Saturated Fat Pushes Liver Cancer Cells Toward a Growth Signaling Pathway

Two fatty acids can load liver cells with fat in almost equal measure yet leave their cancer-relevant signaling machinery in strikingly different states. That is the core message of a new study published in Cancer Reports, which examined how palmitate, a saturated fatty acid abundant in Western diets, and oleate, the monounsaturated fatty acid characteristic of olive oil and Mediterranean dietary patterns, modulate molecular components of the canonical WNT/β-catenin pathway in lipid-loaded HepG2 cells, a widely used human hepatocellular carcinoma-derived line. The work, led by researchers at Iran University of Medical Sciences, suggests that the quality of dietary fat, not merely the quantity of intracellular lipid, may shape the signaling environment of liver cancer cells under metabolic stress.

The WNT/β-catenin pathway is one of the most intensively studied signaling networks in liver cancer biology. Under basal conditions, cytoplasmic β-catenin is held in check by a destruction complex containing APC, Axin, and GSK-3β, which tags the protein for phosphorylation and degradation. When WNT ligands engage their receptors, this process is inhibited, allowing β-catenin to accumulate and translocate to the nucleus, where it partners with TCF/LEF transcription factors to drive the expression of target genes controlling proliferation, differentiation, migration, and survival. Dysregulation of this pathway is a well-established molecular feature of hepatocellular carcinoma, and because it integrates signals governing cell fate, alterations in its activity may represent an important molecular interface between metabolic stress and cancer-associated phenotypes in the liver.

Non-alcoholic fatty liver disease, recently redefined as metabolic dysfunction-associated steatotic liver disease, is the most prevalent chronic liver disease worldwide and carries an increased risk of hepatocellular carcinoma. Disease progression is accompanied by excessive lipid accumulation, oxidative stress, chronic inflammation, mitochondrial dysfunction, and metabolic disturbances that can impair hepatic homeostasis. Lipid overload promotes the production of pro-inflammatory cytokines and reactive oxygen species, contributing to lipid peroxidation that can damage cellular macromolecules and membrane integrity. Persistent oxidative and inflammatory stress may also contribute to DNA damage and genomic instability, providing a mechanistic bridge between metabolic injury and altered cellular signaling. Defining how specific fatty acids alter cancer-relevant signaling pathways may therefore illuminate the molecular connection between metabolic liver disease and hepatocellular carcinoma.

To establish a reproducible lipid-loading model, the researchers first used the MTT assay to determine suitable exposure conditions. Palmitate reduced HepG2 cell viability in a concentration-dependent manner, with estimated IC50 values of approximately 0.8 millimolar after 24 hours and 0.7 millimolar after 48 hours. Oleate was somewhat better tolerated, with IC50 values of approximately 1.0 and 0.9 millimolar at the same time points. Because no significant difference emerged between the 24- and 48-hour values, the shorter exposure was selected for all subsequent experiments. The IC50 concentrations were treated as standardized, experimentally defined exposure levels associated with a measurable cellular response rather than as representations of physiological fatty acid concentrations in vivo.

Combining the two fatty acids proved more challenging than combining their individual tolerances would suggest. When the IC50 concentrations of palmitate and oleate were simply added together, metabolic viability collapsed to roughly 20 percent of control levels, leaving too few viable cells for molecular analysis. The team therefore evaluated lower combinations and settled on 0.4 millimolar palmitate plus 0.8 millimolar oleate, which maintained approximately 50 percent viability while still delivering a robust cellular response. Oil Red O staining confirmed that all treatments significantly increased intracellular lipid droplet accumulation, with the combined treatment producing the highest lipid content of all, indicating a stronger steatotic effect than either fatty acid alone. Lipid droplet accumulation rose progressively with increasing combined concentrations, and an inverse relationship was observed between cell viability and intracellular lipid deposition.

The transcriptional analysis revealed a strikingly asymmetric molecular landscape. Palmitate produced a coordinated activation of canonical WNT/β-catenin signaling components, significantly upregulating WNT expression by 1.53-fold, β-catenin transcript by 3.60-fold, TCF by 1.63-fold, and Axin2, a canonical downstream target gene, by 3.12-fold. In contrast, oleate produced a qualitatively different response, strongly inducing LEF expression by 5.79-fold, the highest transcriptional response among all examined genes, and elevating GSK-3β transcript by 2.27-fold, without significantly altering WNT or TCF expression. Both individual fatty acids suppressed Axin1 transcript levels by approximately 89 percent relative to control, a theoretically significant finding because Axin1 serves as an important scaffold of the β-catenin destruction complex.

