Glyoxalase 2 Drives D-Lactate Oncometabolite Signaling to Promote Prostate Cancer Aggressiveness via FAK/Src Activation

TL;DR

This study shows that the enzyme Glyoxalase 2 (Glo2) promotes prostate cancer aggressiveness by producing D-lactate, which activates cancer-related signaling pathways.

Contribution

The study reveals a novel mechanism where Glo2-generated D-lactate drives tumor aggressiveness via FAK/Src signaling in PTEN-deficient prostate cancer.

Findings

Glo2-dependent D-lactate accumulation promotes EMT-like plasticity in PTEN-deficient prostate cancer cells.

D-lactate enhances migration and invasion through activation of the FAK/Src signaling pathway.

The Glo2–D-lactate axis may contribute to metabolic rewiring in aggressive prostate cancer.

Abstract

Glyoxalase 2 (Glo2) is a key enzyme of the glyoxalase system that catalyzes the conversion of S-lactoylglutathione (LSG) into glutathione (GSH) and D-lactate. In prostate cancer (PCa), we previously demonstrated that the oncogenic PTEN-PI3K–AKT–mTOR–ERα signaling pathway upregulates Glo2, leading to intracellular D-lactate accumulation and enhanced cell migration, invasiveness, and expression of epithelial-to-mesenchymal transition (EMT)-associated markers. However, whether D-lactate acts as a bioactive metabolic signal contributing to tumor aggressiveness remains unclear. Here, after confirming our previous findings, we demonstrate—using Glo2 silencing, ectopic expression, pharmacological inhibitors, and exogenous D-lactate supplementation—that Glo2-dependent D-lactate accumulation promotes EMT-like plasticity, migration, and invasion in PTEN-deficient PCa cells via a functional link with FAK/Src signaling. Collectively, these results suggest that the Glo2–D-lactate axis may contribute to metabolic rewiring associated with aggressive behavior in PTEN-deficient PCa, warranting further in vivo studies to evaluate its potential as a therapeutic target to limit tumor progression.

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1. Introduction

Glyoxalase 2 (Glo2) is an enzyme of the glyoxalase pathway that catalyzes the hydrolysis of S-lactoylglutathione (LSG) into D-lactate and reduced glutathione (GSH) [1]. Through this reaction, Glo2 occupies a strategic, yet still underexplored, position at the interface between metabolic detoxification, antioxidant defense, and intracellular signaling [2,3,4,5], particularly in cancer.

Prostate cancer (PCa) progression is strongly influenced by metabolic and redox adaptations that sustain tumor growth, invasion, and resistance to cellular stress [6,7]. Alterations in oncogenic signaling pathways, most notably loss of PTEN function and hyperactivation of the AKT pathway, are frequent events in advanced PCa and are known to profoundly reshape cellular metabolism and redox homeostasis [8,9,10]. In this context, several studies have identified Glo2 as a downstream target of oncogenic signaling pathway [1,2,11,12]. In particular, in PCa cells we previously demonstrated that activation of the PTEN-PI3K–AKT–mTOR-ERα axis leads to increased Glo2 expression and enzymatic activity [1], resulting in intracellular D-lactate accumulation and enhanced migratory and invasive capacities, as well as modulation of epithelial-to-mesenchymal transition (EMT)-associated markers [1].

EMT is a phenotypic program related to increased cellular plasticity during cancer progression, enabling epithelial tumor cells to acquire mesenchymal-like features that support migration and invasion [13,14]. While EMT has traditionally been viewed as a full transcriptional program driven by extracellular cues [15], in PCa it is increasingly recognized as a partial or hybrid process, often reflected by coordinated changes in epithelial and mesenchymal markers rather than a complete phenotypic conversion.

Growing evidence indicates that intracellular metabolites can function as signaling molecules [16], linking metabolic rewiring to EMT-associated gene expression and cellular plasticity [17,18,19,20,21,22].

Among these metabolites, D-lactate, the downstream product of Glo2-mediated LSG metabolism, has been proposed as an oncometabolite [23]. Once considered merely a byproduct, D-lactate influences mitochondrial redox balance and the NADH/NAD^+^ ratio [24], creating a metabolic context that may favor activation of redox-sensitive pathways such as FAK/Src, central regulators of cell motility and EMT-associated features [25,26,27,28].

