Ubiquitin receptors play redundant roles in the proteasomal degradation of the p53 repressor MDM2.

Much remains to be determined about the participation of ubiquitin receptors in proteasomal degradation and their potential as therapeutic targets. Suppression of the ubiquitin receptor S5A/PSMD4/hRpn10 alone stabilises p53/TP53 but not the key p53 repressor MDM2. Here, we observed S5A and the ubiquitin receptors ADRM1/PSMD16/hRpn13 and RAD23A and B functionally overlap in MDM2 degradation. We provide further evidence that degradation of only a subset of ubiquitinated proteins is sensitive to S5A knockdown because ubiquitin receptor redundancy is commonplace. p53 can be upregulated by S5A modulation while degradation of substrates with redundant receptors is maintained. Our observations and analysis of Cancer Dependency Map (DepMap) screens show S5A depletion/loss substantially reduces cancer cell line viability. This and selective S5A dependency of proteasomal substrates make S5A a target of interest for cancer therapy.

Suppression of mutant Kirsten-RAS (KRASG12D)-driven pancreatic carcinogenesis by dual-specificity MAP kinase phosphatases 5 and 6.

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRASG12D-driven pancreatic cancer. By 56-days, loss of either DUSP5 or DUSP6 causes a significant increase in KRASG12D-driven pancreatic hyperplasia. This is accompanied by increased pancreatic acinar to ductal metaplasia (ADM) and the development of pre-neoplastic pancreatic intraepithelial neoplasia (PanINs). In contrast, by 100-days, pancreatic hyperplasia is reversed with significant atrophy of pancreatic tissue and weight loss observed in animals lacking either DUSP5 or DUSP6. On further ageing, Dusp6-/- mice display accelerated development of metastatic pancreatic ductal adenocarcinoma (PDAC), while in Dusp5-/- animals, although PDAC development is increased this process is attenuated by atrophy of pancreatic acinar tissue and severe weight loss in some animals before cancer could progress. Our data suggest that despite a common target in the ERK MAP kinase, DUSP5 and DUSP6 play partially non-redundant roles in suppressing oncogenic KRASG12D signalling, thus retarding both tumour initiation and progression. Our data suggest that loss of either DUSP5 or DUSP6, as observed in certain human tumours, including the pancreas, could promote carcinogenesis.

The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma.

We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.

Targeting the spliceosome for cutaneous squamous cell carcinoma therapy: a role for c-MYC and wild-type p53 in determining the degree of tumour selectivity.

We show that suppression of the spliceosome has potential for the treatment of cutaneous squamous cell carcinoma (cSCC). The small-molecule inhibitors of the spliceosome at the most advanced stage of development target the splicing factor SF3B1/SF3b155. The majority of cSCC cell lines are more sensitive than normal skin cells to death induced by the SF3B1 inhibitor pladienolide B. Knockdown of SF3B1 and a range of other splicing factors with diverse roles in the spliceosome can also selectively kill cSCC cells. We demonstrate that endogenous c-MYC participates in conferring sensitivity to spliceosome inhibition. c-MYC expression is elevated in cSCC lines and its knockdown reduces alterations in mRNA splicing and attenuates cell death caused by interference with the spliceosome. In addition, this study provides further support for a key role of the p53 pathway in the response to spliceosome disruption. SF3B1 inhibition causes wild-type p53 upregulation associated with altered mRNA splicing and reduced protein expression of both principal p53 negative regulators MDMX/MDM4 and MDM2. We observed that wild-type p53 can promote pladienolide B-induced death in tumour cells. However, p53 is commonly inactivated by mutation in cSCCs and p53 participates in killing normal skin cells at high concentrations of pladienolide B. This may limit the therapeutic window of SF3B1 inhibitors for cSCC. We provide evidence that, while suppression of SF3B1 has promise for treating cSCCs with mutant p53, inhibitors which target the spliceosome through SF3B1-independent mechanisms could have greater cSCC selectivity as a consequence of reduced p53 upregulation in normal cells.

Preclinical comparison of proteasome and ubiquitin E1 enzyme inhibitors in cutaneous squamous cell carcinoma: the identification of mechanisms of differential sensitivity.

Proteasome inhibitors have distinct properties and the biochemical consequences of suppressing ubiquitin E1 enzymes and the proteasome differ. We compared the effects of the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 enzyme inhibitor MLN7243/TAK-243 on cell viability and cell death in normal keratinocytes and cutaneous squamous cell carcinoma (cSCC) cell lines. The effects of both a pulse of treatment and more extended incubation were investigated. This is relevant to directly-delivered therapy (topical treatment/intratumoral injection) where the time of exposure can be controlled and a short exposure may better reflect systemically-delivered inhibitor pharmacokinetics. These agents can selectively kill cSCC cells but there are variations in the pattern of cSCC cell line sensitivity/resistance. Variations in the responses to proteasome inhibitors are associated with differences in the specificity of the inhibitors for the three proteolytic activities of the proteasome. There is greater selectivity for killing cSCC cells compared to normal keratinocytes with a pulse of proteasome inhibitor treatment than with a more extended exposure. We provide evidence that c-MYC-dependent NOXA upregulation confers susceptibility to a short incubation with proteasome inhibitors by priming cSCC cells for rapid BAK-dependent death. We observed that bortezomib-resistant cSCC cells can be sensitive to MLN7243-induced death. Low expression of the ubiquitin E1 UBA1/UBE1 participates in conferring susceptibility to MLN7243 by increasing sensitivity to MLN7243-mediated attenuation of ubiquitination. This study supports further investigation of the potential of proteasome and ubiquitin E1 inhibition for cSCC therapy. Direct delivery of inhibitors could facilitate adequate exposure of skin cancers.

