(714d) Hypoxia Overrides Acidosis-Induced Suppression of Cancer Cell Migration and Dissemination

Solid malignant tumors are characterized by hypoxic and acidic stresses due to compromised vasculature and altered metabolism. While hypoxia contributes to local acidification, tumor acidosis can occur independently through the Warburg effect, whereby cancer cells favor aerobic glycolysis, producing lactic acid even in normoxia. This results in an acidic extracellular pH (pHe) of 6.2–6.9, compared to the physiological pH ~ 7.4. To survive, cancer cells maintain a relatively alkaline intracellular pH (pHi) by upregulating pHi-regulatory mechanisms, supporting proliferation and metastasis. Although hypoxia is known to drive aggressive cancer phenotypes, the role of acidosis in metastasis remains debated. Some studies link acidosis to enhanced metastatic potential, while others report inhibited migration in lung cancer cells and reduced dissociation in melanoma spheroids at pHe ~6.4. Because of the contradictory results, we aimed to systematically investigate how both short-term exposure (24 h) and long-term adaptation to extracellular acidosis (spanning 23 days to 10 weeks) affect cancer cell behavior using comprehensive in vitro and in vivo models. Additionally, we examined the combined impact of hypoxia and acidosis on cellular bioenergetics, migration, and resilience, focusing specifically on the sodium-hydrogen exchanger isoform-1 (NHE1), a critical regulator of pHi, cytoskeletal dynamics, and cell migration.

Our results demonstrate that exposure of cancer cells to a pH_e of 6.4 significantly suppresses cell proliferation and ATP production rate. Acidosis also inhibits the dissociation of tumor cells from spheroids, reduces cellular migration in vitro, and decreases extravasation in vivo, as demonstrated in chick embryo and mouse models. Furthermore, RNA-seq analysis revealed that short-term and long-term adaptation to acidic conditions elicit a similar gene expression profile, characterized by widespread suppression of cellular functions. Mechanistically, acute acidosis (~2 hours) disrupts NHE1 polarization and activity, leading to rapid suppression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Importantly, restoring Akt activity through a constitutively active mutant rescues the migratory impairment under acidic conditions, highlighting a critical bidirectional crosstalk between NHE1 and the PI3K/Akt signaling axis. At later time points, the inhibition of PI3K/Akt prevents Yes-associated protein (YAP) translocation to the nucleus, resulting in decreased expression of NHE1. Cancer cells can rapidly adjust their intracellular pH within minutes upon exposure to acidosis. However, prolonged exposure (≥24 hours) leads to decreased NHE1 expression and delayed recovery when cells are returned to physiological pH_e. The longer the acidic exposure, the more prolonged the delay in recovering NHE1 expression and motility. In line with this memory effect, reduced extravasation capacity was observed when cells preconditioned in acidic environments are injected into chick embryos or mouse models. Importantly, we found that hypoxia prevents acidosis-induced migratory defects by maintaining cellular bioenergetic balance through an overcompensatory increase in glycolytic activity. This heightened glycolysis under hypoxic conditions preserves NHE1 activity and prevents bioenergetic decline, thereby sustaining migratory potential despite the exposure to acidic media. Inhibiting glycolysis under combined hypoxic and acidic conditions suppresses NHE1 activity, while NHE1 inhibition, in turn, reduces glycolysis and impairs migration. These findings reveal a critical feedback regulatory relationship between NHE1 and glycolysis that supports enhanced migration under dual stress conditions.

Our findings highlight that the aggressive phenotype of cancer cells in acidic microenvironments is critically dependent on the presence of hypoxia. While acidosis alone suppresses cancer cell migration and dissemination, hypoxia overrides these inhibitory effects, emerging as the key driver of aggressive tumor phenotype under acidic conditions.