Engineering a synthetic cell panel to identify signalling components reprogrammed by the cell growth regulator anterior gradient-2.

AGR2 forms an ER-resident signalling axis in cell development, limb regeneration, and in human diseases like asthma and cancer, yet molecular mechanisms underlying its effects remain largely undefined. A single integrated Flippase recombination target (FRT) site was engineered within the AGR2-non expressing A375 cell line to allow integration of a constitutively expressed AGR2 alleles. This allows an analysis of how AGR2 protein expression reprogrammes intracellular signalling. The engineered expression of AGR2 had marginal impact on global transcription signalling, compared to its paralogue AGR3. However, expression of AGR2 had a significant impact on remodelling the cellular proteome using a triple-labelled SILAC protocol. 29 045 peptides were detected for the identification and relative quantitation of 3003 proteins across the experimental conditions. Ingenuity Pathway annotation highlighted the dominant pathway suppressed by wt-AGR2 was the p53-signalling axis. DNA damage induced p53 stabilization and p21 induction by cisplatin treatment confirmed that wt-AGR2 expression suppressed the p53 pathway. The furthest outlying SILAC protein expression change induced by AGR2 was the anti-viral and cell cycle regulator tumour susceptibility gene 101 (TSG101), confirmed by immunoblotting. Transfection of TSG101 into MCF7 (AGR2+, oestrogen dependent), A549 (AGR2+, oestrogen independent) or A375 (AGR2-) cells confirmed that TSG101 attenuates p53 signalling. These systems wide screens suggest that the most dominant landscape reprogrammed by low levels of AGR2 protein is the cellular proteome, rather than the transcriptome, and provide focus for evaluating its role in proteostasis.

Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein.

Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay ((2S) MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the (2S) MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2(E60A) revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2(E60A) mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.

Anterior Gradient-3: a novel biomarker for ovarian cancer that mediates cisplatin resistance in xenograft models.

The Anterior Gradient (AGR) genes AGR2 and AGR3 are part of the Protein Disulfide Isomerase (PDI) family and harbour core thioredoxin folds (CxxS motifs) that have the potential to regulate protein folding and maturation. A number of proteomics and transcriptomics screens in the fields of limb regeneration, cancer cell metastasis, pro-oncogenic oestrogen-signalling, and p53 regulation have identified AGR2 as a novel component of these signalling pathways. Curiously, despite the fact that the AGR2 and AGR3 genes are contiguous on chromosome 7p21.1-3, the AGR3 protein has rarely been identified in such OMICs screens along with AGR2 protein. Therefore there is little information on how AGR3 protein is expressed in normal and diseased states. A panel of three monoclonal antibodies was generated towards AGR3 protein for identifying novel clinical models that can be used to define whether AGR3 protein could play a positive or negative role in human cancer development. One monoclonal antibody was AGR3-specific and bound a linear epitope that could be defined using both pep-scan and phage-peptide library screening. Using this monoclonal antibody, endogenous AGR3 protein expression was shown to be cytosolic in four human ovarian cancer subtypes; serous, endometrioid, clear cell, and mucinous. Mucinous ovarian cancers produced the highest number of AGR3 positive cells. AGR3 expression is coupled to AGR2 expression only in mucinous ovarian cancers, whereas AGR3 and AGR2 expressions are uncoupled in the other three types of ovarian cancer. AGR3 expression in ovarian cancer is independent of oestrogen-receptor expression, which is distinct from the oestrogen-receptor dependent expression of AGR3 in breast cancers. Isogenic cancer cell models were created that over-express AGR3 and these demonstrated that AGR3 mediates cisplatin-resistance in mouse xenografts. These data indicate that AGR3 is over-expressed by a hormone (oestrogen-receptor α)-independent mechanism and identify a novel protein-folding associated pathway that could mediate resistance to DNA-damaging agents in human cancers.