence staining for p53 (lower). HN30 cells were treated under neutral or acidic conditions for 24 h. DTT was added to each medium for at least an hour prior to analysis. (G) Quantitative analysis of p53 protein levels. Relative p53 levels were normalized to GAPDH. Means ± S.D. (n = 3 independent experiments). (H) Quantitative analysis of cell proliferation under neutral and acidic conditions in the presence or absence of N‐acetylcysteine (NAC). Cell numbers were counted at 72 h after treatment. Three independent experiments were performed. *p < 0.05, **p < 0.01, two‐tailed Student's t‐test (A–C), Kruskal‐Wallis test followed by Mann‐Whitney U test and false discovery rate correction (E), or one‐way ANOVA with Tukey's multiple comparison test (G, H).

2.6. Neutral pH Promotes Recovery from Acidic pH‐Induced Nucleolar Stress

To determine whether acidic pH‐induced nucleolar stress was reversible, we switched the culture conditions back to neutral pH. Cells were incubated under acidic conditions for 48 h, after which the medium was changed to neutral pH (Figure 5A). Intriguingly, pre‐rRNA transcript levels began to recover within an hour of the medium change (Figure 5B). Similarly, NPM1 localization to the nucleolus was rapidly restored upon neutral pH recovery (Figure 5C,D). Cell proliferation resumed after the pH was restored to neutral, in contrast to the sustained growth arrest under continuous acidic conditions (Figure 5E). Overall, acidic pH‐induced nucleolar stress and growth inhibition are reversible upon normalization of extracellular pH.

Figure 5.

Reversible nucleolar stress after recovery from acidic to neutral pH. (A) Experimental flow to analyze pre‐rRNA levels, NPM1 localization, and cell growth. Forty‐eight hours after cells were treated with acidic pH, the medium was changed to neutral pH or kept at acidic pH. (B) Change in pre‐rRNA levels after recovery treatment, determined by qPCR. HN30 cells were treated with acidic pH for 48 h. Subsequently, the medium was changed to neutral pH. Total mRNA was extracted at each time point after treatment. Means ± S.D. (n = 3 independent experiments). (C) Representative anti‐NPM1 immunofluorescence images of HN30 cells after recovery treatment. Scale bar: 10 μm. (D) Ratio of NPM1 signals in the nucleoli and nucleoplasm after recovery treatment. Each ratio is represented as a box and whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Each plot shows the NPM1 ratio in each cell. Each group contains at least 310 cells. Four independent experiments were performed. (E) Quantitative analysis of the cell proliferation assay after recovery treatment. Cell numbers were counted each time after treatment. The medium was changed to neutral pH at 48 h after acidic treatment or kept at acidic pH. Zero hour represents 48 h after acidic treatment. Means ± S.D. (n = 3 independent experiments). *p < 0.05, **p < 0.01, one‐way ANOVA with Tukey's multiple comparison test (B), Kruskal‐Wallis test followed by Mann‐Whitney U test and false discovery rate correction (D), or two‐way ANOVA followed by Sidak's multiple comparisons post hoc tests (E).

3. Discussion

We demonstrated for the first time that acidic pH downregulates pre‐rRNA levels, resulting in nucleolar stress induction. Impairment of RiBi is known to trigger a nucleolar stress response by promoting the translocation of nucleolar proteins, such as NPM1, into the nucleoplasm (Yang et al. 2018). As rRNA synthesis is a rate‐limiting step in RiBi, high levels of Pol I transcription are required to sustain ribosome production in proliferating cells (Laferté et al. 2006). Indeed, the RNA‐seq results revealed a decrease in Pol I mRNA levels under acidic conditions. The mechanistic target of rapamycin (mTOR) signaling pathway is central to regulating Pol I transcription, primarily via phosphorylation‐dependent activation of the transcription factor TIF‐IA (Mayer and Grummt 2006). In particular, phosphorylation of mTOR downstream targets, such as p70 S6 kinase (S6K) and p85 S6K, is essential for TIF‐IA activity (Chauvin et al. 2014). A previous study reported that acidic extracellular pH inhibits S6K phosphorylation within 30 min in MCF‐7 breast cancer cells (Balgi et al. 2011). Consistently, we observed a marked reduction in S6K phosphorylation in HN30 cells after an hour of acidic pH exposure (Supplementary Information Figure S8A). Likewise, Torin‐1, an mTOR inhibitor, induced NPM1 translocation and inhibited cell growth (Supplementary Information Figures S8B–D). Additionally, nucleolar stress can also be triggered by exposure to cytotoxic agents, ultraviolet radiation, or oxidative stress (Yang et al. 2018). Although DNA damage was not significantly increased following acidic pH treatment, measurement of intracellular glutathione revealed that oxidative stress was elevated under acidic conditions, as evidenced by a decrease in total GSH and an increase in GSSG levels. Treatment with DTT and NAC partially reversed the acidic pH‐induced NPM1 translocation, suggesting that a shift in redox balance toward an oxidized state contributes to nucleolar stress induction. Taken together, acidic pH‐induced nucleolar stress may result from inhibition of mTOR activity and oxidative stress.

