Anti-DUSP4 (aa 339-368) polyclonal antibody

Ochratoxin A (OTA) is a carcinogenic mycotoxin, which is produced by Aspergillus and Penicillium genera of fungi and commonly contaminates food and feed. We and others have previously shown that OTA causes sustained activation of PI3K/AKT and MAPK/ERK1-2 signaling pathways in different cell types and animal models. Given the close relationship between cellular signaling activity and protein stability, we were curious whether increased PI3K/AKT and MAPK/ERK1-2 signaling may be the result of OTA-stimulated alterations in proteolytic activity. We show that both of the major proteolytic systems, autophagy, and the ubiquitin-proteasome system (UPS), are activated upon OTA exposure in human kidney proximal tubule HK-2 and mouse embryonic fibroblast (MEF) cells. OTA stimulates transient autophagic activity at early time points of treatment but autophagic activity subsides after 6 h even in the sustained presence of OTA. Interestingly, OTA exposure also results in increased cell death in wild-type MEF cells but not in autophagy-halted Atg5-deficient cells, suggesting that autophagy exerts a pro-death effect on OTA-induced cytotoxicity. In addition, prolonged OTA exposure decreased ubiquitinated protein levels by increasing proteasomal activity. Using purified and cellular proteasomes, we observed enhanced chymotrypsin-, caspase-, and trypsin-like activities of the 26S but not the 20S proteasome in the presence of OTA. However, in the cellular context, increased proteasomal activity depended on prior induction of autophagy. Our results suggest that autophagy and subsequent UPS activation are responsible for sustained activation of PI3K/AKT and MAPK/ERK1-2 pathways through regulating the levels of critical phosphatases VHR/DUSP3, DUSP4, and PHLPP, which are known to be involved in OTA toxicity and carcinogenicity.

Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2(+) and ERBB2(-) in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2(+) subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2(+), ER- ERBB2(-) and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.