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Abstract

Introduction

Glycolytic phenotype positively supports cancer cell migration and metastasis in various cancers including Triple negative breast cancers (TNBCs). In-depth understanding of molecular pathways associated with increased aerobic glycolysis in TNBCs could provide key insights into the drivers of TNBC progression.

Methods

β-catenin and glycolytic proteins (PFKP, LDHA, MCT1) were assessed by Immunohistochemistry (IHC) in TNBC patients (n = 98), with prognostic value evaluated by Kaplan-Meier and Cox regression. In vitro, the β-catenin inhibitor ie, XAV939 was tested for suppressing β-catenin-driven aerobic glycolysis in TNBC models using MTT for proliferation, Western blotting for protein expression, and wound healing, droplet invasion, and colony formation assays for physiological changes.

Results

β-catenin and glycolytic markers (PFKP, LDHA, MCT1) were overexpressed in >50% of TNBCs. Kaplan-Meier and Cox regression analyses showed that combined expression of β-catenin with glycolytic markers correlated with reduced survival. In vitro, XAV939 suppressed β-catenin-driven aerobic glycolysis in TNBC cells, downregulating β-catenin and glycolytic proteins, reducing glycolytic activity, and impairing aggressive phenotypes (proliferation, migration, invasion, clonogenicity).

Conclusion

Overall, our results highlight the crucial role of β-catenin in controlling aerobic glycolysis via regulation of key glycolytic proteins, thereby positively driving the progression and metastasis of TNBCs. Additionally, our data strongly establish that XAV939 effectively inhibits glycolytic phenotype, thereby suggesting its therapeutic potential in TNBC patients.

Keywords

triple negative breast cancer aerobic glycolysis β-catenin XAV939 migration

Introduction

Triple-negative breast cancers (TNBCs) subtype is distinguished by their aggressive nature, inadequate treatment options, high recurrence rate, and inferior prognosis.¹ Like inflammatory or metaplastic subtypes, TNBC is also challenging to treat and often demands more intensive approaches. In the absence of precision therapy, the therapeutic management of TNBC primarily depends on a multimodal approach that includes chemotherapy, radiation therapy, and surgery. Although initial response rates to adjuvant therapy are satisfactory, TNBC patients frequently demonstrate disease recurrence and early metastatic onset, highlighting the critical need to discover new targets for effective treatment.^2,3 In recent studies, researchers have shown the metabolic dependence (aerobic glycolysis) of TNBC cells thereby opening up a promising therapeutic entry point in the clinical management of TNBC.⁴

To sustain their rapid proliferation, cancer cells reprogram their metabolism to fulfill heightened bioenergetic and biosynthetic requirements. The Warburg effect, or aerobic glycolysis, refers to the characteristic phenomenon in cancer cells where they preferentially generate energy through fermentation (glycolysis) instead of oxidative phosphorylation (OXPHOS), even in the presence of adequate oxygen.⁵ Like other solid cancers, TNBCs are also glycolytic compared to other breast cancer subtypes, as this metabolic shift aids in synchronizing with their accelerated proliferation, migration, and invasion.^6,7 The transition from oxidative phosphorylation (OXPHOS) to glycolysis in TNBCs supports ATP generation and produces glycolytic intermediates that fulfil the anabolic demands of rapid cell proliferation.⁶ The primary factors driving this metabolic reprogramming in TNBCs include the deregulation of various transcription factors and oncogenic signaling pathways, including WNT/β-catenin signaling.^8–10

The Wnt/β-catenin pathway is a canonical signaling cascade in which Wnt ligands promote β-catenin stabilization, allowing its nuclear accumulation and regulation of target genes that govern cell fate, proliferation, and cancer development.⁸ Studies have shown that WNT/β-catenin signaling is upregulated in TNBC patients, and stabilization of β-catenin protein has been linked to poor prognosis.^11–15 However, the mechanisms by which β-catenin regulates glycolytic components in TNBC are still being investigated. Our previous findings revealed that the reinstallation of tumor suppressive WNT5A signaling in TNBCs inhibits β-catenin, phosphofructokinase platelet type (PFKP), monocarboxylate channel 1 (MCT1), and lactate production in TNBC cells, thereby hindering their migration and invasion.¹⁶ In parallel with these findings, we also identified lactate dehydrogenase A (LDHA) as a downstream target of PFKP in TNBC cells, through which these cells regulate lactate production via aerobic glycolysis.¹⁷ These findings led us to hypothesize the existence of the β-catenin as a regulator of the glycolytic phenotype in TNBCs by controlling key glycolytic proteins like PFKP, LDHA and MCT1.

Although targeting metabolic enzymes in cancer offers therapeutic potential, direct inhibition may adversely affect normal cells. Consequently, focusing on the signaling pathways that regulate these metabolic enzymes or proteins could provide a safer and more effective strategy. Metabolic interventions, such as those targeting glycolysis and related pathways, have shown promise against solid tumors, including triple-negative breast cancer (TNBC). However, the high aggressiveness, therapeutic resistance, and stemness of TNBC are largely driven by dysregulated Wnt/β-catenin signaling, which often undermines the efficacy of such metabolic-targeting approaches.^11,13 Thus, novel selective β-catenin inhibitors are urgently needed to target TNBC's oncogenic transcription, stem cells, and metabolic adaptation. In this study, we conducted the immunohistochemical profiling of β-catenin along with glycolytic (PFKP, LDHA, and MCT1) proteins in the TNBC patient's cohort (n = 98). Furthermore, protein expression in TNBC patients was correlated to the clinicopathological traits and survival outcomes to determine its prognostic relevance. In addition, we also assessed the influence of potential β-catenin inhibitor ie, XAV939 by studying its effect on glycolytic proteins and key physiological traits of TNBC cells ie, proliferation, migration, invasion, and colony-forming ability.

