Faculty Bibliography

In response to genotoxic stress, eukaryotic cells activate the DNA damage response (DDR), a series of pathways that coordinate cell cycle arrest and DNA repair to prevent deleterious mutations. In addition, cells possess checkpoint mechanisms that prevent aneuploidy by regulating the number of centrosomes and spindle assembly. Among these mechanisms, ubiquitin-mediated degradation of key proteins has an important role in the regulation of the DDR, centrosome duplication and chromosome segregation. This review discusses the functions of a group of ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) family, in the maintenance of genome stability. Given that general proteasome inhibitors are currently used as anticancer agents, a better understanding of the ubiquitylation of specific targets by specific ubiquitin ligases may result in improved cancer therapeutics.

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene

BACKGROUND: In several human cancers, overexpression of Skp2 (S-phase kinase associated protein 2), which targets p27 for degradation, portends a poorer prognosis. We examined whether Skp2 overexpression is associated with recurrence following radical prostatectomy (RP) for prostate cancer. METHODS: Immunohistochemical staining for Skp2, p27, and MIB-1 was performed on 109 men with node-negative prostate cancer surgically managed from 1985-1996. Associations between the stains were tested and Cox regression was used to determine the association between Skp2 expression and time to biochemical recurrence following RP. RESULTS: The 12 tumors (11%) with Skp2 overexpression all had correspondingly low p27 expression (P=0.006), and a similar inverse Skp2/p27 relationship was seen in vitro in LNCap cells. Skp2 overexpression in tissue was associated with higher Gleason score (P=0.002), more advanced pathological stage (P=0.01), and higher MIB-1 index (P=0.03), but a more favorable PSA profile (P=0.04). Five men received a TURP. Among 104 who received RP, median follow-up was 67 months (range: 0.2-218). After adjusting for PSA, pathologic stage, and Gleason score, Skp2 overexpression remained significantly associated with a shorter time to biochemical recurrence (adjusted hazard ratio 4.8 (95% C.I. 1.6-14, P=0.004)). The median time to recurrence with high vs. low Skp2 was 4 vs. 54 months. CONCLUSIONS: Skp2 overexpression was seen in a significant minority of surgically-managed men and was independently associated with a higher risk of recurrence, raising the possibility that Skp2 could be useful as a prognostic biomarker and as a potential molecular target for novel systemic agents in prostate cancer.

Regulatory mechanisms to prevent centriole overduplication during the cell cycle are not completely understood. In this issue, FBXW5 is shown to control the degradation of the centriole assembly factor HsSAS-6. Moreover, the study proposes that FBXW5 is a substrate of both PLK4 and APC/C, two established regulators of centriole duplication

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C(Cdh1). USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C(Cdh1) inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C(Cdh1) in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry

Nuclear expression of the cell cycle inhibitor p27(KIP1) is reduced in a variety of human malignancies, including breast cancer. Loss of nuclear p27(KIP1) during tumor progression, documented by immunohistochemistry (IHC), has been studied for its potential prognostic implication. We examined by IHC the association between nuclear p27(KIP1) expression and hormone receptor status in T1N0M0 breast cancer. PATIENTS AND METHODS: The correlation between nuclear p27(KIP1) expression and estrogen (ER) and progesterone (PR) hormone receptor status was analyzed in 122 human T1N0M0 (68 T1a/b, 54 T1c) breast cancer specimens. All patients were staged as N0 by axillary node dissection. RESULTS: A statistically significant reduction in p27(KIP1) expression was observed as tumor size increased from T1a/b (7%) to T1c (22%). The proportion of tumors with low nuclear p27(KIP1) expression was higher in the ER-negative/PR-negative group compared to the ER-positive/PR-positive group, but this difference was only statistically significant in the T1a/b subgroup (p=0.0007). CONCLUSION: Further investigations into causes of p27(KIP1) deregulation and their relationship to hormone receptor expression in T1N0M0 breast ductal carcinomas are warranted. Such studies may help identify prognostic, as well as predictive, markers of therapy resistance

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein betaTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the betaTrCP degron in DEPTOR is executed by CK1alpha after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the betaTrCP-dependent degradation of DEPTOR via betaTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind betaTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1alpha and betaTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways