Abstract 4711: Novel mechanism involved in the regulation ofoncogenic Axl receptor tyrosine kinase in cancer

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Abstract Background: Oncogenic receptor tyrosine kinase Axl is overexpressed and plays an important role in multiple human cancers. However the mechanisms of Axl overexpression in cancer remain unclear. This study is to investigate the mechanisms that regulate Axl expression in pancreatic cancer. Experimental procedures: The interactions between HPK1, Axl and c-Cbl were examined using co-immunoprecipitation and immunoblotting. Inhibitors of endocytic pathway and immunofluorescence were used to examine the underlying mechanisms of HPK1-mediated Axl degradation. The functional significance of HPK1-mediated Axl degradation in the invasive capability of pancreatic ductal adenocarcinoma (PDAC) cells and it downstream signaling pathways were examined using HPK1 stable PDAC cells. Immunohistochemistry was used to examine the in vivo correlation between HPK1 and Axl in human pancreatic intraepithelial neoplasia. The RNA sequencing data of HPK1 and Axl expression and survival data of 176 pancreatic cancer patients in the Cancer Genome Atlas (TCGA) database were downloaded from the Human Protein Atlas (https://www.proteinatlas.org). The expression of HPK1 and Axl was categorized as low or high using the cutoff values set by TCGA (3.42 for HPK1 and 14.09 for Axl). Survival analysis were performed using the Kaplan-Meier method and the log-rank test was used to evaluate the statistical significance of differences. Results: We identified Axl as a novel HPK1-interacting protein and demonstrated for the first time that HPK1 down-regulated Axl and decreased the half-life of Axl protein. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1 and monensin which inhibit endocytic pathway. HPK1 accelerates the movement of Axl from the plasma membrane onto endosomes in pancreatic cancer cells after treated with Gas6. HPK1 increased the binding of Axl to c-Cbl, promoted Axl ubiquitination, decreased Axl signaling including phospho-Akt and phospho-Erk, and decreased the invasion capability of pancreatic cancer. More importantly, we showed that Axl expression inversely correlated with HPK1 expression in human pancreatic intraepithelial neoplasia (P=0.005). Low expression of HPK1 and high expression of Axl correlated significantly with poor survival in patients with PDAC (P<0.001). Conclusions: Our results suggest a novel tumor suppressor mechanism of HPK1 in which it targets Axl for degradation via the endocytic pathway. Loss of HPK1 may contribute to Axl overexpression and enhance its downstream signaling and tumor invasion in pancreatic cancer. Citation Format: Hua Wang, Xianzhou Song, Hironari Akasaka, Reza Abbasgholizadeh, Ji-Hyun Shin, Craig D. Logsdon, Anirban Maitra, Andrew J. Bean, Huamin Wang. Novel mechanism involved in the regulation ofoncogenic Axl receptor tyrosine kinase in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4711.