Materials and Methods

Cell Culture and Reagents

MDA-MB-468 [#CL-0290; RRID: CVCL_0419] cells were procured from Elabsciences Bionovation Inc. (TX, USA); while MDA-MB-231 [RRID: CVCL_0062] cells were purchased directly from National Centre for Cell Science (NCCS, Pune, India). All cells were acquired within the last 3 years, and an authentication report detailing the respective cell lines was provided at the time of procurement. All cell lines were maintained in DMEM (Gibco, USA), 10% FBS, 2 mM glutamine, and 1% Pen-strep. All cell lines were regularly tested for mycoplasma contamination using an EZ-PCR kit (HaEmek, Israel). All cell cultures used were confirmed to be free of mycoplasma contamination. Experiments were conducted with cells up to passage twenty.

Clinical Breast Cancer Samples

Present study was approved by Institute Ethics Committee (IEC). As the planned study was retrospective (January 2015-December 2023); TNBC patient's (n = 98) paraffin blocks were traced back, according to the study criterion, and respective tissue sections were cut. The patient cohort consisted exclusively of female patients over 18 years of age with a confirmed TNBC diagnosis, who had not received neoadjuvant chemotherapy prior to surgery, and had no history of other malignancies or significant comorbidities (like hypertension, diabetes, Liver function disorders etc; cases with prior/or secondary malignancies were explicitly excluded. Clinical characteristics of triple-negative breast cancer patients ie, age at diagnosis, skin involvement, distant metastasis, TNM staging, etc, were obtained from the medical records of the Department of Pathology. The histopathological grading was conducted using the Nottingham grading system (Elston and Ellis modification of Scarff-Bloom and Richardson).

PPI Interaction Studies

The STRING database was used to analyze protein-protein interactions (PPIs) between β-catenin, PFKP, LDHA, and MCT1 in order to find both functional and physical relationships. Key hub proteins were identified using the CytoHubba plugin based on network topology after the resultant interaction network was visualized in Cytoscape. The methodology was based on the strategy outlined by Santos et al (2022),¹⁸ emphasizing proteins that are essential to the structure of the network and may have regulatory functions.

β-Catenin Inhibitor

For the present study, XAV939 [Mol. Wt. 312.3 g/mol; C₁₄H₁₁F₃N₂OS; #HY15147, MedChemExpress (NJ, USA)] was used to inhibit β-catenin. XAV939 targets tankyrase (TNKS) 1 and 2, two enzymes involved in the regulation of key cellular processes. By inhibiting the activity of TNKS, XAV939 upregulates the protein expression of the axin-GSK3β complex. This accumulation of the complex promotes the enhanced degradation of β-catenin, a critical regulator in various signaling pathways.¹⁹

Immunohistochemical Staining for β-Catenin and Glycolytic Proteins

Respective monoclonal antibodies were procured for β-catenin (β-catenin-1, mAb#M3539; Agilent (CA, USA); PFKP (mAb# 12746; Cell Signaling Technology (MA, USA); LDHA (OTI2D11, mAb# TA500568; Origene (MD, USA); and MCT1 (H-1, mAb#sc-365501; Santa Cruz Biotech Inc (TX, USA). As outlined, immunohistochemical analysis for all glycolytic proteins was conducted on paraffin-embedded breast carcinoma sections.¹⁷ Briefly, antigen retrieval of tissue sections was performed in citrate buffer (0.01 M) for 20 min at pH 6.0, followed by rinsing with PBS, followed by endogenous peroxidase activity blockage for 45 min in H₂O₂. Using 1% BSA in PBS, non-specific binding was blocked followed by incubation with primary antibodies (β-catenin and other glycolytic proteins) for overnight at 4 °C. Later, sections were incubated with UltravisionTM Quanto Detection System (ThermoFisher, MA, USA). DAB (# 71897, MilliporeSigma, MO, USA) was used as substrate for color, and counterstaining was performed with Mayer's hematoxylin. For the negative controls, primary antibody was substituted with isotype-specific IgG.

Scoring Criteria for IHC Staining

Slides were evaluated independently and blinded by two investigators, one of whom was a professor of pathology. Tumors were categorized on the basis of the percentage and intensity of DAB-stained cancer cells. H-score was then computed by considering the intensity of staining (negative = 0, faint = 1, moderate = 2, strong = 3) and the proportion of cells exhibiting DAB immunoreactivity (≤10% = 0, 10-20% = 1, 20-40% = 2, 40-60% = 3, 70-80% = 4, 80-100% = 5). H-score was calculated using the formula: H-score = I × P{H-score} = I × P.

