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The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

ABSTRACT: Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ?1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with ?2?1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with ?2?1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.

SUBMITTER: Swayampakula M

PROVIDER: S-EPMC5684442 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

Swayampakula M M McDonald P C PC Vallejo M M Coyaud E E Chafe S C SC Westerback A A Venkateswaran G G Shankar J J Gao G G Laurent E M N EMN Lou Y Y Bennewith K L KL Supuran C T CT Nabi I R IR Raught B B Dedhar S S

Oncogene 20170710 45

Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidenc ...[more]

PMID: 28692057

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Project description:BackgroundThe 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.MethodsUsing 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.ResultsAMAP1 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.ConclusionThe 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.

Dataset Information

The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

Publications

The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

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