Based on our previous evidence that the PTEN-PI3K–AKT–mTOR-ERα axis regulates Glo2 expression and D-lactate production in PCa cells [1], and considering the emerging role of D-lactate as a potential oncometabolite, we hypothesized that Glo2-derived D-lactate may function as a metabolic signal promoting aggressive phenotypes, via a functional link with FAK/Src signaling, a possibility that has not been previously investigated. To address this hypothesis, we first confirmed the PTEN-PI3K–AKT–mTOR–ERα–Glo2–D-lactate axis in our experimental models [1], and subsequently investigated the role of Glo2-dependent D-lactate accumulation in promoting EMT-associated cellular plasticity, migration, and invasion via a functional link with FAK/Src pathway in PTEN-deficient PCa cells. Together, these observations support a role for D-lactate as an emerging metabolite linking Glo2-dependent metabolic rewiring to biologically aggressive tumor behavior and provide a rationale for further investigation of the Glo2–D-lactate axis in advanced PCa.

2. Materials and Methods

2.1. Use of AI Tools

Generative artificial intelligence (GenAI) tools were used solely to improve the clarity, readability, and grammatical correctness of the English language. The use of these tools did not affect the study design, data collection, analysis, interpretation of results, or the scientific content of the manuscript. All responsibility for the final content lies with the authors.

2.2. Materials

Cell culture medium, fetal bovine serum (FBS), and penicillin/streptomycin, as well as LY294002 (LY; 50 μM in DMSO, 72 h), MK2206 (MK; 10 μM in DMSO, 48 h), rapamycin (Rapam; 100 nM in DMSO, 48 h) and D-lactic acid (2.5 mM, 4 or 24 h) were purchased from Thermo Fisher Scientific (Milan, Italy). The concentration of exogenous D-lactic acid was selected based on previous in vitro studies investigating the signaling-related effects of lactate isomers, in which millimolar concentrations are commonly required to achieve reproducible intracellular signaling responses in cultured cells over short-term exposure periods [29,30,31]. ICI 182,780 (50 nM in DMSO, 5 h) and PF-573228 (5 µM, 24 h) were purchased from Sigma-Aldrich (Milan, Italy). For all compounds prepared in DMSO, the final solvent concentration did not exceed 0.01%. Control cells received the corresponding volume of DMSO alone.

2.3. Immunohistochemistry

Formalin-fixed, paraffin-embedded (FFPE) prostate adenocarcinoma specimens (n = 60), obtained as residual archival material from the Division of Pathology of the University of Perugia, were analyzed [32]. Approval for the use of human samples was obtained from the Ethics Committee of the University of Perugia (protocol no. 2019-30), and all procedures complied with the Declaration of Helsinki. Tissue specimens were collected from patients undergoing radical prostatectomy. Histopathological evaluation was performed using the Gleason scoring (GS) system, and tumor staging was assigned according to the pTNM classification.

Formalin-fixed paraffin-embedded tissue sections (4 μm) were deparaffinized in xylene and rehydrated through graded ethanol. Immunohistochemical staining was conducted using a Bond III automated platform (Leica Biosystems, Newcastle Upon Tyne, UK) with the Bond™ Polymer Refine Detection kit. Sections were incubated for 30 min with an anti-human Glo2 antibody (1:40 dilution) without prior antigen retrieval, followed by nuclear counterstaining with hematoxylin. Immunoreactivity was independently assessed by two experienced pathologists (AS and GB). Staining was semi-quantitatively evaluated based on both signal intensity (0, 1+, 2+, 3+) and the percentage of positive tumor cells (0–25%, 25–50%, 50–75%, 75–100%). An overall immunoreactivity score was calculated by multiplying intensity and extent scores, resulting in values of 0, 1+, 2+, 3+, 6+, or 9+ [32,33]. Scores between 0 and 2+ were considered negative or weak, whereas scores ≥ 3+ were classified as moderate to strong expression.

2.4. Cell Lines

The human prostate cancer cell lines LNCaP, DU145, and PC3, which differ in their degree of aggressiveness, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The corresponding database access numbers are: LNCaP (ATCC^®^ CRL-1740™), DU145 (ATCC^®^ HTB-81™), and PC-3 (ATCC^®^ CRL-1435™). LNCaP, derived from a lymph node metastasis, are androgen-sensitive and exhibit a low-aggressive phenotype. DU145 cells, originating from a brain metastasis, are androgen-independent and display an intermediate level of aggressiveness. PC3, derived from a bone metastasis, are androgen-independent and represent a highly aggressive PCa model. All cell lines were cultured in accordance with the manufacturer’s protocol at 37 °C in 5% CO_2_.

