The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

PURPOSE: Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A2b adenosine receptor (A2b-AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A2b-AR to the behavior of colorectal cancer cells. PRINCIPAL RESULTS: The A2b-AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. MAJOR CONCLUSIONS: PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A2b-AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic.

Immunopurification of adenomatous polyposis coli (APC) proteins.

BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of ß-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.

Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of ß-catenin.

ß-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although ß-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of ß-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular ß-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of ß-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of ß-catenin within this complex. Each component of ß-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential ß-catenin-interacting proteins, and define when and where a specific complex forms.

Independent interactions of phosphorylated ß-catenin with E-cadherin at cell-cell contacts and APC at cell protrusions.

BACKGROUND: The APC tumour suppressor functions in several cellular processes including the regulation of ß-catenin in Wnt signalling and in cell adhesion and migration. FINDINGS: In this study, we establish that in epithelial cells N-terminally phosphorylated ß-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated ß-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected ß-catenin, GSK3ß and CK1α, but not axin. The APC/phospho-ß-catenin complex in cell protrusions appears to be distinct from the APC/axin/ß-catenin destruction complex. GSK3ß phosphorylates the APC-associated population of ß-catenin, but not the cell junction population. ß-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated ß-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-ß-catenin accumulation in cell protrusions. CONCLUSIONS: We conclude that N-terminal phosphorylation of ß-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-ß-catenin complexes may contribute to the tumour suppressor activity of APC.