MTT Assay

TNBC cells were grown in increasing concentrations of XAV939 (0-50 µM) [for 48 h], and MTT was used to assess cell viability at 570 nm. MTT was also performed on non-malignant MCF10A cells (0-120 µM; for 48 h). For the assay, For the experiments, cells were suspended at a definitive concentration of 2.5 × 10⁵ cells/ml, as described before.²⁰ Cell viability was measured as per the protocol established by Carmichael et al.²¹

Western Blot Analysis

For Immunoblotting, TNBC cells (ie, 2 × 10⁵/well) were plated in 6-well plate, and when the cell reached confluency of 80–90% they were treated with β-catenin inhibitor ie XAV 939 for next 48 h at the suboptimal dose. Cell lysates were made in ice-cold PLB lysis buffer, as described by us previously.¹⁶ Total protein was estimated using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA, USA), and 20–25 µg of samples were prepared with 4× Laemmli buffer. Following SDS-PAGE and transfer, the PVDF membranes were incubated with primary antibodies such as β-catenin (BD-610152, at 1:1000), PFKP (NBP2-01539, at 1:2000), LDHA (3582S, at 1:1000), and MCT1 (SC-365501, at 1:1000) overnight at 4 °C, and subsequently with the corresponding HRP-conjugated secondary antibodies [Anti-mouse at 1:10000 (# P0447) or Anti-rabbit at 1:5000 (# P0448)] from Agilent (CA, USA). Chemiluminescence substrate (CYANAGEN, BO, IT) was used to detect protein bands and images were evaluated with the Chemi Doc™ imaging system (from Bio-Rad, CA, USA).

Cell Migration/Wound Healing Assay

As described before,²⁰ uniform wound was generated using micropipette tip, and the scrapped off cells were rinsed with PBS. Next, treatment was performed with XAV939 for 48 h at 37 °C). After the treatment, the cells were PBS washed and the wound was captured with microscope (Leica DM6 Microscope, Wetzlar, Germany), and wound closure was analyzed using ImageJ software (version 1.52, National Institute of Health, MD, USA).

Matrigel Droplet Invasion Assay

For the assay, cells at a density of eight to ten thousand cells (mixed with the EHS matrix in a 1:1 ratio) per droplet were plated (in 24-well plates). Matrigel was permitted to solidify by keeping the plates at 37 °C for about 20 min. Afterward, media containing vehicle, or XAV939 (for 48 h) and cell invasion was monitored for next one week. The migrating edge beyond the Matrigel drop were captured by Olympus CKX53 microscope (Tokyo, Japan), and analyzed using ImageJ software (version 1.52, National Institute of Health, MD, USA). Using the freehand tool, one outline was drawn to measure the total area encompassing both the Matrigel drop and the migrated cells. A second outline was drawn to measure only the area of the Matrigel drop. The Matrigel drop area was then subtracted from the total area to determine the region occupied by migrated cells. The results are expressed as the extent of cell invasion relative to controls.

Colony Formation Assay

For experiments, cells were treated with either XAV939 (for 48 h with a specific dose) or DMSO (for control). After treatment, the cells were trypsinized, washed and seeded at the density of five hundred cells/well in 6-well plates. After a week (7 days), colonies were fixed (4% formaldehyde) and stained with 0.4% crystal violet. The colonies were then counted, and the data were analyzed as previously described.²²

Extracellular Lactate Assay

Briefly 5 × 10⁴ cells were seeded per well in a 24-well plate and treated with XAV939 or DMSO vehicle for 72 h. After incubation, the conditioned media was harvested for lactate quantification via a colorimetric kit (BioVision, CA, USA), and the remaining adherent cells were lysed for total protein measurement by BCA assay (Thermo Fisher Scientific, MA, USA). Lactate levels were normalized to both viable cell number and protein content (ng/μg protein), with all steps conducted in triplicates. All the experiments involving the lactate measurements were performed under low serum conditions.¹⁷

3D Tumour Spheroid Formation Assay

MDA-MB-468 cells were used for spheroids generation using the method previously described.²³ In short, a single-cell suspension of cells in the complete medium was plated onto ultralow-attachment plates (#4515, CORNING, MA, USA) and kept at 37 °C with 5% CO₂. After 72 h, the spheroids were treated with DMSO, or XAV939 for an additional 48 h. After the treatment, the media was discarded, and fresh media with Matrigel (EHS matrix, Cat # 356234, CORNING, MA, USA) were added. The spheroids were observed under a microscope (Olympus CKX53, Tokyo, Japan) after 5 days, to measure the extent of area.

Statistical Analysis

Descriptive statistics (proportions, mean ± SD, median [IQR]) summarized baseline demographics, clinical features, and lab data. Associations between categorical variables were analyzed using χ² or Fisher's exact test (IBM SPSS Statistics for Windows, Version 31.0 (IBM Corp, Armonk, NY, USA). The Wilcoxon rank-sum test compared continuous variables. Kaplan-Meier plots with log-rank tests illustrated survival, and Cox proportional hazards models assessed event risk between groups. Data were censored as of June 30, 2022. All analyses were conducted using STATA 14 (StataCorp. 2015, TX, USA). For the in vitro experiments, data is presented as the mean ± standard error, with each experiment being repeated at least three times (n = 3). All p-values were corrected for multiple comparisons by Bonferroni Correction as there were multiple primary outcomes in the study. Statistical evaluation and graph making were carried out using GraphPad Prism 8.0 software (CA, USA). A p-value of <0.05 was considered statistically significant.