2.5. Cell Extracts

Total proteins lysates were prepared by lysing cells in RIPA buffer (Thermo Fisher Scientific, Milan, Italy) as reported previously [32]. Protein concentrations were quantified using the Lowry assay, with bovine serum albumin used as the calibration standard [34].

2.6. Glo2, E-Cadherin, Vimentin, PTEN, p-AKT, p-mTOR, p-FAK and p-Src Evaluation

Glo2, E-cadherin, Vimentin, PTEN, p-AKT, p-mTOR, p-FAK, and p-Src levels were evaluated using specific ELISA kits, according to the manufacturer’s instructions. The kits employed were: human Glyoxalase II ELISA Kit (cat. ELH-GLYOX2-1), human E-cadherin ELISA Kit (cat. ab233611), human Vimentin ELISA Kit (cat. ab246526), human PTEN ELISA Kit (cat. ab206979), human AKY3(pS473) ELISA Kit (cat. ab270887) and human phospho-mTOR(S2448) ELISA Kit (cat. ab279868), all from Prodotti Gianni (Milan, Italy). Phosphorylated FAK levels were measured using the Human FAK [Phospho] [pY397] ELISA Kit (cat. KHO0441, Thermo Fisher Scientific, Milan, Italy), while phosphorylated Src levels were determined using PathScan^®^ Phospho-Src [Tyr416] Sandwich ELISA Kit (cat. 7953, Cell Signaling Technology, Danvers, MA, USA). Phosphorylation levels of FAK (Tyr397) and Src (Tyr416) were quantified in total cell lysates and therefore reflect global changes in protein phosphorylation rather than subcellular or spatially resolved signaling dynamics.

2.7. Glo2 Enzyme Activity Assay

Glo2 activity was measured using Glyoxalase II Activity Assay Kit (cat. ab273319, Cambridge, MA, USA) [35].

2.8. RNA Extraction, cDNA Sythesis and qRT-PCR

RNA isolation, cDNA synthesis and qRT-PCR were measured as detailed previously [32,36,37]. Briefly, total cellular RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific, Milan, Italy) and cDNA was synthesized from 1 µg of total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Milan, Italy). qRT-PCR was performed using TaqMan chemistry (Glo2) or SYBR Green chemistry (E-cadherin, vimentin) on a MX3000P Real-Time PCR System (Agilent Technology, Milan, Italy). Gene expression levels were normalized to β-actin. When SYBR Green detection was used for gene expression analysis, β-actin was also detected using SYBR Green chemistry with the same primers employed for TaqMan assays. The sequences of oligonucleotide primers and probes were as follows: Glo2 (Forward 5′-AGAAAGCACGGGGTGAAACTG-3′, Reverse 5′-TACACCTTCAGTCCCGACTCC-3′, probe: 5′-CCACAGTGCTCACCACCCACCACC-3′, FAM-labeled); β-actin (Forward 5′-CACTCTTCCAGCCTTCCTTCC-3′, Reverse 5′-ACAGCACTGTGTTGGCGTAC-3′, probe 5′- TGCGGATGTCCACGTCACACTTCA-3′, FAM-labeled); E-cadherin (Forward 5′- TTGCGGAAGTCAGTTCAG-3′, Reverse 5′-CAGAGCCAAGAGGAGACC-3′), and vimentin (Forward 5′-GCACACAGCAAGGCGATGG-3′, Reverse 5′-GGAGCGAGAGTGGCAGAGG-3′). PCR reactions were performed in a total volume of 20 µL containing 25 ng of cDNA, TaqMan™ Universal PCR Master Mix (Thermo Fisher Scientific, Milan, Italy) or Brilliant II SYBR^®^ Green QPCR Master Mix (Agilent Technology, Milan, Italy), ROX Reference Dye (Agilent Technology, Milan, Italy), and 600 nM of each primer. Thermal cycling conditions consisted of 1 cycle at 95 °C for 5 min, followed by 45 cycles at 95 °C for 20 s and 60 °C for 30 s. To verify the absence of nonspecific amplification in SYBR Green assays, melting curve analyses were performed for all primer pairs under standard conditions. Relative gene expression was calculated using the 2^−(ΔΔCT)^ method [38].