We found that acidic pH induced accumulation of p53 and upregulation of p21. G1 arrest occurs under acidic conditions in HN30 cells. Acidic pH induced accumulation of p53 and upregulation of p21, supporting the observed changes in cell cycle kinetics. However, p53‐depleted HN30 cells still exhibited growth inhibition under acidic conditions, indicating that p53 accumulation alone is insufficient to account for acidic pH–induced growth inhibition. Moreover, NAC treatment failed to restore cell proliferation despite a reduction in p53 levels and partial recovery of NPM1 localization. Similarly, Torin‐1 treatment inhibited cell growth while reducing p53 and p21 levels (Supplementary Information Figure S8E). These findings suggest that p53 levels are not only the master regulator of cell proliferation under acidic conditions. Although p53 contributes to the cellular response to acidic stress, p53‐independent mechanisms also appear to play a critical role in acidic pH–induced growth inhibition. Notably, RPL11 is reported to promote the degradation of E2F1 by binding to MDM2 under nucleolar stress conditions (Donati et al. 2011; Maehama et al. 2023). As E2F1 plays a critical role in G1‐to‐S phase transition, its downregulation may lead to G1 arrest independently of p53. Further studies will be required to elucidate the signaling pathways that regulate cell growth under acidic conditions in this cellular context.

We demonstrated for the first time that acidic pH induces nucleolar stress in HN30 cells with impairment of RiBi and a shift in the redox balance, ultimately leading to growth inhibition. To assess whether this phenomenon is cell line‐dependent, we examined HSC3 and CAL33, human oral cancer cell lines, as well as HeLa, which is a human cervical cancer cell line, and A549, a human lung cancer cell line. NPM1 translocation and reduced cell proliferation were observed under acidic conditions in all tested cell lines, although the extent varied (Supplementary Information Figure S9A,B), suggesting that nucleolar stress responses may be broadly activated within the acidic TME of solid tumors. Notably, the growth‐inhibitory effects of acidic pH were reversible, as returning the culture conditions to neutral pH restored pre‐rRNA expression and NPM1 localization, accompanied by resumption of cell proliferation. This finding implies that cancer cells can adapt to fluctuating TME conditions. Anticancer treatments, such as radiotherapy, have been reported to remodel the TME by reoxygenation and other changes (Hosonuma and Yoshimura 2023; Ma et al. 2024; Moeller et al. 2004; Ohta et al. 2010). Although TME remodeling may enhance treatment efficacy, it may also create permissive conditions for tumor cell repopulation, ultimately contributing to recurrence. Thus, targeting the nucleolar stress response may represent a novel and promising therapeutic strategy. However, further studies are essential to determine how nucleolar stress response modulation within the TME can be leveraged to prevent tumor repopulation and enhance treatment efficacy.

4. Materials and Methods

A complete description of the materials and methods is provided in the Supporting Information.

4.1. NPM1 Signal Quantification

NPM1 localization was quantified using automated image analysis. Fluorescence images were processed in MATLAB (R2024b; MathWorks, Natick, MA) by converting the DAPI and NPM1 channels into separate 8‐bit grayscale images. Nuclear segmentation was achieved using the “nuclei” neural network model from Cellpose (https://www.cellpose.org/). Nucleolar segmentation in the NPM1 channel was performed using a custom pipeline combining classical image‐processing techniques, including Bilateral filtering, Gaussian Laplacian filtering, and Adaptive Thresholding. Segmented nucleoplasm and nucleolar masks were used to measure the mean pixel intensity per cell. For normalization, at least 171 cells per group were analyzed in triplicate.

4.2. Proximity Ligation Assay (PLA)

The assay was performed using the Duolink™ In Situ Starter Set ORANGE (Millipore Sigma, St. Louis, MO) according to the manufacturer's instructions.

4.3. RNA Sequencing

RNA‐seq analysis was performed as described in Supporting Information.

4.4. Statistical Analysis

A two‐tailed Student's t‐test, one‐way ANOVA with Tukey's multiple comparisons test, the Kruskal–Wallis test followed by the Mann–Whitney U test and false discovery rate correction, or two‐way ANOVA with Tukey's or Sidak's post‐hoc test was used. Statistical significance was set at p < 0.05. Detailed information on the statistical analyses is provided in the Supporting Information.

Author Contributions

Carulina Bueno de Mesquita: conceptualization, data curation, formal analysis, investigation, methodology, validation, writing – original draft, review and editing. Atsushi Kaida: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, validation, writing – original draft, review and editing. Hitomi Nojima: validation, writing – original draft, review and editing. Masahiko Miura: funding acquisition, supervision, validation, writing – original draft, review and editing.

Ethics Statement

This study did not involve human participants or animals. Only established human/mammalian cell lines were used, and therefore, institutional review board (IRB) approval was not required.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1

Supporting File 2

Supporting File 3

Data Availability Statement

Data will be made available upon reasonable request.