The RING-finger protein Pirh2 is a p53 family-specific E3 ubiquitin ligase. Pirh2 also ubiquitinates several other important cellular factors and is involved in carcinogenesis. However, its functional role in other cellular processes is poorly understood. To address this question, we performed a proteomic search for novel interacting partners of Pirh2. Using the GST-pulldown approach combined with LC-MS/MS, we revealed 225 proteins that interacted with Pirh2. We found that, according to the GO description, a large group of Pirh2-associated proteins belonged to the RNA metabolism group. Importantly, one of the identified proteins from that group was an RNA-binding protein ELAVL1 (HuR), which is involved in the regulation of splicing and protein stability of several oncogenic proteins. We demonstrated that Pirh2 ubiquitinated the HuR protein facilitating its proteasome-mediated degradation in cells. Importantly, the Pirh2-mediated degradation of HuR occurred in response to heat shock, thereby affecting the survival rate of HeLa cells under elevated temperature. Functionally, Pirh2-mediated degradation of HuR augmented the level of c-Myc expression, whose RNA level is otherwise attenuated by HuR. Taken together, our data indicate that HuR is a new target of Pirh2 and this functional interaction contributes to the heat-shock response of cancer cells affecting their survival.

Ecdysterone (Ecdy) is a hormone found in arthropods, which regulates their development. It is also synthesized by a number of plants to combat insect pests. It provides a number of beneficial pharmacological effects including the anabolic and adaptogenic ones. Ecdysterone is widely marketed as food supplement to enhance the physical performance of athletes. In addition to the estrogen receptor beta (ERbeta)-dependent anabolic effect of Ecdy in muscles, the molecular mechanisms of the plethora of other Ecdy-induced pharmacological effects remain unknown. The aim of this study was to investigate the pharmacological effect of ecdysterone on human breast cancer cell lines of different molecular subtypes. Surprisingly, in contrast to the anabolic effect on muscle tissues, we have revealed a tumor suppressive effect of Ecdy on a panel of breast cancer cell lines studied. Using the SeaHorse-based energy profiling, we have demonstrated that Ecdy dampened glycolysis and respiration, as well as greatly reduced the metabolic potential of triple negative breast cancer cell lines. Furthermore, we have revealed that Ecdy strongly induced autophagy. As part of the combined treatment, based on the Combination Index (CI) and Dose Reduction Index (DRI), Ecdy synergized with doxorubicin to induce cell death in several breast cancer cell lines. In contrast, Ecdy had only minor effect on non-transformed human fibroblasts. Collectively, our results indicate that ecdysterone can be considered as a new potential adjuvant for genotoxic therapy in treatment of breast cancer patients.