Base Editing Reveals Context-Dependent Regulation of Adhesion, Anoikis, and Motility by BAP1 in Renal Cell Models

Background BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that precise correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 activity, reinstated anchorage-dependent growth, and re-sensitized cells to anoikis. Here, we asked the converse question: whether disrupting Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Methods Using the same adenine base-editing strategy, we introduced an inactivating Glu31Gly mutation into HK-2 normal kidney epithelial cells, generating two independent isogenic BAP1-mutant clones. As an additional control, we created a BAP1-knockout HK-2 clone via CRISPR/Cas9. Parental, mutant, and knockout cells were assessed for BAP1 enzymatic activity, DNA repair capacity, viability, proliferation, cell cycle status, anchorage-independent growth, and anoikis resistance. Migration and invasion of HK-2 mutants and knockouts were compared with KMRC-20 revertant clones in which endogenous Glu31Lys had been corrected. Results The Glu31Gly HK-2 mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair—phenotypes comparable to BAP1-knockout cells—confirming successful functional inactivation. Despite this, both mutant and knockout HK-2 cells maintained parental-like morphology, viability, and proliferation. Surprisingly, Glu31Gly did not confer anchorage-independent growth or anoikis resistance: upon detachment, both mutant and knockout cells showed increased apoptosis. In contrast, in KMRC-20 cells, restoration of BAP1 activity enhanced both migration and invasion. Conversely, BAP1 inactivation or loss in HK-2 cells increased invasion but paradoxically reduced migration. These opposite outcomes indicate that BAP1 regulates motility through distinct mechanisms in normal versus malignant renal cells, likely reflecting differences in lineage state, cytoskeletal organization, and downstream signaling. Conclusions Although BAP1 restoration suppresses anchorage-independent growth and anoikis resistance in KMRC-20 ccRCC cells, BAP1 inactivation alone is insufficient to induce these oncogenic traits in normal HK-2 epithelial cells, implying that additional oncogenic alterations are required for anchorage-independent survival during kidney tumorigenesis. The divergent effects of BAP1 gain versus loss on migration and invasion further underscore the context-dependent nature of BAP1 function. These base-editing studies demonstrate that BAP1 differentially regulates adhesion, anoikis, and motility in normal and malignant renal cells and highlight the utility of precise base editing for dissecting clinically relevant mutations.