Genomic and phenotypic analysis reveals a key role for CCN1 (CYR61) in BAG3-modulated adhesion and invasion.

Chaperone protein quantity may regulate the balance of proteins involved in invasion and malignancy. BAG3 is a co-chaperone and pro-survival protein that has been implicated in adhesion, migration, and metastasis. We reported that BAG3 overexpression in MDA435 human breast cancer cells results in a significant decrease in migration and adhesion to matrix molecules that is reversed upon deletion of the BAG3 proline-rich domain (dPXXP). We now hypothesize that transcriptional analysis would identify proteins involved in matrix-related processes that are regulated by BAG3 and/or its PXXP domain mutant. Expression array analysis of MDA435 cells overexpressing either wild-type BAG3 (FL) or dPXXP identified CCN1 as a BAG3 target protein. CCN1 is a known AP-1 target. Increased AP-1 transcriptional activity and AP-1 DNA-binding was found in MDA435 dPXXP cells. Consistent with these findings, CCN1 quantity and secretion were increased in dPXXP mutants but suppressed in FL cells; both BAG3 forms resulted in up-regulated CCN1 in HeLa cells. CCN1 silencing in the BAG3 FL overexpressors reduced the already low phospho-integrin beta1 in response to attachment on collagen IV. Matrigel invasion of HeLa cells engineered with the BAG3 constructs was enhanced in FL cells and minimal in dPXXP cells. CCN1 silencing blocked a greater percentage of the serum-induced invasion in FL cells than in dPXXP cells. This implies a context-dependent function of BAG3 on CCN1 and thus mesenchymal behaviour. CCN1 may be necessary for adhesion and matrix-related signalling in FL cells, abrogating a negative signal of the PXXP domain when BAG3 is intact. We propose that BAG3 regulates CCN1 expression to regulate tumour cell adhesion and migration.

CAIR-1/BAG-3 modulates cell adhesion and migration by downregulating activity of focal adhesion proteins.

CAIR-1/BAG-3 is a stress and survival protein that has been shown to bind SH3 domain-containing proteins through its proline-rich (PXXP) domain. Because stress and survival pathways are active during invasion and metastasis, we hypothesized that CAIR-1 is a regulator of signaling pathways that modulate cell adhesion and migration. MDA-435 human breast carcinoma cells were stably transfected with full-length CAIR-1 (FL) or a proline-rich domain deleted mutant (dPXXP). FL cells migrated poorly through collagen IV-coated filters to serum (14% of control, p=0.0004), whereas migration of dPXXP cells was more robust (228%, p=0.00001). Adhesion to collagen IV-coated surfaces was reduced in FL cells and augmented in dPXXP cells (FL 64%, p=0.03; dPXXP 138%, p=0.01). Rhodamine-phalloidin staining highlighted more stress fibers and thicker filopodial protrusions in dPXXP cells. Fewer focal adhesions were also seen in FL cells. A reduction in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin occurred in FL cells under these conditions. In contrast, increased FAK and paxillin phosphorylation was documented in dPXXP cells. Differential FAK phosphorylation occurred at the major autophosphorylation site Y(397) and Src phosphorylation site Y(861). Concordant with these findings, there was decreased interaction between FAK and its downstream partners p(130)Cas and Crk observed in FL cells but not in dPXXP cells. These results collectively indicate that CAIR-1 may negatively regulate adhesion, focal adhesion assembly, signaling, and migration via its PXXP domain.