2.9. D-Lactate Detection

D-lactate levels were determined with the D-lactate Assay Kit (Colorimetric) (cat. ab83429, Abcam, Cambridge, MA, USA) [39,40].

2.10. Migration and Invasion Assays

Migration was assessed using the CytoSelect 24-Well Cell Migration Assay kit (cat. CBA-100-5, DBA Italia S.r.l, Milan, Italy) while invasion was evaluated with the CytoSelect 24-Well Cell Invasion Assay kit (cat. CBA-110, DBA Italia S.r.l, Milan, Italy), as per the manufacturer’s guidelines. Briefly, cells were seeded at a density of 5 × 10^4^ cells per insert in serum-free medium, and migration toward 10% FBS was allowed to proceed for 10 h. To minimize potential confounding effects of cell proliferation, migration assays were performed over short time windows under serum-free conditions in the upper chamber. For invasion assays, cells were seeded at the same density and allowed to invade through matrix-coated inserts for 16 h under identical conditions.

2.11. siRNA Transfection

PCa cells were transfected with ON-TARGET plus SMARTpool siRNAs targeting Glo2 or PTEN, or with a non-targeting ON-TARGET plus siCONTROL pool as a negative control. All siRNAs were obtained from Dharmacon RNA Technologies (Carlo Erba, Milan, Italy). Transfections were carried out using DharmaFect 1 reagent, following the manufacturer’s protocol.

2.12. Ectopic Expression of Glo2 and PTEN

PCa cells were transfected with pCMV-Glo2 or pCMV-PTEN expression vectors, as recommended by the manufacturer (Origene, Tema Ricerca, Bologna, Italy). For PTEN restoration or knockdown experiments, cells were harvested 48 h after transfection for mRNA analysis, whereas enzymatic activity and D-lactate measurements were performed at 72 h to allow sufficient time for metabolic adaptation.

2.13. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism software (version 10.4.2). Comparisons between two groups were performed with Student’s t-test, while multiple groups comparisons were evaluated by one-way ANOVA followed by Tukey’s post hoc test. Immunohistochemistry results were assessed using Fisher’s exact test. Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Glo2 Is Upregulated in Aggressive PCa Tissues and Cell Models

To validate and extend our previously published findings, we first examined Glo2 expression in human PCa tissues. Glo2 protein level was assessed by immunohistochemistry in PCa specimens obtained from 60 patients who underwent radical prostatectomy for localized (T2) or locally advanced (T3) disease. Based on Gleason score (GS), patients were stratified into low-grade (LG; GS 6, n = 33) and high-grade (HG; GS ≥ 8, n = 27) groups. The two cohorts were age-matched (p > 0.05) [32]. Glo2 expression was significantly higher in HG tumors compared with LG tumors (Figure 1a). Specifically, the majority of LG cases (93.9%, 31/33) exhibited negative-to-weak Glo2 staining, whereas only 6.1% (2/33) showed moderate-to-strong expression. In contrast, 74.1% (20/27) of HG tumors displayed moderate-to-strong immunoreactivity, while 25.9% (7/27) showed negative-to-weak staining. No significant association was observed between Glo2 expression and clinical stage according to TNM classification (p > 0.05), indicating that Glo2 expression correlates with tumor grade and differentiation status rather than with anatomical tumor extension.

These observations were independently confirmed in vitro using PCa cell lines characterized by increasing biological aggressiveness. Consistent with tissue data, Glo2 protein expression and enzyme-specific activity progressively increased across LNCaP (low), DU145 (moderate), and PC3 (high) cells (Figure 1b) [1]. In agreement with our previous reports, enhanced migratory and invasive capabilities (Figure 1c), together with the activation of an EMT-like program (Figure 1d), were also confirmed [1]. This was evidenced by the reduced E-cadherin expression and concomitant upregulation of vimentin (Figure 1d and Figure S1). Based on these results, subsequent analyses were focused on DU145 and PC3 cells as representative models of distinct biological aggressiveness states. In line with Glo2 enzymatic activity, intracellular D-lactate levels were significantly higher in PC3 cells compared with DU145 cells (Figure 1e) [1].

