ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

The ubiquitin-proteasome system is vital for cell growth and homeostasis, but for most cancers proteasomal inhibition has not been effective as a therapy. Normal and cancer cells adapt to proteasomal stress through an evolutionarily conserved recovery response, in which the transcription factor NRF1 upregulates proteasome subunit genes. Starting with a C. elegans screen to identify regulators of the recovery response, here we show that this response depends upon phosphorylation of NRF1 on a single residue by the growth factor-activated kinase ERK1/2. Inhibition of this phosphorylation impairs NRF1 nuclear localization and proteasome gene activation, sensitizes C. elegans and cancer cells to proteasomal stress, and synergizes with proteasome inhibition to retard human melanoma growth in vivo in a mouse model. The evolutionarily conserved ERK1/2-NRF1 axis couples proteasome production to growth signaling, and represents a promising new strategy for expanding the range and efficacy of proteasomal inhibition therapy in cancer.

Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins – EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lsyosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, increases transcriptional activation of both latent and lytic gene expression and induces viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.Author SummaryEpstein-Barr virus (EBV) establishes latent infection in B-lymphocytes and is associated with a number of human malignancies, both of epithelial and lymphoid origin. EBV encoded EBNA3 family of nuclear latent antigens comprising of EBNA3A, EBNA3B, and EBNA3C are unique to immunoblastic lymphomas. While EBNA3A and EBNA3C are involved in blocking many important tumor suppressive mechanisms, EBNA3B exhibits tumor suppressive functions. Although EBNA3 proteins, in particular EBNA3C, interact with and employ different protein degradation machineries to induce B-cell lymphomagenesis, these viral proteins are extremely stable in growing B-lymphocytes. To this end, we now demonstrate that proteasomal inhibition leads to specifically degradation of oncogenic EBNA3A and EBNA3C proteins, whereas EBNA3B remains unaffected. Upon proteasomal inhibition, EBNA3C degradation occurs via autophagy-lysosomal pathway, through labeling with K63-linked polyubiquitination and participating in p62-LC3B complex involved in ubiquitin-mediated autophagy substrate selection and degradation through autolysosomal process. We also demonstrate that the N-terminal domain is responsible for autophgy-lysosomal mediated degradation, while the C-terminal domain plays a crucial role in cytoplasmic localization. Fascinatingly, while proteasomal inhibition reduces EBNA3C’s oncogenic property, it induces both latent and lytic gene expressions and promotes viral reactivation from EBV transformed B-lymphocytes. This is the first report which demonstrates a viral oncoprotein degrades through autophagy-lysosomal pathway upon proteasomal inhibition. In sum, the results promise development of novel strategies specifically targeting proteolytic pathway for the treatment of EBV associated B-cell lymphomas, particularly those are generated in immunocompromised individuals.