Neutrophil extracellular traps (NETs) has been demonstrated to regulate the metastasis of breast cancer. In this study, we showed that de novo cholesterol biosynthesis induced by ASPP2 depletion in mouse breast cancer cell 4T1 and human breast cancer cell MDA-MB-231 promoted NETs formation in vitro, as well as in lung metastases in mice intravenously injected with ASPP2-deficient 4T1 cells. Simvastatin and berberine (BBR), cholesterol synthesis inhibitors, efficiently blocked ASPP2-depletion induced NETs formation. Cholesterol biosynthesis greatly enhanced Coiled-coil domain containing protein 25 (CCDC25) expression on cancer cells as well as in lung metastases. CCDC25 expression was co-localized with caveolin-1, a lipid raft molecule, and was damped by inhibitor of lipid rafts formation. Our data suggest that cholesterol biosynthesis promotes CCDC25 expression in a lipid raft-dependent manner. Clinically, the expression of CCDC25 was positively correlated with the expression of 3-hydroxy-3-methylglutaryl-CoAreductase (HMRCG), and citrullinated histone H3 (H3cit), in tissues from breast cancer patients. High expression of CCDC25 and HMGCR was related with worse prognosis in breast cancer patients. In conclusion, our study explores a novel mechanism for de novo cholesterol biosynthesis in the regulation of CCDC25 expression, NETs formation and breast cancer metastasis. Targeting cholesterol biosynthesis may be promising therapeutic strategies to treat breast cancer metastasis.

Ursolic acid (UA), a pentacyclic triterpenoid that has been found in a broad variety of fruits, spices and medicinal plants, has various biological effects such as reducing inflammation, protecting cells from damage, and preserving brain function. However, its impact on ferroptosis in cancer stem‑like cells remains unexplored. The present study investigated the effect of UA on MDA‑MB‑231 and BT‑549 cell‑derived triple‑negative breast CSCs (BCSCs) and its potential ferroptosis pathway. The effects of ferroptosis on BCSCs were demonstrated by the detection of ferroptosis‑related indexes including the intracellular level of glutathione, malondialdehyde, reactive oxygen species and iron. The effects of UA on the biological behaviors of BCSCs were analyzed by Cell Counting Kit‑8, stemness indexes detection and mammosphere formation assay. The mechanism of UA induction on BCSCs was explored by reverse transcription‑quantitative PCR and western blotting. BALB/c‑nude mice were subcutaneously injected with MDA‑MB‑231‑derived BCSCs to establish xenograft models to detect the effects of UA in vivo. The results revealed that BCSCs have abnormal iron metabolism and are less susceptible to ferroptosis. UA effectively reduces the stemness traits and proliferation of BCSCs in spheroids and mice models by promoting ferroptosis. It was observed that UA stabilizes Kelch‑like ECH‑associated protein 1 and suppresses nuclear factor erythroid‑related factor 2 (NRF2) activation. These findings suggested that the ability of UA to trigger ferroptosis through the inhibition of the NRF2 pathway could be a promising approach for treating BCSCs, potentially addressing metastasis and drug resistance in triple‑negative breast cancer (TNBC). This expands the clinical applications of UA and provides a theoretical basis for its use in TNBC treatment.

Cancer is, fundamentally, a disorder of cell growth and proliferation, which requires adequate supplies of energy and nutrients. In this study, we report that the haplo-insufficient tumor suppressor ASPP2, a p53 activator, negatively regulates the mevalonate pathway to mediate its inhibitory effect on tumor growth in hepatocellular carcinoma (HCC). Gene expression profile analysis revealed that the expression of key enzymes in the mevalonate pathway were increased when ASPP2 was downregulated. HCC cells gained higher cholesterol levels and enhanced tumor-initiating capability in response to the depletion of ASPP2. Simvastatin, a mevalonate pathway inhibitor, efficiently abrogated ASPP2 depletion-induced anchorage-independent cell proliferation, resistance to chemotherapy drugs in vitro, and tumor growth in xenografted nude mice. Mechanistically, ASPP2 interacts with SREBP-2 in the nucleus and restricts the transcriptional activity of SREBP-2 on its target genes, which include key enzymes involved in the mevalonate pathway. Moreover, clinical data revealed better prognosis in patients with high levels of ASPP2 and low levels of the mevalonate pathway enzyme HMGCR. Our findings provide functional and mechanistic insights into the critical role of ASPP2 in the regulation of the mevalonate pathway and the importance of this pathway in tumor initiation and tumor growth, which may provide a new therapeutic opportunity for HCC.

Lenvatinib resistance remains a major challenge in hepatocellular carcinoma (HCC) treatment, highlighting the need for novel therapeutic strategies. Cuproptosis, a copper-dependent form of cell death, has been increasingly implicated in cancer progression. Quercetin, a naturally occurring bioactive flavonol known to modulate mitochondrial metabolism and multiple oncogenic signaling pathways, has emerged as a potential sensitizer. This study aimed to elucidate the molecular mechanisms by which lenvatinib-resistant HCC cells evade copper-induced cell death and to evaluate whether quercetin enhances ES-Cu-induced cuproptosis by targeting FDX1 and reprogramming mitochondrial metabolism. Integrated methodologies including bioinformatics analysis, clinical specimen profiling, qRT-PCR, cell proliferation assays, intracellular copper quantification, and Western blot were employed to investigate (1) the insensitivityof lenvatinib-resistant hepatocellular carcinoma (HCC) to cuproptosis but target FDX1 can reverse it(2) the potential of quercetin-ES-Cu combinatorial treatment to reverse drug resistance. Furthermore, subcutaneous xenograft models, mitochondrial OCR measurements, mitochondrial enzyme activity/functional assays, and bio-quercetin administration analyses (immunofluorescence, co-immunoprecipitation) were systematically implemented in both in vitro and in vivo settings to delineate the FDX1-mediated cuproptosis activation mechanism and validate therapeutic efficacy. Lenvatinib-resistant HCC cells displayed downregulation of cuproptosis-related genes (FDX1, DLAT) and impaired copper accumulation. Quercetin bind with FDX1, enhanced mitochondrial OCR, and synergistically increased intracellular copper accumulation with ES-Cu, leading to lipoylated protein aggregation, mitochondrial dysfunction, and copper-induced cell death. In vivo, quercetin plus ES-Cu significantly suppressed tumor growth without evident toxicity. This study elucidates the mechanisms by which lenvatinib-resistant HCC cells evade cuproptosis, highlighting the therapeutic potential of quercetin and ES-Cu combination treatment as a novel strategy to overcome lenvatinib resistance in HCC.

Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their self‐renewal and bipotential differentiation properties. However, the efficiency of bidifferentiation and repopulation efficiency of iHepSCs is relatively low. Recent evidence shows that physiological hypoxia, a vital factor within stem cell “niche” microenvironment, plays key roles in regulating tissue stem cell biological behaviors including proliferation and differentiation. In this study, we found that physiological hypoxia (10% O₂) enhanced the stemness properties and promoted the proliferation ability of iHepSCs by accelerating G1/S transition via p53‐p21 signaling pathway. In addition, short‐term hypoxia preconditioning improved the efficiency of hepatic differentiation of iHepSCs, and long‐term hypoxia promoted cholangiocytic differentiation but inhibited hepatic differentiation of iHepSCs. These results demonstrated the potential effects of hypoxia on stemness preservation, proliferation, and bidifferentiation of iHepSCs and promising perspective to explore appropriate culture conditions for therapeutic stem cells.