Chapter 5: Preferential cullin neddylation and dysregulation of the WNT pathway by
5.5 Results
5.5.1 DCUN1D1 knockdown in PCa cells decreased global neddylation, ubiquitination and decreased expression of neddylation components
The neddylation pathway is a posttranslational modification process that mediates transfer of 9kDa NEDD8 to target proteins (Kumar, Yoshida and Noda, 1993; Soucy et al., 2010). It also plays a role in ubiquitination through NEDD8 modification of the cullin proteins, which are scaffolding molecules in the cullin RING E3 ligases (CRLs) (Hori et al., 1999; Petroski and Deshaies, 2005). Although initially thought to be an enhancer of neddylation, in vivo studies demonstrated that DCUN1D1 is essential for neddylation, in part, due to its role in the nuclear translocation of cullin 1 (Kim et al., 2008; G. Huang et al., 2011). We performed proteomics analysis to identify DCUN1D1 substrates and to determine its mechanism of action in PCa. To explore this further, we began by testing the global neddylation and ubiquitination status of PCa following knockdown of DCUN1D1 by performing western blot analysis of DU145 and DU145 DCUN1D1 knockdown cells. We observed significant reduction in DCUN1D1 expression levels in the DU145 DCUN1D1 knockdown cells relative to the control cells (Figure 43A).
Additionally, immunoblotting of whole-cell lysates using anti-NEDD8 antibody showed decreased neddylation in the knockdown cells, particularly in the high molecular weight region (Figure 43B). We also observed decreased anti-ubiquitin immunoreactivity in the DU145 DCUN1D1 knockdown cells relative to the control DU145 cells (Figure 43C).
Figure 43. Showing western blot analysis of DCUN1D1, NEDD8 and ubiquitin expression. Protein extracts were obtained from DU145 and DU145 DCUN1D1 knockdown cell lines and tested for A) DCUN1D1 expression, B) neddylation status using anti-NEDD8 antibody and C) the ubiquitination status using the anti-ubiquitin antibody. The GAPDH loading control was probed using the anti- GAPDH antibody.
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Having observed the above, we evaluated the expression level of the components of the neddylation pathway. Neddylation is a multi-step process that is mediated by an E1, E2 and an E3 ligase. It begins with the activation of NEDD8 by the E1 neddylation activating enzyme (NAE) heterodimer, APPBP1/UBA3, followed by transfer of the activated NEDD8 to the E2 neddylation conjugation enzyme (UBC12) (Osaka et al., 1998; Gong and Yeh, 1999; Huang et al., 2004, 2005). UBC12 then transfers NEDD8 to the neddylation E3 ligase for substrate modification (Figure 15) (Kim et al., 2008). We wanted to determine whether DCUN1D1 knockdown and the subsequently observed reduction in neddylation was due to a dysregulation in the expression of the other components of the neddylation pathway. This was also of interest because although we identified components of the neddylation pathway following IPMS analysis in HEK293TT, we did not identify any in the SILAC proteomics analysis in DU145 versus DU145 DCUN1D1 knockdown cells. Significantly, knockdown of DCUN1D1 in DU145 PCa cells led to decreased expression of the E1 NAE APPBP1/UBA3 and the neddylation conjugation enzyme, UBC12 (Figure 44A). This data demonstrates that dysregulation of DCUN1D1 led to significant disruption of the neddylation pathway as observed by reduced expression of the components that mediate neddylation. It also indicates that dysregulation of the neddylation pathway was key to the mechanism of action DCUN1D1 in PCa.
Figure 44. Blockage of DCUN1D1 decreased expression of neddylation pathway components and cullin-associated proteins. Protein extracts containing DU145 and DU145 DCUN1D1 knockdown cell lines were analysed using western blot analysis. A) Blockage of DCUN1D1 decreased the expression of the neddylation pathway components including the E1 NAE heterodimer APPB1 (top panel), UBA3 (middle panel) and the neddylation conjugating enzyme, UBC12 (bottom panel). B) Western blot showing decreased expression of the cullin-associated proteins RBX1 (top panel) and CAND1 (bottom panel). The GAPDH loading control was probed using the anti-GAPDH antibody.
