Project A

Targeting Oncogenic Endocytosis to Combat Liver Cancer Plasticity and Therapy Resistance

     Hepatocytes are metabolic factories and membrane trafficking machines that continuously filter nutrients and xenobiotics form the bloodstream, store glycogen and lipids, and release glucose, cholesterol, and protein products like albumin (Fig. 1A)(1).  Genomic insults that transform hepatocytes can give rise to hepatocellular carcinoma (HCC), presenting the most common form of primary liver cancer and the 6th most common and 2nd most deadly malignancy worldwide (2, 3).  HCCs are so deadly because they are often diagnosed at advanced stages when cancer cells have become metastatic and extremely therapy resistant, driving the frequent relapse after surgical resection or ablation of tumors, and rendering systemic therapies largely ineffective (4-10).  

     We discovered that HCC cells exploit the endocytic machinery to activate diverse developmental and survival pathways that promote epithelial-mesenchymal plasticity (EMP) and therapy resistance (11-15).  Specifically, we found that HCC cells highjack protein interaction networks controlled by endocytic adaptor-associated kinase 1 (AAK1)–adapter protein 2 (AP2) complexes (Fig. 1B and 1C).  We showed that in mesenchymal-like HCC and neuroblastoma cells, AAK1–AP2 complexes recruit specific cargo adaptors that control the endocytosis and trafficking of multiple EMP-driving receptor kinases (Fig. 1D)(15).  Knocking down AAK1–AP2 complex members, including AAK1, the Ral-binding protein 1 (RALBP1), and the RalBP1-associated and EPS domain-containing proteins REPS1 and REPS2, reduced the expression of EMP markers (Fig. 1E), and sensitized HCC cells to cell cycle checkpoint kinase inhibition up to 20-fold, suggesting that AAK1-AP2 complexes may be targeted to inhibit EMP and survival signaling (Fig. 1F).  MS-based proteome profiling and gene set enrichment analysis (GSEA) of RNAi lines confirmed that AAK1–AP2 complex knockdown deactivated multiple developmental pathways, including NOTCH, WNT/β-catenin, and JAK-STAT signaling, as well as PI3K-AKT and NF-κB survival pathways (Fig. 1G).  

       Mechanistically, how AAK1–AP2 complexes control these pathways remains unknown, hindering their development as therapeutic targets.  We hypothesize that the oncogenic rewiring of AAK1–AP2 complexes alters the fate of multiple EMP-driving receptors by promoting receptor recycling over lysosomal degradation (Fig. 1H).  Going forward, we will 1) use MS-based interactomics, CRISPR technologies, multiplex immunohistochemistry (IHC), and immunofluorescence (IF) to determine the molecular mechanisms of oncogenic AAK1–AP2 signaling in vitro and in vivo, and 2) develop proteolysis-targeting chimeras (PROTACs) that degrade AAK1-AP2 complex components and disrupt their oncogenic functions.  Long-term, we seek to develop AAK1-AP2 complexes as drug targets to combat HCC metastasis and therapy resistance in the clinic.

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