Phosphoproteomic profiling reveals signaling pathways modulated by Annona muricata leaf extract in oral adenosquamous carcinoma cells

Abstract Phosphorylation driven dysregulation of intracellular signaling networks is a central feature of cancer initiation, progression, and therapeutic resistance. Although Annona muricata leaf extracts have demonstrated anticancer activity across multiple experimental models, the underlying molecular mechanisms particularly at the level of phosphorylation dependent signaling remain poorly understood. In this study, we employed a tandem mass tag TMT-based quantitative phosphoproteomic approach to systematically characterize signaling alterations induced by methanolic Annona muricataleaf extract (AME) in oral squamous cell carcinoma (OSCC) CAL-27 cells. Functional assays revealed that AME significantly inhibited cell proliferation, migration, and clonogenic survival. Global phosphoproteomic profiling identified 6,362 phosphopeptides corresponding to 1,964 unique phosphorylation sites across nearly 7,000 phosphoproteins. AME treatment induced widespread, time-dependent hypophosphorylation, indicating a coordinated suppression of oncogenic signaling networks. Pathway and network analyses revealed marked inhibition of signaling pathways associated with key oncogenic kinases, including cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), and signaling modules linked to EGFR and mTOR pathways. Kinase-substrate enrichment and kinome mapping further demonstrated reduced inferred activity of CDK and MAPK driven signaling, accompanied by suppression of cell cycle progression, mitosis, and checkpoint regulation. Collectively, these findings demonstrate that AME induces systems-level remodeling of phosphorylation dependent signaling networks, enforcing a growth-restrictive cellular state in OSCC cells. This study highlights quantitative phosphoproteomics as a powerful strategy for dissecting natural product mediated regulation of oncogenic signaling and provides mechanistic insight into the anticancer potential of Annona muricata. Competing Interest Statement The authors have declared no competing interest. Footnotes Institutional Email IDs Mohd Altaf Najar mohammadanajar{at}yenepoya.edu.in, Devasahayam Arokia Balaya Rex rexprem{at}yenepoya.edu.in, Prashant Kumar Modi prashantmodi{at}yenepoya.edu.in, Suchitha G. P suchithajrf{at}yenepoya.edu.in, Amrutha, S amrutha{at}yenepoya.edu.in, T. S. Keshava Prasad Keshava.prasad{at}nitte.edu.in, Shobha Dagamajalu shobha_d{at}yenepoya.edu.in Abbreviations - BCA - bicinchoninic acid - CDK - cyclin dependent kinase - CSNK1A1 - casein kinase 1 alpha 1 - DPPH - 2,2-diphenylpicrylhydrazyl - EGFR - epidermal growth factor receptor - FBS - fetal bovine serum - LC-MS/MS - liquid chromatography-mass spectrometry/mass spectrometry - MAPK - mitogen-activated protein kinase - MTOR - mammalian target of rapamycin - MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide - PKC - protein kinase C - PRKCD - protein kinase C delta - ROCK1 - Rho associated coiled-coil containing protein kinase 1 - TEABC - triethyl ammonium benzyl chloride.