小分子化合物yxx1121通过靶向降解CALR抑制EGFR/STAT3/PI3K/AKT信号级联并激活CASP9通路发挥抗胃癌作用OA

Objective Gastric cancer(GC)is the fifth most common malignancy worldwide,and the disease burden is particularly prominent in China.Identifying novel pathogenic drivers and their inhibitors is crucial for advancing precision treatment of GC.This study aimed to investigate the function and molecular mechanisms of calreticulin(CALR),which plays key roles in multiple cellular processes such as calcium homeostasis,protein folding,and immune responses,in GC,and to screen small-molecule compounds targeting CALR and evaluate their anti-GC effects.Methods ①CALR was knocked down or overexpressed in GC cells via lentivirus,and the effects of CALR on GC cell proliferation,apoptosis,and cell cycle in vitro and in vivo were analyzed by EdU assay,flow cytometry,and xenograft tumor model in nude mice;2The mechanism by which CALR regulates the proliferation of GC cells using transcriptome sequencing,RT-qPCR,Western blotting,and flow cytometry;③Candidate small-molecule compounds targeting CALR were screened and validated by CCK-8 assay,surface plasmon resonance(SPR),molecular dynamics,molecular docking,and cellular thermal shift assay;④ To clarify the degradation mechanism of the candidate small-molecule compounds,its inhibitory effect on gastric cancer cell proliferation,and its toxicity were analyzed by RT-qPCR,Western blotting,cycloheximide(CHX)chase assay,in vitro and in vivo experiments,HE staining,and blood cells and biochemical indicators.Results ①EdU assay showed that CALR expression significantly affected the proliferation of GC cells(P<0.05).Flow cytometry analysis revealed that inhibition of CALR expression significantly promoted apoptosis in GC cells(P<0.05);2 Mechanistic studies demonstrated that CALR regulated apoptosis by synergistically modulating the caspase 9(CASP9)apoptotic pathway and phosphorylation-dependent oncogenic signaling cascades(EGFR/STAT3/PI3K/AKT).In CALR-knockdown model,treatment with a CASP9 inhibitor significantly suppressed apoptosis(P<0.05)and cleaved-CASP9 expression in MGC-803 cells;③The small-molecule compound yxx1121 primarily targeted CALR protein degradation via the lysosomal pathway,and this inhibitor exhibited low toxicity and effective proliferation inhibition in CALR-overexpressing GC cells in vitro and in vivo(P<0.05).Conclusion CALR is upregulated in gastric cancer and promotes cell proliferation,and its mechanism involves the EGFR/STAT3/PI3K/AKT signaling cascade and the CASP9 apoptotic pathway.The novel small-molecule compound yxx1121 can target CALR for degradation through the lysosomal pathway,inhibit the above oncogenic signaling cascades,and thereby exert anti-GC effects.