Background

Phosphatase of Regenerating Liver-3 (PRL-3) is frequently upregulated in cancers, particularly in multiple myeloma (MM). MM cells are highly dependent on the bone marrow microenvironment, which is notably more acidic than the physiological pH of blood. The Warburg effect, where cancer cells favor glycolysis despite sufficient oxygen, is induced by PRL-3 at neutral pH, but its role in adapting MM cells to acidic conditions remains underexplored. PRL-3 has been implicated in increasing intracellular pH in kidney cells, potentially supporting survival in hostile, acidic microenvironments. Lactate export, essential for sustaining high glycolytic flux in cancer, is mediated by monocarboxylate transporters, predominantly MCT1 and MCT4, which co-transport protons and lactate, thereby influencing pH balance. Other proton transporters, such as sodium-hydrogen exchanger 1 (NHE1) and v-ATPase, are hypothesized to help maintain optimal intracellular pH. Understanding how PRL-3 interacts with these transporters may reveal novel metabolic vulnerabilities in MM.

Methods

MM cell lines INA-6, JJN3, and RPMI8226 were transduced to overexpress PRL-3 (PRL-3 cells) or to serve as controls (mock cells). Expression of NHE1, v-ATPase, MCT1, and MCT4 was assessed by immunoblotting. Cell viability and ATP levels were measured using Annexin V/PI flow cytometry and CellTiter-Glo. Glycolytic and mitochondrial activity were evaluated using Seahorse Glycolysis Stress and Cell Mito Stress tests. Pharmacologic inhibition of lactate and proton transport was performed using Syrosingopine (dual MCT inhibitor), AZD0095 (MCT4 inhibitor), AZD3965 (MCT1 inhibitor), Bafilomycin A1 (v-ATPase inhibitor), and ethyl isopropyl amiloride (EIPA; NHE1 inhibitor).

Results

PRL-3 cells demonstrated both increased glycolysis and basal respiration compared to mock cells, particularly under acidic conditions. At neutral pH, PRL-3 cells exhibited reduced MCT1 and elevated MCT4 expression. MCT1 levels were unchanged in both cell lines across pH 6.0–7.5. While MCT4 expression was higher in PRL-3 cells at all pH levels, it dropped sharply in both PRL-3 and mock cells at pH values below 6.5. NHE1 expression was maintained in PRL-3 cells at pH 6.5 and 6.0 but diminished in mock cells. V-ATPase expression remained constant in both cell types and at all pH levels tested.

Inhibition assays at pH 6.5 and 7.5 revealed that Syrosingopine reduced ATP levels in both cell types. AZD0095 had no blocking effect but paradoxically elevated ATP levels at pH 6.5. Combining AZD0095 and AZD3965 was harmless to PBMCs but decreased ATP levels and viability in both PRL-3 and mock cells, with the greatest effect observed in PRL-3 cells.

At pH 7.5, 4 µM EIPA had no effect on PRL-3 or mock cells. At pH 6.0, PRL-3 conferred a marked proliferative advantage, which was abolished by treatment with EIPA. Bafilomycin A1 was cytotoxic at pH 7.5 (IC50: 3 nM) but inactive at pH 6.5. Combined with EIPA, Bafilomycin A1 regained potency at pH 6.5.

Conclusions

These findings highlight PRL-3 as a regulator of metabolic adaptation in MM, promoting both glycolysis and oxidative phosphorylation, particularly under acidic conditions that mimic the bone marrow microenvironment. Lactate export via MCT1 and MCT4 appears critical for maintaining energy production and pH balance in myeloma cells but not in PBMCs. Thus, dual MCT1 and MCT4 inhibition may be a promising target for clinical treatment with minimal hematological toxicity. Additionally, NHE1 seems to be critical for survival in acidic conditions, while v-ATPase is more important at neutral pH. This suggests compensatory roles among transporters and pathways whose importance varies depending on the pH level. Increased metabolism and proton transport are associated with high PRL-3 expression, supporting PRL-3 as a key protein for cell survival in acidic conditions and a potential therapeutic target in myeloma treatment.

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2025
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