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5.5.2 DCUN1D1 shows preferential neddylation activity of cullin proteins in PCa
We explored this further by evaluating the status of proteins that play a role in the neddylation of cullin proteins, which included the cullin-associated RBX1 and the negative regulator, CAND1. RBX1 (RING box protein 1) is a ubiquitin E3 ligase component of the CRLs that interacts with cullins (Kamura et al., 1999; Chen et al., 2000; Petroski and Deshaies, 2005). The cullin-RBX interaction is considered to be the core complex of the CRLs and when unneddylated it is normally bound to CAND1 (J. Zheng et al., 2002; Wu et al., 2016). CAND1 on the other hand binds to cullin-RBX complexes creating sterical hindrance to CRL assembly, acting as a CRL assembly factor (Min et al., 2003; Goldenberg et al., 2004;
Wu et al., 2016). It functions as an exchange factor for the F box substrate recognition protein during CRL assembly, to facilitate substrate ubiquitination. It interacts specifically with unneddylated cullin- RBX complexes and is associated with DCUN1D1 (J. Zheng et al., 2002; Kim et al., 2008; Wu et al., 2016). Therefore, to determine the extent of the effect of DCUN1D1 knockdown on the neddylation pathway components involved in cullin neddylation, we evaluated the expression levels of RBX1 and CAND1. We observed decreased expression of RBX1 and CAND1 following knockdown of DCUN1D1 in DU145 PCa cells in comparison to the control (Figure 44B). This demonstrated the dysregulation of cullin-neddylation associated proteins following knockdown of DCUN1D1 in PCa cells. It also provided further evidence for the association of DCUN1D1 with RBX1 and CAND1, as observed previously and as demonstrated following IPMS analysis in this study (Kim et al., 2008).
We then determined whether the dysregulation in the expression of modulators of cullin neddylation had an impact on the status of cullin proteins, upon markedly reduced DCUN1D1 expression. Although cullin 3, cullin 4B and cullin 5 were identified as DCUN1D1 substrates in this study, studies performed previously have demonstrated that RBX1 and DCUN1D1 interact with cullin 1, cullin 2, cullin 3, cullin 4A, cullin 4B and cullin 5 (Ohta et al., 1999; Petroski and Deshaies, 2005; Kim et al., 2008). Therefore, to validate the above as DCUN1D1 substrates and to evaluate the neddylation status of the potential DCUN1D1 cullin substrates in PCa, we performed western blot analysis on cullin 1 - 5. Significantly, we did not observe any changes in the expression levels of these cullins following western blot analysis of DU145 versus DU145 DCUN1D1 knockdown cells, however, we observed selective decreases in NEDD8 modification (Figure 45). We observed decreased NEDD8 modification in cullin 1, cullin 3, cullin 4A, cullin 4B and cullin 5 following DCUN1D1 knockdown. We did not observe this effect in cullin 2 (Figure 45). This data further validates cullin 3, cullin 4B and cullin 5 as DCUN1D1 substrates as observed previously and identified in IPMS performed in this study. It also suggests preferential cullin neddylation of DCUN1D1 in its mechanism of action in PCa. Although DCUN1D1 has been demonstrated to neddylate cullin 1 - 5, this is the first description of DCUN1D1-mediated preferential neddylation of cullin proteins in PCa.
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5.5.3 The WNT pathway was inhibited following knockdown of DCUN1D1 in PCa
In order to determine the broader mechanism of action of DCUN1D1 in PCa we explored the WNT pathway which was dysregulated following proteomics analysis in this study and is subject to regulation by CRL ubiquitination (Stamos and Weis, 2013). It was also of interest because studies performed previously in our lab found it to be dysregulated by DNA microarray analysis of DU145 versus DU145 DCUN1D1 knockdown cells (Vava and Zerbini, 2014).
