Chapter 6: General Discussion, Conclusion and Future Work
6.1 General Discussion
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(ELONGIN BC-CUL2/5-SOCS box) (Petroski and Deshaies, 2005). Based on this work and evidence published previously on DCUN1D1 substrates, we suggest that DCUN1D1 may be “Cullin Neddylase 1”
(Kim et al., 2008; K. Huang et al., 2011; Heir et al., 2013; Keuss et al., 2016; Huang et al., 2017). It appears to specifically target the cullin proteins but to play a role in critical cellular activities. This could be in a manner similar to kinases and phosphatases where the posttranslational modification signals or triggers critical cellular activities. DCUN1D1 may be targeting cullin proteins then based on the functions associated with that cullin and related ubiquitination substrates, impacting cellular functions. This could be attributing to the mechanism of action of DCUN1D1 in PCa based on the proteins targeted downstream of the neddylated cullin RING E3 ligases or through other mechanisms that require in-depth exploration.
Furthermore, following SILAC quantitative proteomics analysis we identified proteins differentially expressed in DU145 DCUN1D1 knockdown cells relative to DU145 PCa cells. The changes in the proteome we quantified classified into cellular components, organelles, structure molecule activity with the biological processes dysregulated including biological regulation, cellular component organization/biogenesis, cellular processes, localization, metabolic processes and response to stimulus. We also observed associations as depicted by STRING analysis, between the components of the 43S preinitiation complex (whose assembly triggers the eukaryotic translation initiation), the ribosome, ribonucleoproteins, the centrosome, the spliceosome and processing in the endoplasmic reticulum. Interestingly, as observed in the dysregulation of the centrosome here, DCUN1D1 has previously been implicated in mitosis, where, DCUN1D1-mediated cullin 3 neddylation promoted Aurora B ubiquitination during mitosis (Huang et al., 2017). Additionally, we found dysregulation in TCP1 which has been demonstrated to be involved in the folding of 10% of the cell proteome and has been found to be upregulated in prostate tissue samples using tissue microarray analysis (Leitner et al., 2012). We also found RPS10 which is a component of the 40S ribosomal subunit and the eukaryotic translation initiation factor 3 subunit B (EIF3B) which both play a role in protein synthesis through protein translation (Pestova et al., 2001). This suggested that the knockdown of the protein- degradation linked E3 ligase of neddylation (DCUN1D1) in PCa, led to alterations in translation-related and protein-processing activities.
Similar to the IPMS analysis, we observed the implication of metabolic processes and inflammatory responses. However, the metabolic pathways pertained to glycolysis and pyruvate metabolism as opposed to lipid metabolism and changes in cellular energetics is a known “Hallmark of Cancer”.
Inflammatory responses were also identified through dysregulation in the inflammation mediated by chemokine and cytokine biological processes. There was also dysregulation in cellular signalling as observed with the dysregulation of cytoskeletal regulation of Rho GTPase signalling, FGF signalling and integrin signalling. Significantly, we found the dysregulation of the WNT pathway specifically through ACTB and potentially, HNRNPA2B1 which has been shown to be upregulated in PCa and to regulate the WNT pathway component, β-catenin (Stockley et al., 2014). Moreover, although the substrates of DCUN1D1 identified in the pulldown assay such as the cullins and the cullin-neddylation components were not identified, we found the ubiquitin activating enzyme UBA1, providing further evidence for the association of UBA1 with the neddylation pathway as demonstrated previously by Leidecker et al., 2012. We also observed the dysregulation of potential neddylation substrates in the form of ribosomal proteins including, RPS6, RPS16, RPL27, where, RPS6 was also identified following IPMS analysis of DCUN1D1 pulldown products.
Interestingly, DCUN1D1 has a C terminus UBA domain which is normally found in proteins involved in the UPP, DNA excision repair, cell signalling via kinases and in proteins with elongation factor binding domains and these encompass the above mentioned activities (L. Chen et al., 2001; Hartmann-
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Petersen et al., 2003; Myung, Kim and Crews, 2008; Huang et al., 2015; Wu et al., 2015; Wang et al., 2018). The UBA domain-associated activity could also allude to the insights obtained from the targets and the mechanisms of the compounds strongly connected to the DCUN1D1 knockdown signature following CMap analysis.
We found compounds related to perturbagen classes that were strongly connected to the DCUN1D1 knockdown signature which included: leucine rich repeat kinase inhibitors, PKC activators, DNA dependent protein kinase inhibitors, bromodomain inhibitors, IGF-1 inhibitors, bile acid and HMGCR inhibitors. We also found anti-inflammatory compounds, such as talniflumate and palmitoylethanolamide as well as chemical messenger agonists such as the androgenic steroid mestanolone and dopamine agonists, ergocryptine and carmoxirole. Interestingly, blockage of DCUN1D1 in PCa cells produced a signature that strongly connected to kinase inhibitors.