Protein-level measurements added crucial nuance to the transcriptional picture. Palmitate significantly increased WNT-1 protein expression by 1.30-fold and β-catenin protein by 1.19-fold, consistent with the corresponding mRNA changes. Oleate and the combined treatment also significantly elevated β-catenin transcript levels, by 2.87-fold and 2.88-fold respectively, but neither produced a corresponding increase in β-catenin protein abundance, which remained comparable to or slightly lower than control levels. Palmitate also significantly elevated Glypican-3 protein expression by 1.35-fold, whereas oleate reduced GPC3 protein levels, although not significantly. These findings demonstrate that palmitate promotes WNT and β-catenin expression at both transcriptional and protein levels, whereas oleate partially attenuates this response despite producing a common phenotype of intracellular lipid accumulation.

The authors interpret these results as evidence that palmitate generates a molecular environment more compatible with enhanced canonical WNT/β-catenin signaling, whereas oleate does not produce the same coordinated pattern. Mechanistically, the simultaneous suppression of Axin1 and induction of Axin2 in palmitate-treated cells is compatible with a shift toward enhanced pathway signaling, because reduced Axin1 may favor β-catenin stabilization while increased Axin2 may reflect activation of a downstream transcriptional response and an accompanying negative-feedback mechanism. The oleate-associated increase in β-catenin transcript without corresponding protein accumulation suggests that transcriptional regulation alone is insufficient to infer sustained canonical WNT activity, and that posttranscriptional and posttranslational mechanisms may contribute to the dissociation between mRNA and protein abundance. The pronounced induction of LEF by oleate should not be interpreted as direct evidence of canonical WNT activation, because TCF and LEF can participate in overlapping as well as context-dependent transcriptional programs.

The combined palmitate/oleate treatment generally produced intermediate molecular responses, attenuating several of the effects induced by each fatty acid alone while maintaining significant activation of selected pathway components. However, the authors caution that because the palmitate concentration differed between the palmitate-alone and combined-treatment conditions, these differences cannot be interpreted as direct evidence of an oleate-mediated protective or antagonistic effect. Rather, they indicate that combined fatty acid exposure produced a molecular response distinct from that observed with palmitate alone. This distinction may be particularly relevant in metabolic liver disease, where hepatocytes are exposed to mixtures of fatty acids rather than to individual lipid species, and where the balance between pathway-promoting and pathway-restraining signals may depend on fatty acid composition.

Several limitations frame the conclusions. The study was performed exclusively in HepG2 cells, a hepatocellular carcinoma-derived model with a transformed phenotype and potentially altered WNT/β-catenin regulation, so the findings cannot be directly extrapolated to normal hepatocytes or to the early stages of metabolic dysfunction-associated steatotic liver disease. Functional WNT/β-catenin activation was not directly assessed through β-catenin nuclear localization, active non-phosphorylated β-catenin measurement, or β-catenin-dependent transcriptional assays, and GSK-3β phosphorylation status and enzymatic activity were not evaluated. Lipidomic and detailed metabolic analyses were not performed, so specific intracellular lipid species, oxidative stress responses, endoplasmic reticulum stress, mitochondrial function, and inflammatory mediators remain unexplored. The MTT assay provides an indirect assessment of metabolic activity and cannot independently distinguish changes in metabolic activity from alterations in cell number or specific cell death mechanisms. Future studies incorporating functional assays, primary or non-transformed hepatocyte models, and appropriate in vivo approaches will be required to validate these findings and clarify the specific contribution and interaction of these fatty acids.

Subject of Research: Fatty acid composition and WNT/β-catenin signaling in lipid-loaded HepG2 cells

Article Title: Differential Modulation of WNT/β‐Catenin Pathway‐Associated Molecular Responses by Palmitate and Oleate in Lipid‐Loaded HepG2 Cells

Article References: Al‐Haeri, M., Mohammadi, Y., Pashaei‐Asl, R., & Bahreini, E. (2026). Differential Modulation of WNT /β‐Catenin Pathway‐Associated Molecular Responses by Palmitate and Oleate in Lipid‐Loaded HepG2 Cells. Cancer Reports, 9(10), Article e70703. https://doi.org/10.1002/cnr2.70703

Image Credits: AI Generated

DOI: 10.1002/cnr2.70703

Keywords: Differential, Modulation, Catenin, Pathway, Associated, Molecular, Responses, Palmitate, Oleate, Lipid, Loaded, HepG2