Results

PPI (Protein-Protein Interaction) Using STRING Database and Immunohistochemical Analysis for β-Catenin, and Glycolytic Proteins in TNBC Patients (n = 98)

Protein-protein interaction (PPI) analysis using the STRING database revealed β-catenin connectivity with all three glycolytic targets (PFKP, LDHA, and MCT1), indicating that β-catenin plays a role in network-wide metabolic regulation. We used the degree ranking method, which counts the number of direct interactions (edges) that each protein (node) has, in the CytoHubba plugin in Cytoscape to further clarify important regulatory nodes (Supplementary Figure S1A; Embedded Table). The fact that LDHA was the most highly ranked hub protein suggests that it plays a crucial part in the interaction network (Supplementary Figure S1A). These results point to LDHA as a possible downstream effector in β-catenin-mediated metabolic reprogramming in triple-negative breast cancer (TNBC). After in-silico validation of β-catenin-regulated metabolic network, we next carried out protein profiling of β-catenin and other glycolysis-linked proteins ie, PFKP, LDHA, and MCT1 in a retrospective cohort of 98 TNBC cases by performing immunohistochemistry (IHC). Of the 98 TNBC patients analysed, β-catenin expression was found to be overexpressed in 64% (63/98) of cases. For β-catenin immunostaining cytoplasmic and nuclear staining was considered as positive²⁴ (Figure 1; panel 1). A representative image of TNBC patient exhibiting negative β-catenin expression has been shown in Supplementary Figure S1B. For other glycolytic proteins ie, PFKP, LDHA, and MCT1 immunopositivity of 53%, 58%, and 51% was observed respectively (Table 1). For PFKP and LDHA immunostaining, cytoplasmic localization was considered as positive,^17,25 while for MCT1, membranous expression was considered²⁶ (Figure 1; panel 2, 3 & 4). On analysis, no statistical association was observed between any of the proteins with clinicopathological parameters analysed (Table 1).

Figure 1.

Immunohistochemical analysis for β-catenin and PFKP/LDHA/MCT1 axis proteins in TNBC patients (n = 98). First panel: β-catenin immunostaining in pancreas tissue served as positive control. In TNBC patients, cytoplasmic and nuclear β-catenin expression was scored as positive. Second panel: For PFKP, immunostaining in placental tissue served as the positive control. In TNBC patients, cytoplasmic PFKP expression was classified as positive. Third panel: For LDHA, immunostaining in breast cancer tissue was used as positive control. For the LDHA expression in patients, cytoplasmic expression was scored as positive. Fourth panel: For MCT1, immunostaining in colon tissue served as a control. In patients, MCT1 membranous expression was scored as positive. Arrows show cytoplasmic ‘C’, membranous ‘M’, and nuclear ‘N’ staining. Magnification X 200.

Table 1.

Expression Patterns and Clinico-Pathological Parameters of β-Catenin and Glycolytic Markers (PFKP, LDH-A and MCT-1) in TNBC Patients (n = 98).

Parameters	TNBC Cases (N = 98)	β-Catenin (Cyto/Nucl)		PFKP (Cyto)		LDHA (Cyto)		MCT-1 (Memb)
		Neg	Pos	Neg	Pos	Neg	Pos	Neg	Pos
		(-)	(+)	(-)	(+)	(-)	(+)	(-)	(+)
		35	63	46	52	41	57	48	50
Age
≤50	68	24	44	29	39	28	40	31	37
>50	30	11	19	17	13	13	17	17	13
		p = 1.00		p = 0.27		p = 1.00		p = 0.382
Tumor Grade
T₁ + T₂	45	17	28	22	23	20	25	22	23
T₃ + T₄	53	18	35	24	29	21	32	26	27
		p = 0.833		p = 0.84		p = 0.684		p = 1.00
Lymphatic Involvement
N₀	48	17	31	21	27	19	29	21	27
N_1–2	50	18	32	25	25	22	28	27	23
		p = 1.00		p = 0.551		p = 0.687		p = 0.322
Stage
I + II	48	18	30	20	28	18	30	24	24
III + IV	50	17	33	26	24	23	27	24	26
		p = 0.833		p = 0.32		p = 0.420		p = 1.00

Next, we analyzed protein-protein associations between β-catenin, PFKP, LDHA, and MCT1 expression (Table 2). Our analysis demonstrated that β-catenin expression was positively associated with key glycolytic markers like PFKP (p = 0.002; OR: 4.06) and MCT1 expression (p = 0.020; OR: 2.913) in TNBC patients thereby suggesting a potential role of β-catenin in driving glycolysis in TNBC patients via positive regulations of these proteins (Table 2). The data also confirm our previous in vitro findings in TNBC cells, where reduction of β-catenin led to decrease in PFKP and MCT1 proteins, upon WNT5A stimulation.¹⁶

Table 2.

Correlation Between the Expression Pattern of β-catenin and Glycolytic Markers (PFKP, LDH-A, and MCT-1) in the TNBC Cohort (n = 98).