3.2. Glo2 Silencing and Overexpression Confirm a Causal Role for This Enzyme in PCa Cell Aggressiveness

To further substantiate the functional role of Glo2 in PCa cell aggressiveness, we performed complementary loss- and gain-of-function approaches in PCa cell models. Consistent with our previous observations [1], silencing of Glo2 in highly aggressive PC3 cells (Figure S2) resulted in a significant reduction in cell migration (Figure 2a) and invasion (Figure 2b) compared with control siRNA-transfected (siCtr) cells. Glo2 depletion was accompanied by modulation of EMT-associated markers, as evidenced by increased E-cadherin and decreased vimentin expression (Figure 2c), supporting a causal role of Glo2 in promoting an EMT-like phenotype. This functional involvement was further corroborated by gain-of-function experiments in DU145 cells. Ectopic overexpression of Glo2 (Figure S3) significantly enhanced cell migration (Figure 2e) and invasion (Figure 2f) compared with pCMV-Ctr cells and promoted EMT-like features, as indicated by reduced E-cadherin and increased vimentin expression (Figure 2g). Importantly, D-lactate levels were modulated in accordance with Glo2 activity, being significantly reduced in Glo2-silenced PC3 cells (Figure 2d) and increased in Glo2-overexpressing DU145 cells (Figure 2h). Collectively, these results further confirm the contribution of the Glo2/D-lactate metabolic axis to EMT-associated marker regulation and to the migratory and invasive behavior of PCa cells [1].

3.3. The PTEN/PI3K/AKT/mTOR/ERα Axis Coordinately Regulates Glo2 Expression and D-Lactate Production in PCa Cells

We next sought to confirm and mechanistically extend our previous findings indicating that the PTEN/PI3K/AKT/mTOR/ERα signaling axis contributes to the differential regulation of Glo2 observed between DU145 (PTEN wild-type) and PC3 (PTEN-null) PCa cells [1]. After validating the cell-specific activation status of the PTEN/PI3K/AKT/mTOR/ERα pathway in these models (Figure S4) [32], we assessed the impact of genetic modulation of PTEN on Glo2 expression and metabolic output. Restoration of wild-type PTEN in PTEN-null PC3 cells resulted in a marked reduction in Glo2 mRNA expression and enzymatic activity (Figure 3a), accompanied by a significant decrease in intracellular D-lactate levels (Figure 3b). Conversely, PTEN knockdown in DU145 cells led to a substantial upregulation of Glo2 expression at both transcript and enzyme activity levels (Figure 3c), paralleled by a significant increase in D-lactate accumulation (Figure 3d).

Moreover, pharmacological inhibition of PI3K (by LY294002, LY), AKT (by MK2206, MK) or mTOR (by Rapamycin, Rapam) in PC3 cells (Figure 4a), as well as inhibition of ERα (by ICI 182,780) (Figure 4b), significantly abrogated Glo2 upregulation (Figure 4a,b) and reduced intracellular D-lactate levels (Figure 4c,d). Collectively, these results further confirm that Glo2 expression and D-lactate production are coordinately regulated by the PTEN/PI3K/AKT/mTOR/ERα signaling axis in aggressive PCa cells [1].

3.4. Glo2-Derived D-Lactate Activates FAK/Src Signaling and Promotes EMT-Associated Phenotypic Changes, Enhancing Migratory and Invasive Behavior in PC3 Cells

Having established that Glo2 regulates intracellular D-lactate accumulation in PC3 cells (Figure 2d) and sustains their aggressive phenotype (Figure 2a–c), we next investigated the previously unexplored functional role linking Glo2-dependent metabolic reprogramming to tumor cell motility. We hypothesized that Glo2 promotes PCa cell aggressiveness through a D-lactate-dependent activation of pro-migratory signaling pathways, focusing on the central focal adesion kinase (FAK)/Src signaling hub [41]. As a first step, we assessed FAK/Src activation in Glo2-silenced PC3 cells. Notably, silencing of Glo2 resulted in a significant reduction in the phosphorylation of both FAK (Tyr397) and Src (Tyr416), as determined by ELISA using two independent kits on total cell lysates (Figure 5a) [42,43]. These findings provide the first evidence that Glo2 activity is required for sustained FAK/Src signaling in aggressive PCa cells, identifying a novel functional link between Glo2-dependent D-lactate accumulation and activation of pro-migratory pathways.