We explored the expression level of β-catenin, which is a widely characterized transcriptional co- activator of the canonical WNT pathway (MacDonald, Tamai and He, 2009). The WNT/β-catenin pathway has also been implicated in a number of pathways/processes that span the mechanism of action of DCUN1D1 as proposed in this study. These include the regulation of transcription, cell differentiation, development and the regulation of inflammation (Masckauchán et al., 2005; Yaguchi et al., 2012; Steinhart and Angers, 2018). β-catenin has also been shown to be associated with PPAR signalling which was dysregulated following IPMS analysis of DCUN1D1 pulldown products (Sharma et al., 2004; Liu et al., 2006). Significantly, we observed increased phosphorylation of β-catenin and a subsequent reduction in the expression level of total β-catenin in the DU145 DCUN1D1 knockdown cells relative to the control DU145 cells (Figure 46). This indicated the deactivation of the WNT signalling pathway following knockdown of DCUN1D1. In the absence of stimulation of the WNT pathway by ligand binding, cytoplasmic β-catenin is phosphorylated then targeted for proteasomal
Figure 45. Blockage of DCUN1D1 showed preferential NEDD8-modification of the cullin family of proteins. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using anti-cullin 1, anti-cullin 2, anti-cullin 3, anti-cullin 4A, anti-cullin 4B and anti-cullin 5. The GAPDH loading control was probed using the anti-GAPDH antibody.
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degradation, preventing its entry into the nucleus and repressing the expression of WNT target genes (MacDonald, Tamai and He, 2009; Stamos and Weis, 2013). Therefore, blockage of DCUN1D1 led to the deactivation of the WNT pathway, validating its role in the mechanism of action of DCUN1D1.
5.5.4 Blockage of DCUN1D1 activated the AKT pathway in PCa
In order to further elucidate the mechanism of action of DCUN1D1, we tested the status of the AKT pathway due to the known association between the WNT and AKT pathway and their key roles in cancer. The dysregulation of the PI3K/AKT/mTOR pathway has been extensively associated with the development and progression of PCa including metastatic PCa (Edlind and Hsieh, 2014). Additionally, we identified PI3K and mTOR inhibitors as the top 8 and top 17 scoring perturbagen classes in sample LH3 following CMap analysis of the DCUN1D1 knockdown signature. Particularly, we identified compounds that target components of the PI3K/AKT/mTOR in sample LH3 including sirolimus (mTOR inhibitor), everolimus (mTOR inhibitor), PI-828 (PI3K inhibitor), deforolimus (mTOR inhibitor) and AZD- 8055 (mTOR inhibitor). Therefore, to determine the status of the AKT pathway, we immunoblotted whole-cell lysates using anti-AKT (Ser473) antibody and observed increased phosphorylation of AKT at Ser473, which is the phosphorylation step that completes activation of AKT (Figure 47). This indicates that blockage of DCUN1D1 leads to the activation of the AKT signalling pathway and although the AKT pathway is a pro-survival and proliferation pathway, studies performed in our lab found that knockdown of DCUN1D1 in PCa cells decreased proliferation and migration (Fresno Vara et al., 2004;
Figure 46. Inhibition of DCUN1D1 deactivated the WNT signalling pathway. Protein extracts containing DU145 and DU145 DCUN1D1 knockdown cell lines were analysed using western blot analysis. Western blot analysis showing increased phosphorylation of β-catenin and decreased expression of total β-catenin. The GAPDH loading control was probed using the anti-GAPDH antibody.
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Vava and Zerbini, 2014). Therefore, it could be that the activation of the AKT pathway is a response by the cancer cells to the effects of DCUN1D1 knockdown but is not sufficient to impede the inhibition of PCa growth.
Figure 47. Showing western blot analysis of the AKT pathway. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using anti-p-AKT, anti-AKT. The GAPDH loading control was probed using the anti-GAPDH antibody.
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