Protein Expression	PFKP (Cyto)		LDHA (Cyto)		MCT-1 (Memb)
Protein Expression	Neg (-) 46	Pos (+) 52	Neg (-) 41	Pos (+) 57	Neg (-) 48	Pos (+) 50
β-catenin (Cyto/Nucl)
Neg (-) 35	24	11	16	19	23	12
Pos (+) 63	22	41	25	38	25	38
	p = 0.002* OR = 4.066 χ² = 10.230		p = 0.670 OR = 1.280 χ² = 0.336		p = 0.020* OR = 2.913 χ² = 6.101

Prognostic Potential of β-Catenin and its Associated Glycolytic Proteins in TNBC Patients (n = 98)

Kaplan-Meier survival estimates revealed distinct survival probabilities associated with β-catenin and its downstream aerobic glycolytic components expression (viz. PFKP, LDHA, and MCT1) in TNBC patients. Patients with high expression (≥10%) of LDHA (p = 0.02), MCT1 (p = 0.04), and β-catenin-LDHA-MCT1 combination (p = 0.004) showed significantly reduced survival compared to those with low expression (<10%). Additionally, the combined high expression of β-catenin, PFKP, LDHA, and MCT1 was strongly associated with the worst survival outcomes in TNBC patients (p < 0.001) (Figure 2).

Figure 2.

Kaplan-Meier analysis of TNBC patients by using individual as well as combined expression of β-catenin and other glycolytic proteins for Overall Survival (OS). A, β-catenin^(<10%,low)/β-catenin^{(≥10%,high)}; B, PFKP^(<10%,low)/PFKP^{(≥10%,high)}; C, LDHA^(<10%,low)/LDHA^{(≥10%,high)}; D, MCT1^(<10%,low)/MCT1^{(≥10%,high)}; E, β-catenin + PFKP^(<10%,low)/β-catenin + PFKP^{(≥10%,high)}; F, β-catenin + LDHA^(<10%,low)/β-catenin + LDHA^{(≥10%,high)}; G, β-catenin + MCT1^(<10%,low)/β-catenin + MCT1^{(≥10%,high)}; H, β-catenin + PFKP + LDHA^(<10%,low)/β-catenin + PFKP + LDHA^{(≥10%,high)}; I, β-catenin + PFKP + MCT1^(<10%,low)/β-catenin + PFKP + MCT1^{(≥10%,high)}; J, β-catenin + LDHA + MCT1^(<10%,low)/ β-catenin + LDHA + MCT1^{(≥10%,high)}; K, β-catenin +PFKP + LDHA + MCT1^(<10%,low)/β-catenin +PFKP + LDHA + MCT1^{(≥10%,high)}. p values were determined by log-rank testing and values were significant when p ≤ 0.05.

Cox Proportional Hazard Analysis

Univariate analysis identified LDHA (HR = 2.01; 95% CI: 1.05-3.83; p = 0.034), MCT1 (HR = 1.88; 95% CI: 1.02-3.47; p = 0.041), β-catenin-MCT1 (HR = 2.09; 95% CI: 1.15-3.10; p = 0.015), β-catenin-PFKP-LDHA (HR = 2.02; 95% CI: 1.10-3.70; p = 0.023), β-catenin-PFKP-MCT1 (HR = 2.40; 95% CI: 1.31-4.40; p = 0.005), β-catenin-LDHA-MCT1 (HR = 2.54; 95% CI: 1.37-4.78; p = 0.004), and β-catenin-PFKP-LDHA-MCT1 (HR = 3.05; 95% CI: 1.62-5.74; p = 0.001) as significant predictors of reduced survival (Table 3). Multivariate analysis showed LDHA (adjusted HR = 1.99; 95% CI: 1.04-3.81; p = 0.036) and β-catenin-MCT1 (adjusted HR = 2.07; 95% CI: 1.14-3.77; p = 0.016) as standalone prognostic indicators for overall survival (Table 3). These results indicate that increased expression of β-catenin and its downstream glycolytic components, particularly LDHA and MCT1, contributes to poorer prognosis in TNBC patients, and combined effect of these markers indicates a more aggressive phenotype in TNBCs, highlighting the potential of β-catenin as a driver of TNBC progression.

Table 3.

Factors Affecting Death. Univariate and Multivariate Analysis of Prognostic Factors by Cox Proportional Hazard Model (n = 98).

Variables	Unadjusted HR (95% CI)	p-Value	Adjusted HR (95% CI)	p-Value
β-catenin	1.07 (0.58 to 1.95)	0.825	-	-
PFKP	1.37 (0.76 to 2.48)	0.292	-	-
LDHA	2.01 (1.05 to 3.83)	0.034*	1.99 (1.04 to 3.81)	0.036
MCT1	1.88 (1.02 to 3.47)	0.041*	-	-
β-catenin-PFKP	1.36 (0.75 to 2.46)	0.296	-	-
β-catenin-LDHA	1.72 (0.95 to 3.10)	0.069	-	-
β-catenin-MCT1	2.09 (1.15 to 3.10)	0.015*	2.07 (1.143to 3.77)	0.016
β-catenin-PFKP-LDHA	2.02 (1.10 to 3.70)	0.023*	-	-
β-catenin-PFKP-MCT1	2.40 (1.31 to 4.40)	0.005*	-	-
β-catenin-LDHA-MCT1	2.54 (1.37 to 4.78)	0.004*	-	-
β-catenin-PFKP-LDHA-MCT1	3.05 (1.62 to 5.74)	0.001*	-	-

XAV939 Inhibits β-Catenin and its Downstream Glycolytic Proteins, Extracellular Lactate, and Migration of Human TNBC Cells