To directly test whether D-lactate mediates the effects of Glo2 on FAK/Src signaling and tumor cell aggressiveness, we performed rescue experiments in Glo2-silenced PC3 cells by exogenous D-lactate supplementation. Exogenously supplied D-lactate is efficiently taken up by cells through monocarboxylate transporters (MCTs) [44,45,46] and, once internalized, localizes to the cytosolic compartment similarly to endogenously produced D-lactate [47]. Accordingly, exogenous D-lactate is widely used as a functional surrogate to investigate intracellular D-lactate-dependent signaling events [30,47,48]. Notably, treatment of Glo2-silenced PC3 cells with D-lactate (2.5 mM) resulted in a marked reactivation of both FAK (Tyr397) and Src (Tyr416) phosphorylation (Figure 6a). Consistent with these signaling changes, D-lactate supplementation significantly rescued the migratory and invasive capabilities of Glo2-depleted cells (Figure 6b), functionally supporting a key role for D-lactate as a downstream effector of Glo2-dependent PCa cell aggressiveness.

Finally, to directly assess the requirement of FAK signaling in mediating D-lactate-induced aggressiveness, Glo2-silenced PC3 cells supplemented with D-lactate were treated with the selective FAK inhibitor PF-573228 [49,50,51]. Pharmacological inhibition of FAK abrogated D-lactate-induced Src activation (Figure 7a) and significantly reduced cell migration and invasion (Figure 7b), demonstrating that FAK/Src signaling is essential for mediating the pro-aggressive effects of D-lactate in PC3 cells.

4. Discussion

Prostate cancer (PCa) is highly prevalent in men, and clinical outcomes worsen significantly as the disease acquires a more aggressive biological phenotype [52,53,54]. Understanding the processes that drive disease progression are therefore critical [55].

In this study, building on previous evidence that the PTEN-PI3K-AKT–mTOR–ERα axis regulates Glo2 expression and D-lactate production in PTEN-deficient PCa cells [1], we identified a previously unexplored functional link between Glo2-derived D-lactate and FAK/Src signaling that promotes migration, invasion and EMT-associated phenotypic traits.

Glo2, together with glyoxalase 1 (Glo1), forms the glyoxalase system, a conserved cellular pathway responsible for the metabolism of reactive dicarbonyl compounds, particularly methylglyoxal (MG), primarily produced during glycolysis [1]. MG can readily modify proteins forming advanced glycation end products (AGEs) such as MG-derived hydroimidazolones (MG-H1). Accumulation of MG and its adducts can lead to cellular dysfunction, oxidative stress, and inflammation [56,57]. MG removal proceeds through two sequential steps: Glo1 catalyzes the conversion of MG into SLG and Glo2 hydrolyzes SLG to D-lactate, in a glutathione (GSH)-dependent manner. In cancer, glyoxalase enzymes are often dysregulated, contributing to altered metabolism and enhanced survival [58]. While Glo2 has traditionally been viewed as a detoxifying enzyme within the glyoxalase system, our findings expand this paradigm by demonstrating that its metabolic product, D-lactate, acts as a bioactive mediator linking oncogenic metabolic rewiring to pro-migratory and pro-invasive signaling. Hence, beyond the established regulation of Glo2 by PTEN-dependent signaling, the present study shows that D-lactate accumulation is not merely a metabolic consequence of Glo2 upregulation but is functionally required to sustain FAK and Src activation, thereby driving migration, invasion and an EMT-like phenotype in PTEN-deficient PCa cells.

At the same time, while our study focused on Glo2 regulation and downstream signaling, D-lactate production is ultimately dependent on the coordinated activity of Glo1 and Glo2, as described above. Moreover, in our recent work, we demonstrated that PTEN-PI3K-AKT–mTOR–ERα-driven Glo1 overexpression sustains PC3 PCa cell growth through MG-H1/RAGE pathway desensitization, and that correlates with PCa aggressiveness [32]. Hence, it is plausible that Glo1 may indeed act in concert with Glo2 to contribute to D-lactate-dependent signaling in PC3 cells.

