Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a 'good' transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2-small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.

In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulklysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival. Here, we report the identification of brusatol as a unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug.

In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.

Nrf2 is a transcription factor that has emerged as the cell's main defense mechanism against many harmful environmental toxicants and carcinogens. Nrf2 is negatively regulated by Keap1, a substrate adaptor protein for the Cullin3 (Cul3)-containing E3-ligase complex, which targets Nrf2 for ubiquitination and degradation by the ubiquitin proteasome system (UPS). Recent evidence suggests that constitutive activation of Nrf2, due to mutations in Keap1 or Nrf2, is prominent in many cancer types and contributes to chemoresistance. Regulation of Nrf2 by the Cul3-Keap1-E3 ligase provides strong evidence that tight regulation of Cullin-ring ligases (CRLs) is imperative to maintain cellular homeostasis. There are seven known Cullin proteins that form various CRL complexes. They are regulated by neddylation=deneddylation, ubiquitination=deubiquitination, CAND1-assisted complex assembly=disassembly, and subunit dimerization. In this review, we will discuss the regulation of each CRL using the Cul3-Keap1-E3 ligase complex as the primary focus. The substrates of CRLs are involved in many signaling pathways. Therefore, deregulation of CRLs affects several cellular processes, including cell cycle arrest, DNA repair, cell proliferation, senescence, and death, which may lead to many human diseases, including cancer. This makes CRLs a promising target for novel cancer drug therapies. Antioxid. Redox Signal. 13, 1699-1712.