IHC analysis of β-catenin and PFKP/LDHA/MCT1 glycolytic axis revealed a β-catenin-driven aerobic glycolysis linked to poor prognosis in TNBC patients. These findings establish the concept of the β-catenin-PFKP-LDHA-MCT1 axis in TNBCs, which drives aerobic glycolysis and might contribute to the aggressive phenotype of TNBCs (ie, increased cancer migration and metastasis). In the next set of experiments, we used human TNBC cell lines to assess the potential of the β-catenin inhibitor (i.e., XAV939) in inhibiting the β-catenin regulated PFKP/LDHA/MCT1 axis. In order to evaluate the IC₅₀ for XAV939 on TNBC cells, MTT assays were performed. TNBC cells were treated with various concentrations of XAV939 (5-50 µM) for 48 h. IC₅₀ dose for XAV939 in MDA-MB-231 was found to be 50 µM, while for MDA-MB-468 it was 40 µM (Figure 3A). Compared to malignant TNBC cells, IC₅₀ for XAV939 in non-malignant MCF10A was found to be 110 µM (Supplementary Figure S2). For downstream experiments, an optimum dose of 20 µM was used. Subsequently, both TNBC cells were exposed for 24 & 48 h, and expression of β-catenin and PFKP/LDHA/MCT1 proteins were analysed by western blotting (Figure 3B,C). We found significant downregulation of β-catenin, PFKP, and LDHA proteins in MDA-MB-231 cells treated with XAV939 for 48 h. However, no expression for MCT1 protein was found in MDA-MB-231 cells (Figure 3B). Similar to our findings, researchers have previously reported that MCT1 protein is not expressed in MDA-MB-231 cells due to MCT1 gene promoter hypermethylation.^27,28

Figure 3.

Evaluation of XAV939 on the proliferation, β-catenin and its downstream targets, lactate production, and migration of TNBC cells. A, MDA-MB-231 and MDA-MB-468 cells were exposed to different concentrations of XAV939 (5-50 μM) for 48 h. B, MDA-MB-231 and C, MDA-MB-468 cells were exposed to XAV939 (20 μM) for 24 h and 48 h, and β-catenin, PFKP, LDHA, and MCT1 protein expression was assessed. The protein bands were quantified by calculating the densitometric values and normalizing them to the actin levels.

Parallelly in MDA-MB-468 cells, significant downregulation in the β-catenin, PFKP, and LDHA (including MCT1) proteins was also observed, when treated with XAV939 for 24 & 48 h (Figure 3C). All these experiments suggested that PFKP, LDHA, and MCT1 proteins are present downstream of β-catenin, as inhibition of β-catenin by XAV939 resulted in downregulation of all the glycolytic gene. To further validate our results, we silenced β-catenin using siRNA in MDA-MB-231 cells and found a marked decrease in PFKP and LDHA proteins (Supplementary Figure S3).

As XAV939 significantly suppressed the β-catenin regulated glycolytic markers, we next evaluated its effect on extracellular lactate. We found significant downregulation in the lactate production in XAV939 treated MDA-MB-231 cells for 72 h (Figure 4A). Furthermore, we also observed downregulation in the migration of treated TNBC cells with XAV939, compared to controls. The results demonstrate statistically significant inhibition in the relative migration rate of MDA-MB-231 and MDA-MB-468 cells when treated with XAV939 (Figure 4B). Overall, our experiments demonstrated that like TNBC patients, active β-catenin-mediated aerobic glycolysis signaling is also present in human TNBC cell lines, and treatment with a β-catenin inhibitor ie, XAV939 impaired the β-catenin metabolic axis thereby inhibiting the cell migration, and lactate production in TNBC cells.

Figure 4.

XAV939 impairs extracellular lactate and migration of TNBC cells. A, Extracellular Lactate estimation was performed in MDA-MB-231 cells treated with XAV939 for 48 h. Graphs display the relative lactate levels in MDA-MB-231 cells treated with XAV939 and the vehicle (DMSO). B, A wound-healing assay was conducted on MDA-MB-231 and MDA-MB-468 cells treated with XAV939 or DMSO control for 24 h. All bars represent at least three independent experiments (n = 3).

XAV939 Effective Inhibition on the Colony Formation, Invasion Potential and Spheroid Formation of Human TNBC Cells

As both WNT/ β-catenin and extracellular lactate are associated with tumor initiation capacity, stem cells, and chemoresistance in TNBCs we proceeded to evaluate the effect of XAV939 on the clonogenic potential of both human TNBC cells. In support to previous results, XAV939 substantially hinders the clonogenic capacity of both TNBC cells, compared to control (Figure 5A). Next, we investigated the TNBC-cell invasion potential using Matrigel droplet invasion assay (as described in Materials and Methods). Both the TNBC cell lines showed inhibition of invasion after the treatment with XAV939 (Figure 5B). Our present set of experiments highlights that XAV939 supress β-catenin regulated aerobic glycolysis thereby inhibiting the colony formation potential and invasion of TNBC cells.

Figure 5.

Assessment of the inhibitory effect of XAV939 on the clonogenic and invasion potential of TNBC cells. The clonogenic potential of the cells was assessed using a colony formation assay, which was conducted over a period of 2 weeks. This assay evaluates the cell's ability to proliferate and form colonies under the given experimental conditions. A, XAV939 treated TNBC cells exhibited substantial impairment in colony formation capacity, compared with controls. B, Droplet Matrigel invasion assay showed significant inhibition in the invasion potential of MDA-MB-231 & MDA-MB-468 cells when treated with XAV939. All bars represent at least three independent experiments (n = 3).