Finally, as described above, enhanced glycolytic flux, a hallmark of highly proliferating systems such as cancer, increases MG production, thereby activating Glo1/Glo2 and promoting D-lactate generation. In addition, our in vitro work has shown that ERα regulates Glo2-dependent D-lactate accumulation. Therefore, it would be of interest to investigate in future studies whether both glycolytic flux and ERα expression correlate with tumor aggressiveness in clinical PCa samples, since both increased glycolysis and elevated ERα expression are features of aggressive disease, and the existing literature supports the biological plausibility of these associations [59,60,61]. In this context, measuring glycolytic flux and ERα expression (alone or in combination with D-lactate levels) may serve as biomarkers of aggressive disease or identify patient subgroups that could benefit from targeting the glyoxalase pathway.

Overall, our findings open new avenues for investigation, particularly in in vivo models, to assess the potential relevance of the Glo2/D-lactate/FAK–Src axis in PCa progression. Moreover, they extend our understanding of the pathways through which PTEN loss promotes cell plasticity, migration and invasion, highlighting a downstream metabolic–signaling mechanism that may warrant consideration in therapeutic strategies targeting PTEN-related signaling. PTEN loss is a frequent event in advanced PCa and is associated with resistance to upstream pathway inhibition. Indeed, previous studies have shown that pharmacological inhibition of PI3K/AKT/mTOR or experimental restoration of PTEN may not fully reverse aggressive traits such as migration, invasion, or EMT-associated features [62,63,64], underscoring the contribution of downstream and compensatory pathways. In this scenario, PTEN loss can be viewed as an initiating event that establishes a metabolic–signaling program capable of maintaining aggressive phenotypes independently of upstream PTEN activity. The identification of a downstream axis involving Glo2-derived D-lactate and FAK/Src suggests that targeting this pathway may represent a complementary strategy to limit aggressive traits that persist despite upstream oncogenic modulation. Future in vivo validation and pharmacological targeting studies will be necessary to assess the translational potential of this approach.

Historically considered a mere by-product of metabolism, D-lactate is now recognized as a signaling metabolite that can influence mitochondrial redox balance and the NADH/NAD^+^ ratio [23,24]. Thus, our results support the emerging concept that specific metabolites act as bioactive messengers in cancer progression [22,65,66]. Here, we demonstrate that these metabolic effects are coupled to activation of redox-sensitive signaling pathways, including FAK/Src, directly linking metabolic rewiring to cellular phenotypes associated with tumor aggressiveness. These results support the notion that metabolic rewiring in cancer is not merely a consequence of tumor growth, but is actively driven by oncogenic signaling to promote aggressive phenotypes. Complementary evidence from sorafenib-treated hepatocellular carcinoma demonstrates that therapy-induced metabolic remodeling, including altered glycerolipid and redox metabolism, supports survival and correlates with plasma metabolite signatures such as D-lactate and glycerol [65]. Together, these observations underscore the central role of metabolic signaling as both a functional driver of tumor aggressiveness and a potential source of clinically relevant biomarkers.

Our observations are consistent with previous reports highlighting FAK/Src as key regulators of cancer cell motility, EMT-like phenotype, and metastasis [25,26,27,28,67].

An additional implication of our findings concerns the tumor microenvironment. As a diffusible metabolite, D-lactate may exert paracrine effects on neighboring stromal or immune cells, potentially amplifying tumor-promoting signaling beyond cancer cells themselves [16,18]. Although direct evidence for D-lactate acting in a paracrine manner remains to be fully established, evidence from studies on L-lactate, a closely related diffusible oncometabolite, supports the concept that extracellular metabolites can significantly impact the tumor microenvironment. Tumor-derived lactate has been shown to be taken up by stromal cells such as mesenchymal stem cells and cancer-associated fibroblasts, stimulating their migration and metabolic activity and facilitating crosstalk that promotes tumor progression [68]. Moreover, elevated extracellular lactate modulates immune cell function by inhibiting effector T and natural killer cells while supporting immunosuppressive cell populations, thereby contributing to tumor-promoting immunosuppression [69]. Lactate also influences the phenotype and function of fibroblasts within the tumor niche, enhancing extracellular matrix remodeling, EMT, and angiogenesis [70]. Together, these studies show how diffusible metabolites such as lactate can exert paracrine effects on stromal and immune cells, supporting the plausibility that D-lactate may similarly act beyond cancer cells to shape the tumor microenvironment. This represents an interesting avenue for future investigation.

