A partial epithelial-mesenchymal transition signature for highly aggressive colorectal cancer cells that survive under nutrient restriction.

Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model. An EMT-specific quantitative polymerase chain reaction (qPCR) array was used to screen for deregulated genes, leading to the establishment of an in silico gene signature that was correlated with poor disease-free survival in CRC patients along with the CRC consensus molecular subtype CMS4. Among the significantly deregulated p-EMT genes, a triple-gene signature consisting of SERPINE1, SOX10, and epidermal growth factor receptor (EGFR) was identified. Starvation-induced p-EMT was characterised by increased migratory potential and chemoresistance, as well as E-cadherin processing and internalisation. Both gene signature and E-cadherin alterations could be reversed by the proteasomal inhibitor MG132. Spatially resolving EGFR expression with high-resolution immunofluorescence imaging identified a proliferation stop in starved CRC cells caused by EGFR internalisation. In conclusion, we have gained insight into a previously undiscovered EMT mechanism that may become relevant when tumour cells are under nutrient stress, as seen in early stages of metastasis. Targeting this process of tumour cell dissemination might help to prevent EMT and overcome drug resistance. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Benja-ummarit induces ferroptosis with cell ballooning feature through ROS and iron-dependent pathway in hepatocellular carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Benja-ummarit (BU), a traditional Thai herbal formula, has been prescribed by traditional Thai practitioners for the treatment of liver cancer. Clinical trials of BU have shown an increase in overall survival in hepatocellular carcinoma (HCC) patients, including stage 1-3 (with or without prior standard chemotherapy) and terminal stage. The clinical outcomes differ from those of other apoptosis-based conventional chemotherapies. The molecular mechanisms underlying the anti-cancer properties of BU remain unclear. AIM OF STUDY: To investigate BU-induced ferroptosis through morphological and molecular analyses of HCC cell lines and HCC rat tissues. METHODOLOGY: Cytotoxicity of BU extract in HepG2 and HuH-7 cells, with or without LX-2 in 2D and 3D cultures, was determined through MTT assay and by observing spheroid formation, respectively, as compared to sorafenib. Morphological changes and the cellular ultrastructure of the treated cells were evaluated by light microscopy and transmission electron microscopy (TEM), respectively. In addition, alterations in ferroptosis protein markers in both cell lines and rat liver tissue were determined using western blot analysis and immunohistochemical staining, respectively. To investigate the pathways mediating ferroptosis, cells were pretreated with an iron chelator to confirm the iron-dependent ferroptosis induced by the BU extract. Intracellular ROS, a mediator of ferroptosis, was measured using a scanning ion conductance microscope (SICM). SICM was also used to determine cellular stiffness. The lipid profiles of BU-treated cells were studied using LC-MS/MS. RESULTS: The BU extract induced cell death under all HCC cell culture conditions. The BU-IC50 in HepG2 and HuH-7 were 31.24 ± 4.46 µg/mL and 23.35 ± 0.27 µg/mL, respectively as determined by MTT assay. In co-culture with LX-2, BU exhibited a similar trend of cytotoxicity in both HepG2 and HuH-7 cells. Light microscopy showed cell ballooning features with intact plasma membranes, and TEM microscopy showed mitochondrial swelling and reduced mitochondrial cristae in BU-treated cells. BU promotes intracellular iron levels by increasing DMT1 and NCOA4 expression and decreasing FTH1 expression. BU also suppressed the cellular antioxidant system by lowering CD98, NRF2, and GPX4 expression, and promoting KEAP1 expression. IHC results of HCC rat liver tissues showed the absence of DMT1 and high expression of GPX4 in the tumor area. Pre-treatment with an iron chelator partially restored cell viability and shifted the mode of cell death to a more apoptosis-like morphology in the BU-treated group. The SICM showed increased intracellular ROS levels and cellular stiffness 24 h after BU treatment. In more detail of BU-mediated ferroptosis, cellular lipid profiling revealed increased expression of 3 polyunsaturated lipids, which are highly susceptible to lipid peroxidation, in BU-treated cells. DISCUSSION: Alterations in intracellular iron levels, ROS levels, and cellular lipid composition have been previously reported in cancer cells. Therefore, targeting the iron-dependent ROS pathway and polyunsaturated lipids via BU-induced ferroptosis may be more cancer-specific than apoptosis-based cancer drugs. These observations are in accordance with the clinical outcomes of BU. The ferroptosis-inducing mechanism of BU makes it an extremely promising novel drug candidate for the treatment of HCC.