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

ABSTRACT: Highlights • The AMAP1-PRKD2 pathway regulates the intracellular trafficking of CAIX.• The AMAP1-PIAS3 pathway stabilizes HIF-1α and promotes the transcription of CAIX.• High ASAP1 together with high PIAS3 correlates with high CA9 in breast cancer.• High ASAP1 with high PIAS3 predicts the unfavorable prognosis of breast cancer.• Regulation of CAIX transcription and trafficking may be interconnected via AMAP1.

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.

SUBMITTER: Horikawa M 

PROVIDER: S-EPMC8577137 | biostudies-literature |

REPOSITORIES: biostudies-literature