Cell Communication and Signaling最新文献

Background: Triple-negative breast cancer (TNBC) is an aggressive subclass of breast cancer with limited treatment options and a strong tendency to metastasize to the lung. Tumor-derived extracellular vesicles (EVs) are key mediators of stromal reprogramming in the pre-metastatic niche. Annexin A2 (AnxA2) is overexpressed in TNBC and linked to signal transduction, cytoskeletal remodeling, and poor prognosis, but its role in shaping EV cargo composition and consequent stromal behavior remains unclear.

Methods: EVs were isolated from the conditioned media of MDA-MB-4175 (LM2) TNBC cells with stable AnxA2 knockdown and control cells using differential ultracentrifugation. EVs were characterized and validated using nanoparticle tracking analysis, Western blotting, and cryo-electron microscopy. EV proteomes were profiled using quantitative proteomics, followed by bioinformatic analyses. Functional and pathway enrichment was performed to identify proteome signatures altered by AnxA2 depletion. The internalization of EVs by recipient lung fibroblasts (MRC-5 and WI-38 cells) was assessed using confocal imaging, and EV-mediated effects on fibroblast behavior were evaluated through migration assays and immunoblotting for proteins associated with fibroblast activation.

Results: AnxA2 knockdown reduced pro-metastatic cellular properties in LM2 cells. EVs from AnxA2-deficient cells displayed reduced abundance of proteins involved in cytoskeletal regulation, adhesion, and vesicle trafficking, with enrichment of immune-related proteins. Pathway analyses predicted reduced motility and vesicle trafficking signaling. PKH26 labeled EVs derived from AnxA2 expressing cells exhibited increased internalization by lung fibroblasts as detected by confocal imaging. Functionally, AnxA2-enriched EV-mediated interactions enhanced fibroblast migration, proliferation, and activation marker expression compared with AnxA2-deficient EVs, consistent with the predicted proteomic changes.

Conclusions: AnxA2 emerges as a regulator of the protein composition of TNBC-derived EVs, enhancing EV-mediated interactions with lung fibroblasts and modulating fibroblast motility and activation, linking tumor cell protein expression to stromal remodeling in the lung microenvironment. This work establishes a framework suggestive of an EV cargo-driven mechanism through which EVs derived from AnxA2-expressing cells may influence lung-specific metastatic colonization in TNBC and highlights the importance of evaluating EV-mediated communication in cancer progression.

Background: Retinoblastoma Transcriptional Corepressor 1 (RB1) is a critical tumor suppressor restricting the malignant progression of cancer cells. Emerging evidence indicates that RB1 mutations typically promote tumorigenesis through loss of its tumor-suppressive functions. Yet, the biological significance of mutated RB1, specifically certain rare variants, in non-small cell lung cancer (NSCLC) remains elusive. Here, we first reported a rare and previously uncharacterized missense mutation in RB1, the 680th residue isoleucine replaced by threonine (RB1-I680T), in NSCLC.

Methods: To investigate the functional and mechanistic consequences of the RB1-I680T mutation in NSCLC, we first used CRISPR-Cas9 to knock out endogenous RB1 in NSCLC cells. Then, we generated cell models harboring the RB1-I680T mutation by infecting these knockout cells with lentivirus carrying either wild-type RB1 (RB1-WT) or RB1-I680T expression constructs. The biological phenotypes mediated by RB1-I680T were investigated using in vitro and in vivo experiments. The exploration of the molecular mechanism was performed primarily through co-immunoprecipitation, immunofluorescence, dual-luciferase reporter assays, western blot analysis, and protein docking and dynamics simulation.

Results: Our study demonstrated that the I680T mutation caused faster tumor growth and potentiated chemotherapy-induced tumor regression compared to RB1-WT control. Mechanistic studies illustrated that the I680T mutation in RB1 disrupted its inhibition of E2F1 transcriptional activity by weakening the physical interaction between RB1 and E2F1 in a manner dependent on conformational flexibility of RB1 pocket B domain, which is essential for sustaining the enhanced proliferation and chemosensitivity in NSCLC cells.

Conclusion: Our findings elucidate that the I680T mutation-induced loss-of-function of RB1 simultaneously confers invasive proliferation and chemotherapeutic vulnerability to tumor cells, suggesting that RB1-I680T could serve as a predictive biomarker for chemotherapy response in NSCLC. Stratifying patients based on the RB1-I680T mutation status may enable personalized therapeutic strategies, particularly for tumors with E2F1 dysregulation.