Targeting the ubiquitin-proteasome system to activate wild-type p53 for cancer therapy.

Ubiquitination plays a key role in regulating the tumour suppressor p53. It targets p53 for degradation by the 26S proteasome. The ubiquitin pathway also regulates the activity and localisation of p53. Ubiquitination requires ubiquitin-activating and -conjugating enzymes and ubiquitin ligases. In addition, ubiquitination can be reversed by the action of deubiquitinating enzymes. Here we give an overview of the role of components of the ubiquitin-proteasome system in the regulation of p53 and review progress in targeting these proteins to activate wild-type p53 for the treatment of cancer.

Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53.

Both p53 and its repressor Mdm2 are subject to ubiquitination and proteasomal degradation. We show that knockdown of the deubiquitinating enzyme USP5 (isopeptidase T) results in an increase in the level and transcriptional activity of p53. Suppression of USP5 stabilizes p53, whereas it has little or no effect on the stability of Mdm2. This provides a mechanism for transcriptional activation of p53. USP5 knockdown interferes with the degradation of ubiquitinated p53 rather than attenuating p53 ubiquitination. In vitro studies have shown that a preferred substrate for USP5 is unanchored polyubiquitin. Consistent with this, we observed for the first time in a mammalian system that USP5 makes a major contribution to Lys-48-linked polyubiquitin disassembly and that suppression of USP5 results in the accumulation of unanchored polyubiquitin chains. Ectopic expression of a C-terminal mutant of ubiquitin (G75A/G76A), which also causes the accumulation of free polyubiquitin, recapitulates the effects of USP5 knockdown on the p53 pathway. We propose a model in which p53 is selectively stabilized because the unanchored polyubiquitin that accumulates after USP5 knockdown is able to compete with ubiquitinated p53 but not with Mdm2 for proteasomal recognition. This raises the possibility that there are significant differences in proteasomal recognition of p53 and Mdm2. These differences could be exploited therapeutically. Our study reveals a novel mechanism for regulation of p53 and identifies USP5 as a potential target for p53 activating therapeutic agents for the treatment of cancer.

The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2.

Mdm2 is an E3 ubiquitin ligase that promotes its own ubiquitination and also ubiquitination of the p53 tumour suppressor. In a bacterial two-hybrid screen, using Mdm2 as bait, we identified an Mdm2-interacting peptide that bears sequence similarity to the deubiquitinating enzyme USP2a. We have established that full-length USP2a associates with Mdm2 in cells where it can deubiquitinate Mdm2 while demonstrating no deubiquitinating activity towards p53. Ectopic expression of USP2a causes accumulation of Mdm2 in a dose-dependent manner and consequently promotes Mdm2-mediated p53 degradation. This differs from the behaviour of HAUSP, which deubiquitinates p53 in addition to Mdm2 and thus protects p53 from Mdm2-mediated degradation. We further demonstrate that suppression of endogenous USP2a destabilises Mdm2 and causes accumulation of p53 protein and activation of p53. Our data identify the deubiquitinating enzyme USP2a as a novel regulator of the p53 pathway that acts through its ability to selectively target Mdm2.

Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules.

MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposi's sarcoma-associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3-associated class I by lysine-63-linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine-63-linked polyubiquitination requires both UbcH5b/c and Ubc13-conjugating enzymes for initiating mono- and subsequent polyubiquitination of class I, and the clathrin-dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine-63-linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.

p53 isoforms can regulate p53 transcriptional activity.

The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53beta can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while Delta133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.

p14 Arf promotes small ubiquitin-like modifier conjugation of Werners helicase.

Here we demonstrate a novel p53-independent interaction between the nucleolar tumor suppressors, p14 Arf and Werners helicase (WRN). Binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2. Residues 2-14 and 82-101 of p14 Arf and residues in the central region and C terminus of WRN have particular importance for binding. p14 Arf promotes small ubiquitin-like modifier (SUMO) modification of WRN in a synergistic manner with the SUMO-conjugating enzyme, UBCH9. p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-specific protease, thus implicating the SUMO conjugation pathway in WRN re-localization. We establish that the ability to promote SUMO conjugation is a general property of the p14 Arf tumor suppressor.

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo.

p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity.

Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. Here, we show that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), we demonstrate that Mdm2-dependent NEDD8 modification of p53 inhibits its transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins.

B-Myb overcomes a p107-mediated cell proliferation block by interacting with an N-terminal domain of p107.

B-Myb is a cell-cycle regulated transcription factor which is implicated in cell proliferation and has an essential role in early embryonic development. In this study we examined the functions of B-Myb required to overcome G1 arrest in Saos-2 cells induced by the retinoblastoma-related p107 protein. Our results demonstrated that this activity was independent of B-Myb transactivation function, but correlated with its capacity to form an in vivo complex with p107. A large proportion of B-Myb formed complexes with p107 in cotransfected cells, however, B-Myb bound weakly to the related p130 protein and not at all to pRb. In contrast to the E2F transcription factors, which bind the p107 C-terminal pocket domain, B-Myb recognizes an N-terminal p107 region which overlaps the larger cyclin-binding domain. B-Myb and cyclin A2 formed mutually exclusive complexes with p107, and B-Myb enhanced the activity of co-transfected cyclin E kinase activity, implying that B-Myb affects the cell cycle by preventing sequestration of active cyclin/cdk2 complexes. This study defines a novel function of B-Myb and further suggests that the p107 N-terminus provides an interaction domain for transcription factors involved in cell cycle control.