Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage

Abstract

Purpose Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a pro-apoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage. Experimental Design Methods of Immunohistochemistry and Immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by pull-down assay. Poly-ubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays. Results UBE2F is overexpressed in NSCLC (Non-Small-Cell-Lung-Cancer), and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with low level of NOXA and poor patient survival. Conclusion By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target.

SAG/RBX2 E3 ligase complexes with UBCH10 and UBE2S E2s to ubiquitylate β-TrCP1 via K11-linkage for degradation

“…lanes 5 vs. 6), suggesting that it is likely that β-TrCP1 is subjected to ubiquitylation and degradation via both K11 and K48 linkage, although mainly via K11 linkage. Serving as the controls, NOXA is mainly ubiquitylated via K11 linkage for degradation, as we reported recently33, whereas p27 is solely ubiquitylated via K48 linkage for targeted degradation under this experimental conditions (Fig. 4F).…”

“…Establishment of U2OS cells expressing shUb-Ub (WT), -Ub (K11R), -Ub (K48R), and -Ub (K63R) were described previously33. Mouse embryonic stem cells (AB2, wild type and AB1, Sag-null) were cultured as described19.…”

Inhibition of Neddylation Modification Sensitizes Pancreatic Cancer Cells to Gemcitabine

“…However, we did not see further accumulations of these proteins in combinational treatment (Figure 7 A ), suggesting that it is unlikely that these proteins will play a critical role in chemo-sensitization. On the other hand, we did observe combinational treatment caused higher accumulation of (a) NOXA, a pro-apoptotic protein, shown to be a CRL5 substrate [22], [35], and (b) ERBIN, a natural occurring inhibitor of RAS-MAPK pathway [36], [37], a recently characterized substrate of SAG-CRL1 [23] in both pancreatic cancer cell lines (Figure 7 A ). Given the fact that NOXA is a p53 transcriptional target [38], whereas both Panc-1 and Miapaca-2 cells harbors mutant p53 [39], [40], the drug-induced NOXA accumulation is likely p53-independent, rather due to inhibition of cullin-RING ligase activity to block its degradation in case of MLN4924 effect.

Figure 7MLN4924-induced accumulation of NOXA and ERBIN plays a causal role in gemcitabine sensitization.

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“…The siRNAs targeting NOXA, ERBIN and control siRNA were described previously [22], [23]. Immunostaining kit was obtained from DakoCytomation California, Inc. (Carpinteria, CA).…”

Purine metabolism regulates DNA repair and therapy resistance in glioblastoma

“…Immunohistochemical analyses were performed using the ABC Vectastain Kit (Vector Laboratories) as previously described 51 . After deparaffinization, rehydration, antigen retrieval, and blocking, the tumor tissue slides were incubated with primary Ki-67 antibody (Cat# 550609, BD Biosciences) at 4°C overnight.…”