Hypoxia induced responses are reflected in the stromal proteome of breast cancer.

Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses, focusing on secreted proteins from low-grade (luminal-like) and high-grade (basal-like) cell lines before and after hypoxia. We examine the overlap between proteomics data from secretome analysis and laser microdissected human breast cancer stroma, and we identify a 33-protein stromal-based hypoxia profile (33P) capturing differences between luminal-like and basal-like tumors. The 33P signature is associated with metabolic differences and other adaptations following hypoxia. We observe that mRNA values for 33P predict patient survival independently of molecular subtypes and basic prognostic factors, also among low-grade luminal-like tumors. We find a significant prognostic interaction between 33P and radiation therapy.

Gα(12) binds to the N-terminal regulatory domain of p120(ctn), and downregulates p120(ctn) tyrosine phosphorylation induced by Src family kinases via a RhoA independent mechanism.

p120 Catenin (p120(ctn)) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα(12) interacts with p120(ctn). In the present study, we have delineated a region of p120(ctn) that binds to Gα(12). We report that the N-terminal region of p120(ctn) (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120(ctn) binding to Gα(12). This region harbors the phosphorylation domain of p120(ctn) and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120(ctn) was observed in the presence of activated Gα(12). This down-regulation was triggered by three different Gα(12) mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120(ctn). In summary, our results suggest that Gα(12) binds to p120(ctn) and modulates its phosphorylation status through a Rho-independent mechanism. Gα(12) emerges as an important regulator of p120(ctn) function, and possibly of cadherin-mediated adhesion and/or cell motility.

A role for Galpha12/Galpha13 in p120ctn regulation.

The catenin p120 (p120ctn) is an armadillo repeat domain protein that binds to cadherins and has been shown to facilitate strong cell-cell adhesion. We have investigated a possible link between heterotrimeric G proteins and p120ctn, and found that both Galpha12 and Galpha13 can completely and selectively abrogate the p120ctn-induced branching phenotype in different cell types. Consistent with these observations, the expression of Galpha12 or Galpha13 compensates for the reduction of Rho activity induced by p120ctn. On the other hand, p120ctn can be selectively coimmunoprecipitated with Galpha12, and the coimmunoprecipitation was favored by activation of the G protein. A specific interaction between p120ctn and Galpha12Q231L was also observed in in vitro binding experiments. In addition, p120ctn can be immunoprecipitated along with Galpha12Q231L in L cells in absence of E-cadherin. Interestingly, the expression of Galpha12Q231L increases the amount of p120ctn associated with E-cadherin. These findings demonstrate that Galpha12 and p120ctn are binding partners, and they also suggest a role for Galpha12 in regulating p120ctn activity and its interaction with cadherins. We propose that the Galpha12-p120ctn interaction acts as a molecular switch, which regulates cadherin-mediated cell-cell adhesion.