The Tumor-Promoting Role of ADM in Bladder Cancer and its Mediated Mechanisms of Cell Death via PKR and STAT1.

Adrenomedullin (ADM) is an endogenous physiological hormone that has also been identified as a tumor-associated cytokine. This study aims to explore whether ADM can impact on bladder cancer (BC) development, focusing on mechanisms of programmed cell death and relate to protein binding, thereby expanding its diagnostic value of clinical applications. Here we used ELISA and bladder cancer cell lines to detect ADM expression in clinical serum samples and investigate its functional roles. Quantitative PCR, Western blot, and flow cytometry analyses were integrated to evaluate the effects of ADM modulation on cell proliferation and apoptosis. Quantitative proteomics using tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile alterations in protein expression following ADM knockdown in bladder cancer cells. The effect of ADM on cell membrane integrity was assessed using transmission electron microscopy (TEM) and lactate dehydrogenase (LDH) release assays. Furthermore, siRNA-based experiments combined with immunoprecipitation were employed to investigate protein-protein interactions within bladder cancer (BC) cells. Finally, a mouse xenograft model was established to observe tumor growth and progression. Knocking down ADM increases apoptosis and decreases proliferation, while overexpressing ADM reverses these outcomes. Inhibition of ADM-induced LDH release and necroptosis-like cellular changes, observed using TEM, indicates the prevention of cell membrane rupture. Furthermore, ADM activates the PI3K/AKT pathway to phosphorylate STAT1, inhibiting its nuclear translocation; concurrently, ADM directly binds to STAT1 to block its activation. ADM knockdown reverses these dual inhibitory effects, upregulating the STAT1-PKR axis and activating the RIPK1/RIPK3/MLKL cascade to induce necroptosis. In addition, Animal experiments demonstrated a reduction in tumor growth in mice with ADM knockdown. Our study elucidated the novel tumorigenic role and specific mechanism of ADM in bladder cancer, providing new therapeutic insights for its prevention and treatment.