Abstract
Hyperproliferating cancer cells produce energy mainly from aerobic glycolysis, which results in elevated ROS levels. Thus aggressive tumors often possess enhanced anti-oxidant capacity that impedes many current anti-cancer therapies. Additionally, in ROS-compromised cancer cells ubiquitin proteasome system (UPS) is often deregulated for timely removal of oxidized proteins, thus enabling cell survival. Taken that UPS maintains the turnover of factors controlling cell cycle and apoptosis – such as p53 or p73, it represents a promising target for pharmaceutical intervention. Enhancing oxidative insult in already ROS-compromised cancer cells appears as an attractive anti-tumor scenario. TAp73 is a bona fide tumor suppressor that drives the chemosensitivity of some cancers to cisplatin or γ-radiation. It is an important drug target in tumors where p53 is lost or mutated. Here we discovered a novel synergistic mechanism leading to potent p73 activation and cancer cell death by oxidative stress and inhibition of 20S proteasomes. Using a small-molecule inhibitor of 20S proteasome and ROS-inducer – withaferin A (WA), we found that WA-induced ROS activates JNK kinase and stabilizes phase II anti-oxidant response effector NF-E2-related transcription factor (NRF2). This results in activation of Nrf2 target – NQO1 (NADPH quinone oxidoreductase), and TAp73 protein stabilization. The observed effect was ablated by the ROS scavenger – NAC. Concurrently, stress-activated JNK phosphorylates TAp73 at multiple serine and threonine residues, which is crucial to ablate TAp73/MDM2 complex and to promote TAp73 transcriptional function and induction of robust apoptosis. Taken together our data demonstrate that ROS insult in combination with the inhibition of 20S proteasome and TAp73 activation endows synthetic lethality in cancer cells. Thus, our results may enable the establishment of a novel pharmacological strategy to exploit the enhanced sensitivity of tumors to elevated ROS and proteasomal stress to kill advanced tumors by pharmacological activation of TAp73 using molecules like WA.
Highlights
Redox homeostasis is essential to sustain cellular functions and enable cell survival
Withaferin A (WA) was shown to exert anti-tumor activity in cells expressing mutant or wildtype p53.29,47 To address the question whether cancer cells lacking p53 but expressing TA isoform of p73 are dying upon treatment with WA, we performed viability and clonogenic assays in H1299, human lung adenocarcinoma cells and in HCT 116TP53 − / − human colon cancer cell line previously engineered for homozygous deletion of TP53 gene resulting in p53 protein loss of function.[48]
We have found that WA induced oxidative stress and activated the NRF2-related antioxidant response (Figures 2a and c), which is prevented by cells. (e) Co-expression of MDM2 inhibits TAp73-dependent transactivation in a yeast-based reporter assay, which is partially restored by WA (n = 4). (f) In the presence of Jun N-terminal kinase (JNK)
Summary
Redox homeostasis is essential to sustain cellular functions and enable cell survival. In normal cells, elevated levels of ROS often promote an enhanced activity of the proteasome system to remove the oxidized proteins, to prevent their toxicity This phenomenon is observed in many human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.[14,15,16,17,18,19] ROS-compromised cancer cells, for timely removal of oxidized proteins, possess an enhanced system for the protein ubiqutination and an amplified activity of the ubiquitin proteasome pathway (UPP), in particular 20S proteasome particle.[14] The 20S proteasome particle is important for the degradation of the liable tumor suppressors like p53 and TAp73, the process rescued by NADPH quinone oxidoreductase (NQO1).[20] With the approval of bortezomib, to treat multiple myeloma, inhibition of the proteasome activity emerges as a promising anti-cancer therapy.[21]. TA isoforms of p73 can compensate for p53 function in tumors with lost or mutated p53
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