ErbB3 is a receptor tyrosine kinase in the epidermal growth factor receptor (EGFR) family. Like other family members, it has an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. ErbB3 requires interactions with other receptors and dimerizes with ErbB2 and MET, to activate downstream signaling pathways. Mutations in the ErbB3 gene within the extracellular and kinase domains have been identified in many cancer types. To understand the impact of ErbB3 on cancer growth and metastasis, the human ovarian cancer cell line, OVCAR8, was used as a model. Parental OVCAR8 cells that express ErbB2 and ErbB3 were compared to a CRISPR-edited ErbB3 knockout cell line (ErbB3 KO) and ErbB3 KO lines stably expressing ErbB3 mutants (V104L, T355I, V855A, and E928G). To understand how the mutant ErbB3 receptors interact with other receptor tyrosine kinases, cells were stimulated with the ErbB3 ligand, heregulin (HRG), and the phosphorylation of receptors was quantified by fluorescent western blotting. Data shows that expression of ErbB3 V104L or V855A rescues ligand-dependent phosphorylation of ErbB3 and ErbB2. Interestingly, expression of these mutants also leads to ligand-independent activation of MET, but not ErbB2, suggesting that mutations in ErbB3 may enhance interactions with MET. Spheroid formation and spheroid attachment assays were performed to observe how loss or mutation of ErbB3 affects spheroid characteristics. Spheroid area and circumference were measured after 24 and 48 hours of spheroid formation and circularity was calculated. Data shows that loss of ErbB3 compromises spheroid formation resulting in larger, less compact spheroids. Surprisingly, ErbB3 KO has the opposite effect compared to ErbB2 KO, which produces tighter and more circular spheroids. A double ErbB2/ErbB3 KO shows a similar spheroid phenotype to the ErbB3 KO. ErbB3 mutants V104L and V855A partially rescue spheroid formation in this assay. Thus, we suggest ErbB3 may play an important role in spheroid formation and metastasis in ovarian cancer.
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