Additional to these experiments, the capability of cells to undergo anchorage-independent growth was evaluated through a three-dimensional (3D) multicellular spheroid assay using Matrigel. Tumor spheroids serve as a physiologically relevant in vitro model, as they recapitulate key features of avascular tumor regions, including gradients in nutrients and drug diffusion, as well as cell–cell interactions that influence therapeutic response. In this study, MDA-MB-468 cells were allowed to form spheroids, which were treated once with either vehicle control (DMSO) or XAV939 (40 µM) 48 hrs prior to Matrigel embedding. The subsequent expansion of spheroids within Matrigel were observed over a period of five days. Notably, XAV939-treated spheroids exhibited a marked reduction in size compared with controls (Supplementary Figure S4).

Discussion

Cancer cells undergo substantial metabolic changes to support their rapid growth and survival in a nutrient-poor environment. One key example is the shift to ‘aerobic glycolysis’ or the ‘Warburg effect,’ where cancer cells increase their glucose metabolism even when oxygen is plentiful. This shift decreases the production of reactive oxygen species (ROS) by reducing oxidative phosphorylation (OXPHOS), and provides high levels of glycolytic intermediates necessary for biosynthesis, aiding in their increased proliferation. Previously, we demonstrated that restoration of tumor-suppressor WNT5A signaling in TNBC cells inhibits TNBC cell migration and invasion. Through our experiments we further demonstrated that this effect is due to WNT5A's ability to suppress the β-catenin-PFKP-MCT1 axis, resulting in decreased lactate production.¹⁶ In a subsequent study, we also discovered that LDHA operates downstream of PFKP in TNBC cells.¹⁷ These findings led us to hypothesize that β-catenin regulate key glycolytic proteins like PFKP, LDHA, and MCT1 thereby positively driving aerobic glycolysis and progression of TNBCs.

Our results on protein-protein interactions (PPIs) showed that β-catenin may largely control glycolysis in triple-negative breast cancer (TNBC) by modifying LDHA, indicating that LDHA is an important downstream effector in β-catenin-induced metabolic reprogramming. Nevertheless, our previous research demonstrating that β-catenin inhibition lowers PFKP expression and that PFKP inhibition directly downregulate LDHA protein levels suggests another, indirect regulatory pathway relating these proteins. Our Immunohistochemistry results showed elevated expression of β-catenin (64%), and other glycolytic proteins PFKP (53%), LDHA (58%), and MCT1(51%) in TNBC patients. Protein-protein analysis revealed significant associations between β-catenin & PFKP (p = 0.002; OR: 4.06), and β-catenin & MCT1(p = 0.02; OR: 2.913) proteins. These findings are in support of previous studies in TNBCs, where inhibition of β-catenin either by XAV939 or recombinant WNT5A resulted in the decrease of PFKP protein thereby showing the dependence of these proteins on each other in this breast cancer subtype.¹⁶ Furthermore, MCT1 has also been shown as a direct target of WNT signaling in colon cancers, as XAV939 directly inhibits the expression of MCT1 in colon cancer cells.²⁹ Our Kaplan -Meier analysis further revealed that TNBC patients with high expression of LDHA (p = 0.02), MCT1 (p = 0.04), and β-catenin/LDHA/MCT1 combination (p = 0.004) showed significantly reduced survival compared to those with low expression. In support to our findings, previous studies on breast cancer have identified LDHA and MCT1 as independent prognostic factors.^30,31 Our results also imply that Wnt/β-catenin signaling might regulate MCT1 and LDHA expression. Like LDHA, MCT1 is a Wnt-responsive gene whose expression is transcriptionally controlled by c-Myc, a β-catenin downstream target.^10,32 Herein, we like to discuss the implication of loss of MCT1 expression in MDA-MB-231 cells, stemming from promoter hypermethylation, may curtail the efficacy of direct MCT1 inhibitors like AZD3965 due to compensatory MCT4-mediated lactate efflux,³³ while Wnt/β-catenin inhibition (via XAV939) could indirectly suppress aerobic glycolysis associated cancer characteristics. In MCT1-negative TNBC cases (around 49% in our study), demethylating agents such as 5-aza-2′-deoxycytidine offer a viable combination strategy to restore expression and enhance therapeutic response.

Furthermore, we also found that the combined expression of β-catenin, PFKP, LDHA, and MCT1 was strongly associated with the worst survival outcomes in TNBC patients (p < 0.001). The results were further validated by univariate analysis [β-catenin/PFKP/LDHA/MCT1 (HR = 3.05; p = 0.001)]. All these findings highlight the critical role of β-catenin, which may coordinate aerobic glycolysis in TNBC patients by interacting with key metabolic proteins such as PFKP, LDHA, and MCT1. This interaction appears to promote disease progression and adversely impact survival. In recent years, the role of β-catenin has become increasingly evident as a central player in metabolic regulation, not only in cancer but also in various other diseases.^34–36 In the context of cancer, both our work and that of others have extensively documented how β-catenin promotes the glycolytic phenotype across different cancer types.^10,37 Our present study further supports this, highlighting β-catenin's function as a master regulator of aerobic glycolysis in triple-negative breast cancers (TNBCs), and underscoring its potential as a therapeutic target.