A limitation of the present study concerns the concentration of exogenous D-lactate used in rescue experiments. While intracellular D-lactate levels measured in PCa cells were in the sub-millimolar range, higher extracellular concentrations (2.5 mM) were required to reproducibly restore FAK/Src activation and migratory behavior following Glo2 silencing [29,30,31]. This difference likely reflects the need to overcome barriers related to cellular uptake, metabolic turnover, and compartmentalization in short-term in vitro assays, rather than indicating that such concentrations are reached intracellularly under physiological conditions. Accordingly, the rescue experiments are intended to demonstrate the signaling competence of D-lactate rather than to model its precise physiological concentration. Notably, although total lactate levels in the microenvironment of aggressive prostate tumors can reach millimolar to tens-of-millimolar concentrations in vivo, physiological D-lactate concentrations within PCa tissues remain poorly defined. Therefore, the observed effects should be interpreted as mechanistic responses to elevated D-lactate exposure within an in vitro context rather than as direct surrogates of in vivo D-lactate levels.

A further consideration concerns the assessment of FAK and Src activation. In the present study, phosphorylation of FAK (Tyr397) and Src (Tyr416) was quantified by ELISA in whole-cell lysates, providing a measure of global phosphorylation changes. While these sites are widely accepted indicators of FAK/Src activation, this approach does not capture the spatial organization or subcellular localization of signaling events, which are known to be critical for focal adhesion dynamics and directed cell migration. Therefore, our data support a role for D-lactate in promoting overall activation of FAK/Src signaling but do not address the spatial redistribution or focal adhesion-specific activation of these kinases. Consequently, while the results indicate global FAK/Src activation downstream of D-lactate, they do not allow us to distinguish whether D-lactate specifically enhances focal adhesion-localized signaling, which is crucial for directional migration. We cannot exclude the possibility that D-lactate also modulates additional spatially restricted signaling events or focal adhesion turnover, and the observed phenotypic effects may involve broader reprogramming of adhesion dynamics beyond global kinase activation. Future studies employing imaging-based approaches or subcellular fractionation will be required to resolve the spatial dynamics of FAK/Src signaling downstream of D-lactate.

Notably, the lack of correlation between Glo2 expression and clinical stage, despite its association with higher tumor grade, requires careful interpretation. Tumor grade primarily reflects cellular differentiation and intrinsic biological aggressiveness, whereas clinical stage captures anatomical tumor spread, which is influenced by additional factors such as tumor–stromal interactions, microenvironmental constraints, and time-dependent progression. Consistent with this distinction, our functional data indicate that Glo2 and D-lactate signaling enhance migratory and invasive traits at the cellular level, without necessarily implying that Glo2 expression alone is sufficient to drive macroscopic tumor dissemination detectable by TNM staging. Moreover, the relatively limited stage distribution within the analyzed cohort may have reduced the statistical power to detect associations with clinical stage. Collectively, these observations suggest that Glo2 expression is more closely linked to metabolic and phenotypic aggressiveness than to anatomical disease extent.

Importantly, although Glo2 expression was associated with tumor grade, the present study was not designed to evaluate prognostic outcomes. Therefore, the potential impact of Glo2 on patient survival and disease progression warrants investigation in larger, well-characterized cohorts.

5. Conclusions

In conclusion, this study identifies Glo2-derived D-lactate as a previously unrecognized metabolic signal that contributes to PCa cells’ aggressive phenotypic traits, with the involvement of FAK/Src activation, providing new insights into metabolite-mediated regulation of tumor behavior. Although our data establish a functional requirement for FAK/Src signaling downstream of D-lactate, the precise molecular mechanism by which D-lactate activates FAK/Src remains to be determined. Our observations support a model in which oncometabolite-driven signaling cooperates with oncogenic pathways to sustain aggressive characteristics in PTEN-deficient PCa cells. Future studies employing in vivo models and analyses of the tumor microenvironment will be essential to define the physiological relevance and therapeutic potential of targeting this pathway in advanced PCa. Moreover, investigating whether modulation of D-lactate levels or pharmacological inhibition of FAK/Src can synergize with existing therapies may offer novel strategies to control PCa’s intrinsic biological aggressiveness.