PhosphoSitePlus® analysis has demonstrated the presence of two phosphorylation sites (S31 and Y72) in the N terminus of DCUN1D1. Furthermore, transfection of DCUN1D1 in LnCap cells has been shown to inhibit invasion and migration involving the focal adhesion kinase (FAK) and DCUN1D1 has been found to be an endogenous activator of FAK in non-small-cell lung cancer (Zhang et al., 2017; Li et al., 2019). However, we did not identify FAK in our study and there has not been extensive description of the role of kinases in DCUN1D1-mediated activity nor the identification of a kinase or kinases that phosphorylate DCUN1D1. Therefore, the strong connection observed with the DCUN1D1 knockdown signature and kinase inhibitors suggests the potential role of the kinases such as DNA dependent kinases in DCUN1D1-mediated activities and the potential of treatment of DCUN1D1-regulated kinase pathways in PCa.
Interestingly, although we may have expected the identification of the NAE inhibitor among the strongly connected compounds due to linkages to the neddylation pathway, our search did not identify a strong connection with the NAE inhibitor MLN4924. We did however find strong connections to the proteasome inhibitor, calpeptin in one of the samples. It’s important to note that we performed CMap database searches using the minimum acceptable number of gene names, but the range of values accepted by the database is 10 – 150. Therefore, it is possible that a greater sample size may strengthen connections with some lower scoring compounds and provide further insights.
Significantly, although not found in common across our samples, we found two compounds that we identified and screened as potential DCUN1D1 inhibitors following DNA microarray analysis of DCUND1 knockdown in DU145 PCa cells, namely, thapsigargin and podophyllotoxin. We observed podophyllotoxin to decrease mRNA and protein expression of DCUN1D1 and to have an additive inhibitory effect on PCa growth in combination with monensin (Vava and Zerbini, 2014). Identifying them using a proteomics approach and transcriptomics approach strengthens the evidence for their potential as DCUN1D1 inhibitors.
The collective information we obtained following the above-mentioned high throughput data analyses led us to a proposed mechanism of action. Western blot analysis of changes in specific protein expression levels following knockdown of DCUN1D1 determined that blockage of DCUN1D1 led to dysregulation of the neddylation pathway, ubiquitination and deactivation of the WNT/β-catenin pathway. Additionally, although DCUN1D1 has been demonstrated to neddylate cullin 1 - 5, our data suggests selective DCUN1D1-mediated cullin neddylation in PCa (Kim et al., 2008; Keuss et al., 2016).
This would be the first description of DCUN1D1-mediated preferential neddylation of cullin proteins in PCa. Moreover, the observed deactivation in the WNT/β-catenin pathway following blockage of DCUN1D1 strengthens the evidence previously found in our lab around the association between DCUN1D1 and the WNT signalling pathway using transcriptomics and currently using different proteomics approaches (Vava and Zerbini, 2014). The WNT/β-catenin pathway has been shown to play
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a critical role in cell proliferation, embryonic development and the pro-inflammatory phenotype in the tumour environment (Masckauchán et al., 2005; Yaguchi et al., 2012; Steinhart and Angers, 2018).
These activities overlap with the observations that we have made of DCUN1D1 and implicate it in the mechanism of action of DCUN1D1.
This study has provided further insights into the cellular activities into which DCUN1D1 may be playing a role. Analysis of DCUN1D1 binding partners identified cullin proteins as target proteins of DCUN1D1 which along with studies published previously on DCUN1D1, we postulate may be the primary targets of DCUN1D1. We also identified neddylation substrates such as ribosomal proteins of which DCUN1D1 is associated, however, it was not clear whether this could be as an E3 ligase or as part of a broader mechanism such as through the implication in eukaryotic translation or dysregulation of ribosome biogenesis in PCa. In addition, key cellular pathways were implicated including proteasome degradation, transcription, translation, metabolism and inflammation. Analysis of the drugs strongly connected with the DCUN1D1 knockdown signature in PCa showed aspects of the implicated pathways as well as strong connections with kinase inhibitors, raising interesting questions around the role of DCUN1D1-mediated kinase signalling pathways in PCa. We validated the role of cullin 3, cullin 4B and cullin 5 neddylation in the mechanism of DCUN1D1 in PCa, demonstrating preferential decreases in cullin neddylation of cullin 1, 3, 4A, 4B and cullin 5. Following the strength of evidence obtained in our lab at a transcriptomics and proteomics level, the WNT/β-catenin pathway was shown using western blot as playing a role in the mechanism of action of DCUN1D1 in PCa.