Dual roles of AMAP1 in the transcriptional regulation and intracellular trafficking of carbonic anhydrase IX

BACKGROUND: The cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated. METHODS: Using highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients’ prognoses was analyzed using a TCGA breast cancer dataset. RESULTS: AMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer. CONCLUSION: The AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.