Targeting the MLN64/STARD3-cholesterol axis disrupts tight junction remodelling and attenuates gastric cancer aggressiveness
- 1Cardiff University School of Medicine
- 2Karmanos Cancer Institute, Department of Oncology, School of Medicine, Wayne State University
- 3Henry Wellcome Building for Biomedical Research, University Hospital of Wales
Abstract
Introduction: Gastric cancer remains a major cause of cancer-related mortality, largely due to invasion, metastasis, and therapeutic resistance. Dysregulated cholesterol metabolism is increasingly recognised as a driver of gastric cancer progression, but how intracellular cholesterol trafficking contributes to malignant junctional remodelling remains poorly understood. MLN64/STARD3 is a cholesterol-binding lipid transfer protein associated with cancer progression, yet its functional role in gastric cancer cell architecture and aggressiveness has not been fully defined.
Methods: In this study, we investigated whether MLN64 is associated with cholesterol-dependent tight junction organisation and malignant behaviour in gastric cancer cells.
Results: Stable MLN64 knockdown in AGS and HGC-27 cells reduced membrane-associated free cholesterol, as shown by an approximately 50% decrease in the Filipin III membrane/cytoplasm fluorescence ratio in both cell lines. MLN64 depletion was also associated with reduced ZO-1 protein abundance, disrupted junctional ZO-1 localisation, and impaired tight junction integrity, including reduced transepithelial resistance, altered impedance, and increased paracellular permeability. Functionally, MLN64 depletion attenuated gastric cancer cell proliferation and invasive capacity, supporting an association between this cholesterol-trafficking axis, junctional organisation, and aggressive cellular phenotypes. To explore the pharmacological tractability of this pathway, in silico screening of an FDA-approved drug library identified Digoxin and Targretin as candidate compounds predicted to interact with the MLN64 START domain. Digoxin showed a favourable predicted binding energy of -13.73 kcal/mol. Functional validation showed that these agents phenocopied several effects of MLN64 depletion, including altered cholesterol distribution, reduced junctional ZO-1 organisation, impaired adhesion/migration dynamics, and reduced barrier function.
Conclusion: Together, these findings identify MLN64 as a regulator associated with cholesterol-dependent tight junction remodelling in gastric cancer cells. Digoxin and Targretin provide preliminary evidence that this pathway may be pharmacologically tractable; however, direct target-engagement and rescue studies will be required to establish causality and determine whether their effects are mediated through direct interaction with MLN64. The MLN64- cholesterol axis may represent a potential vulnerability in gastric cancer membrane organisation and progression.
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BibTeX
@article{li2026targeting,
title = {{Targeting the MLN64/STARD3-cholesterol axis disrupts tight junction remodelling and attenuates gastric cancer aggressiveness}},
author = {Amber Xinyu Li and Yiran Chen and Xinguo Zhuang and Layla Alshammari and Elyas Khan and Q. Ping Dou and Tracey A. Martin and Wen G. Jiang},
journal = {Frontiers in Oncology},
year = {2026},
month = oct,
doi = {10.3389/fonc.2026.1913732},
url = {https://www.frontiersin.org/articles/10.3389/fonc.2026.1913732/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Targeting the MLN64/STARD3-cholesterol axis disrupts tight junction remodelling and attenuates gastric cancer aggressiveness
AU - Amber Xinyu Li
AU - Yiran Chen
AU - Xinguo Zhuang
AU - Layla Alshammari
AU - Elyas Khan
AU - Q. Ping Dou
AU - Tracey A. Martin
AU - Wen G. Jiang
PY - 2026
DA - 2026/10/15
JO - Frontiers in Oncology
DO - 10.3389/fonc.2026.1913732
UR - https://www.frontiersin.org/articles/10.3389/fonc.2026.1913732/full
AB - Introduction: Gastric cancer remains a major cause of cancer-related mortality, largely due to invasion, metastasis, and therapeutic resistance. Dysregulated cholesterol metabolism is increasingly recognised as a driver of gastric cancer progression, but how intracellular cholesterol trafficking contributes to malignant junctional remodelling remains poorly understood. MLN64/STARD3 is a cholesterol-binding lipid transfer protein associated with cancer progression, yet its functional role in gastric cancer cell architecture and aggressiveness has not been fully defined. Methods: In this study, we investigated whether MLN64 is associated with cholesterol-dependent tight junction organisation and malignant behaviour in gastric cancer cells. Results: Stable MLN64 knockdown in AGS and HGC-27 cells reduced membrane-associated free cholesterol, as shown by an approximately 50% decrease in the Filipin III membrane/cytoplasm fluorescence ratio in both cell lines. MLN64 depletion was also associated with reduced ZO-1 protein abundance, disrupted junctional ZO-1 localisation, and impaired tight junction integrity, including reduced transepithelial resistance, altered impedance, and increased paracellular permeability. Functionally, MLN64 depletion attenuated gastric cancer cell proliferation and invasive capacity, supporting an association between this cholesterol-trafficking axis, junctional organisation, and aggressive cellular phenotypes. To explore the pharmacological tractability of this pathway, in silico screening of an FDA-approved drug library identified Digoxin and Targretin as candidate compounds predicted to interact with the MLN64 START domain. Digoxin showed a favourable predicted binding energy of -13.73 kcal/mol. Functional validation showed that these agents phenocopied several effects of MLN64 depletion, including altered cholesterol distribution, reduced junctional ZO-1 organisation, impaired adhesion/migration dynamics, and reduced barrier function. Conclusion: Together, these findings identify MLN64 as a regulator associated with cholesterol-dependent tight junction remodelling in gastric cancer cells. Digoxin and Targretin provide preliminary evidence that this pathway may be pharmacologically tractable; however, direct target-engagement and rescue studies will be required to establish causality and determine whether their effects are mediated through direct interaction with MLN64. The MLN64- cholesterol axis may represent a potential vulnerability in gastric cancer membrane organisation and progression.
SN - 2234-943X
ER -