CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins

Abstract

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Transport Metabolons and Acid/Base Balance in Tumor Cells

“…The authors showed by PLA that CAIX is closely colocalized with NBCe1 in the lamellipodia of hypoxic A549 adenocarcinomic human alveolar basal epithelial cells [39]. The same group also confirmed the colocalization of CAIX with NBCe1 in the invadopodia of hypoxic HT1080 fibrosarcoma cells [80]. Knockdown of the CAIX protein, as well as pharmacological inhibition of CAIX enzymatic activity or overexpression of a catalytically inactive CAIX mutant, resulted in a decrease in cell migration as well as reduced formation of invadopodia and proteolytic cleavage of the ECM.…”

“…Knockdown of the CAIX protein, as well as pharmacological inhibition of CAIX enzymatic activity or overexpression of a catalytically inactive CAIX mutant, resulted in a decrease in cell migration as well as reduced formation of invadopodia and proteolytic cleavage of the ECM. The authors further showed that application of monoclonal antibodies against the CAIX catalytic domain or the PG domain inhibits the metastatic properties of TE-1 human squamous cell carcinoma cells in a quail chorioallantoic membrane invasion assay and of HT-2018 cells in a mouse lung colonization assay [80]. Based on these results, the authors concluded that CAIX forms a transport metabolon with NBCe1 in the leading edge of migrating and invading tumor cells.…”

“…Interestingly, the transporters mentioned above have been found to interact with CAIX in cancer cells. CAIX itself, which is redistributed to the lamellipodia of migrating cells [39,46], plays a central role in tumor cell migration and invasion [80,142]. CAIX catalytic activity is required for the generation of acidic pH e nanodomains, which are mandatory for proteolytic cleavage of the ECM [80].…”

“…CAIX itself, which is redistributed to the lamellipodia of migrating cells [39,46], plays a central role in tumor cell migration and invasion [80,142]. CAIX catalytic activity is required for the generation of acidic pH e nanodomains, which are mandatory for proteolytic cleavage of the ECM [80]. Furthermore, CAIX directly interacts with different actin-regulating proteins in the cytosol [80] and promotes focal cell adhesion through its extracellular PG domain [46].…”

“…Based on these results, the authors concluded that CAIX forms a transport metabolon with NBCe1 in the leading edge of migrating and invading tumor cells. This transport metabolon facilitates local bicarbonate uptake into the cells and thereby contributes to the formation of local pH i and pH e gradients, which drive remodeling of the actin cytoskeleton and proteolytic cleavage of the ECM [35,80] (Figure 4). CAIX not only colocalizes with NBCe1 but also with the Cl − /HCO 3 − exchanger AE2 as shown by PLA in the lamellipodia of hypoxic SiHa cervical cancer cells [39].…”