Acidic Tumor Microenvironment Promotes 5-Fluorouracil Resistance Via Lactate-Driven HIF-1α/MCT4 Activation and Epigenetic Remodeling in Gastric Cancer

Introduction: Tumor acidosis is associated with poor therapeutic response. Yet, it remains unclear whether 5-fluorouracil (5-FU) resistance in gastric cancer is driven by low extracellular pH per se or by lactate as a signaling metabolite. Methods: The human gastric cancer cell line AGS was exposed to extracellular acidosis (pH 6.6; short-term or chronic) or exogenous lactate at neutral pH. 5-FU sensitivity, apoptosis, and lactate production were assessed. Extracellular pH was buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); lactate metabolism/ export was disrupted by LDHA inhibition (FX11) or siRNA-mediated MCT4 knockdown. Global histone methylation was analyzed by Western blot, and qPCR quantified H3K4me3 and H3K27me3 occupancy at SLC16A3 (MCT4) and LDHA promoters. Results: Acidosis reduced 5-FU sensitivity, increased lactate output, suppressed apoptosis, and activated hypoxia- inducible factor 1 alpha (HIF-1α)/monocarboxylate transporter 4 (MCT4) signaling. Buffering pH or inhibiting lactate synthesis/export restored 5-FU sensitivity, indicating a reversible, metabolism-dependent phenotype. Under neutral pH, exogenous lactate alone induced reduced 5-FU sensitivity and activated HIF-1α signaling, supporting a pH-independent role for lactate. Acid exposure increased global H3K27me3 and H3K9me3, which normalized after buffering, whereas SLC16A3 and LDHA promoters showed H3K4me3 enrichment with reduced H3K27me3. discussion: In this study, we demonstrate that an acidic tumor microenvironment promotes resistance to 5-FU in gastric cancer through a lactate-dependent mechanism that integrates metabolic adaptation with epigenetic regulation. Our data show that acidic exposure suppresses apoptosis and increases 5-FU tolerance, accompanied by enhanced glycolysis, lactate accumulation, and activation of the HIF-1α/MCT4 axis. Importantly, exogenous lactate was sufficient to induce similar resistance under neutral pH conditions, indicating that lactate acts as a key signaling mediator rather than extracellular acidity per se. Tumor acidosis has traditionally been viewed as a secondary consequence of altered metabolism [23]; however, our findings support a more active role for lactate in driving adaptive survival programs. By upregulating MCT4, cancer cells efficiently export lactate, preventing intracellular acidification while sustaining high glycolytic flux. This metabolic configuration confers a survival advantage under chemotherapeutic stress and aligns with previous reports linking lactate metabolism to aggressive tumor behavior and poor prognosis [24]. A major advance of this study is the identification of epigenetic remodeling as a mechanistic bridge between transient metabolic stress and sustained chemoresistance. At the global level, acidic stress induced an increase in repressive histone methylation marks, including H3K27me3 and H3K9me3, consistent with a generalized chromatin response to environmental stress. Notably, this global repression was reversible upon pH buffering, highlighting the dynamic nature of acid-induced epigenetic changes. Despite this global trend toward repression, locus-specific chromatin analysis revealed a contrasting pattern at key metabolic genes. ChIP-qPCR demonstrated selective enrichment of the activating histone mark H3K4me3 and concomitant depletion of H3K27me3 at the promoters of MCT4 and LDHA. These findings indicate that acidic stress and lactate signaling do not uniformly suppress transcription but instead promote targeted epigenetic activation of genes critical for lactate production and export. Such selective remodeling provides a plausible mechanism by which cancer cells stabilize a lactate-driven metabolic state that supports chemoresistance. The coexistence of global chromatin repression with locus-specific activation underscores the complexity of epigenetic adaptation in response to metabolic stress. Rather than inducing a broad transcriptional program, lactate appears to reinforce a focused metabolic axis through epigenetic priming, ensuring sustained expression of genes essential for survival under chemotherapeutic pressure. Disruption of the HIF-1α/MCT4 pathway attenuated both metabolic and epigenetic alterations, further supporting the functional coupling between lactate signaling and chromatin regulation. Several limitations of this study should be acknowledged. While we focused on histone methylation, other epigenetic mechanisms, such as DNA methylation or RNA modifications, may also contribute to acid-induced chemoresistance. In conclusion, our study reveals that an acidic tumor microenvironment promotes 5-FU resistance through lactate-mediated metabolic reprogramming and selective histone methylation remodeling. By epigenetically stabilizing the HIF-1α/MCT4 axis, lactate enables gastric cancer cells to adapt to chemotherapeutic stress. These findings highlight lactate metabolism and its epigenetic consequences as potential therapeutic targets for overcoming chemotherapy resistance in gastric cancer. Discussion: These findings suggest that acidic stress is transduced through lactate signaling to selectively activate chromatin at lactate-metabolism genes, stabilizing an adaptive survival program under chemotherapy. Conclusion: Lactate signaling, rather than low pH alone, underlies a reversible 5-FU-resistant state in gastric cancer cells and may be mitigated by targeting lactate metabolism and/or extracellular pH control.