Tankyrase (TNKS) have been shown to play a vital role in development of various cancers, including breast cancer. One of the mechanisms associated with increased oncogenic activity mediated by TNKS is through activation of Wnt/β-catenin signaling. In direct evidence supporting this notion, XAV939 (TNKS-inhibitor) has been shown to inhibit aerobic glycolysis in ovarian cancer cells through inhibition of pyruvate carboxylase via Wnt/ β-catenin signaling (through stabilization of Axin protein).³⁸ Similarly, in colon cancers, treatment with XAV939 resulted in reduced lactate production and a 30%–40% increase in the ratio of oxidative phosphorylation to glycolysis (OCR/ECR).³⁹ To investigate whether β-catenin metabolic axis can be targeted with XAV939, we next treated TNBC cells ie, MDA-MB-231 and MDA-MB-468 cells. XAV939 treatment led to a significant decrease in the protein levels of β-catenin and glycolytic proteins ie, PFKP, LDHA, and MCT1. In parallel, the decrease in glycolytic proteins sync with decreased extracellular lactate production and migration of TNBC cells suggesting that inhibition of β-catenin by XAV939 alone is sufficient to decrease the aerobic glycolysis in TNBC cells. Additionally, treatment with XAV939 significantly reduced the size of MDA-MB-468 3D spheroids embedded in Matrigel, indicating suppressed anchorage-independent growth relative to DMSO controls. These findings suggest that pharmacological inhibition of Wnt/β-catenin signaling by XAV939 may impair the growth and survival potential of triple-negative breast cancer cells, highlighting its potential as a therapeutic strategy.

In support of our finding researchers have demonstrated XAV939-mediated inhibition on aerobic glycolysis in colon and hepatocellular carcinoma cells.^40,41 A post-hoc power calculation revealed that the study had more than 81% power to detect a difference of 31% (48% vs 79%) in β catenin between those with and without PFKP at a level of significance of 0.02. Recent oncology studies also suggest that β-catenin plays a central role in metabolic regulation by influencing not only aerobic glycolysis, but also oxidative phosphorylation,^42–44 offering a potential therapeutic window. In this study, we focused solely on β-catenin's regulation of aerobic glycolysis in TNBCs, without exploring its role in oxidative phosphorylation, which may be considered a limitation. Schematic representation of our findings is depicted in Figure 6. Overall, in the present study, we have established that β-catenin-driven glycolysis is linked to poor prognosis in TNBC patients and that XAV939 effectively inhibits this pathway. Given the absence of clinically validated β-catenin inhibitors, our findings highlight the potential of XAV939/or similar compounds as a therapeutic option for TNBC. In future, subsequent studies should be conducted to validate the current findings on the effects of XAV939 in TNBCs using in vivo models or 3D organoids to support translational relevance.

Figure 6.

XAV939 target β-catenin induced aerobic glycolysis in TNBC cells. β-catenin positively regulates the glycolytic proteins ie, Phosphofructokinase-Platelet type (PFKP), Lactate Dehydrogenase A (LDHA), and Monocarboxylate transporter 1 (MCT1), contributing to elevated lactate levels in TNBC patients. Lactate, which is the final byproduct of anaerobic metabolism, is transported out of the cells into the extracellular space by the monocarboxylate transporter 1 (MCT1). This release of lactate in tumor microenvironment (TME) led to the acidification, thereby resulting in a lower pH. The acidic environment not only supports the survival and proliferation of tumor cells but aid in the metastatic potential of triple-negative breast cancer (TNBC) cells, facilitating their ability to disseminate to distant sites. As demonstrated in the article, the tankyrase inhibitor XAV939 significantly reduces β-catenin activity and glycolytic proteins, thereby inhibiting aerobic glycolysis in TNBC.

Supplemental Material

sj-pdf-1-tct-10.1177_15330338261425407 - Supplemental material for β-Catenin–Facilitated Glycolytic Reprogramming Fuels TNBC Progression: Therapeutic Blockade with XAV939

Supplemental material, sj-pdf-1-tct-10.1177_15330338261425407 for β-Catenin–Facilitated Glycolytic Reprogramming Fuels TNBC Progression: Therapeutic Blockade with XAV939 by Sheikh Mohammad Umar, Shruti Kahol, Sandeep R. Mathur, Ajay Gogia, S.V.S. Deo, Shivam Pandey and Chandra Prakash Prasad in Technology in Cancer Research & Treatment

Footnotes

Abbreviations

Acknowledgements

Not applicable.

ORCID iD

Chandra Prakash Prasad

Ethics Approval Statement

The study including patients and human tissues was conducted by the guidelines set forth by the Declaration of Helsinki, and the research protocol received approval from the Institute Ethics Committee (IEC) of All India Institute of Medical Sciences (AIIMS) (Reference No: IEC-410/03.08.2018, RP-36/2018).

Informed Consent Statement

This study utilized archival patient samples for which informed consent had been obtained previously in accordance with the protocols approved by the institutional ethics committee.

Author Contributions Statement

SMU performed the experiments and formal analysis, and wrote the manuscript; SK performed the experiments and formal analysis; SRM performed formal analysis and validation; SVSD and AG provided the patient data and follow-up; SP performed statistical analysis, and wrote the manuscript; CPP: conceived and designed the study, wrote the manuscript, supervised and funded the study.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the DST SERB ECR grant (No. 2017/001836), and AIIMS Intramural grants (No. A-515) to C. P. Prasad. S. M. Umar is the recipient of the Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India (09/0006(12965)/2022-EMR-I).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

All data are available from the corresponding author upon reasonable request.

Supplemental Material

Supplemental material for this article is available online.

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