Summary Background HSD3B1(1245A>C) has been mechanistically linked to castration-resistant prostate cancer by encoding an altered enzyme that augments dihydrotestosterone synthesis. We hypothesized that men inheriting the HSD3B1(1245C) allele would exhibit resistance to androgen deprivation therapy (ADT). Methods We determined HSD3B1 genotype retrospectively in men treated with ADT for post-prostatectomy biochemical failure and correlated genotype with long-term clinical outcomes. Patients who received postoperative adjuvant or salvage radiotherapy were eligible, provided they had residual active disease as reflected by continued increase in their PSA after treatment. We analyzed progression-free survival (PFS; primary endpoint), distant metastasis-free survival (DMFS), and overall survival (OS) according to HSD3B1 genotype. Multivariable analyses were performed to assess the independent predictive value of HSD3B1 genotype on outcomes. Results were externally validated in two additional cohorts, including a second post-prostatectomy biochemical failure cohort as well as a metastatic cohort. There was no age limit for eligibility in the primary or validation cohorts. Findings The study included 443 patients: 118 in the primary cohort, 137 in the post-prostatectomy validation cohort, and 188 in the metastatic validation cohort. In the primary study cohort, median PFS diminished as a function of the number of variant alleles inherited: 6.6 years in homozygous wild-type men (95% CI, 3.8 to not reached); 4.1 years in heterozygotes (95% CI, 3.0 to 5.5); and 2.5 years in homozygous variant men (95% CI, 0.7 to not reached); P=0.011. Median DMFS likewise decreased according to the number of variant alleles inherited: 9.1 years (95% CI, 7.4 to not reached); 6.8 years (95% CI, 4.3 to 7.4); and 3.6 years (95% CI, 1.0 to 7.3), respectively; P=0.014. Finally, OS diminished with the number of variant alleles inherited: 5-year and 10-year OS 82% (95% CI, 69 to 94) and 55% (95% CI, 35 to 75) in homozygous wild-type men; 74% (95% CI, 62 to 85) and 35% (95% CI, 21 to 49) in heterozygotes; and 58% (95% CI, 30 to 86) and 0% in homozygous variant men; P=0.0064. On multivariable analysis, the hazard ratio (HR) for progression was 1.6 for men with at least one variant allele (95% CI, 1.0 to 2.7; P=0.074), which compared favorably with Gleason score (HR 1.3 for Gleason score 8–10 vs. 6–7; 95% CI 0.8 to 2.0; P=0.31), though neither factor reached statistical significance with the small sample size. The impact of homozygous variant genotype on metastasis (HR 2.8; 95% CI, 1.1 to 6.7; P=0.025) and death (HR 3.5; 95% CI 1.3 to 9.5; P=0.013) was maintained on multivariable analysis. Findings in the external cohorts independently validated the impact of HSD3B1(1245C) on outcomes. Interpretation Inheritance of the HSD3B1(1245C) allele that enhances dihydrotestosterone synthesis is associated with prostate cancer resistance to ADT. Our findings nominate HSD3B1 as a powerful genetic biomarker capable of distinguishing men who are a priori ...

Components of the TNFR1 complex are subject to dynamic ubiquitination that impacts on their effects as signalling factors. We have found that the ubiquitin-specific protease USP2a has a pivotal role in the decision for cell death or survival by the TNFR1 complex. This enzyme is a novel component of the TNFR1 complex that is recruited upon ligand binding and controls the signalling activity of the TNFR1-interacting protein RIP1 by removing its K63-linked ubiquitin chains. USP2a similarly de-ubiquitinates TRAF2, a ubiquitin-ligase recruited to the TNFR1 complex. During the TNF response the activity of USP2a on RIP1 and TRAF2 is required for the efficient reappearance of IjBa, which is essential to inactivate the anti-apoptotic transcription factor NF-jB. The effects of USP2a culminate in the conversion of the anti-apoptotic TNFR1 complex I into the pro-apoptotic TNFR1 complex II. Consequently, downregulation of USP2a promotes NF-jB activation and protects cells against TNF-induced cell death. Tumour necrosis factor (TNF) is a pleiotropic cytokine that can, through the TNF receptor 1 (TNFR1), elicit various cellular responses ranging from inflammation, cell proliferation, cell survival to apoptosis. The interaction of this cytokine with TNFR1 results in the recruitment of the adaptor TRADD to its intracellular death domain. RIP kinase 1 (RIP1), the ubiquitin ligase TNF receptor-associated factor 2 (TRAF2), and the inhibitors of apoptosis cIAP1 and cIAP2 then associate in the so-called complex I. 1 Following its recruitment to the complex I, RIP1 is ubiquitinated mainly by K63 ubiquitin chains. 2-5 The E3 ubiquitin ligase TRAF2 was the first to be described as being responsible for K63 ubiquitination of RIP1 5-7 together with the lipid shingosine-1-phosphate. 8 Other publications have shown that cIAP1/2 can attach K63 ubiquitin chains to RIP1 [9][10][11][12] or that TRAF2 and cIAPs cooperate for the ubiquitination of RIP1 6,13,14 . These ubiquitin chains bind and associate the TAB/TAK (TAK1/TAB2/TAB3) and NEMO/IKK (IKKa, b, g) kinases complex. 3,4,12,15 By proximity, TAK1 activates IKKb by phosphorylation, which in turn phosphorylates IkBa to promote its K48-linked polyubiquitination and degradation by the proteasome. The antiapoptotic transcription factor NF-kB is then free to translocate to the nucleus and activate gene transcription leading to survival of the cells. 7,[15][16][17][18][19][20] Upon internalisation of the complex and in the absence of K63 ubiquitination, RIP1 is able to form the so-called complex II with FADD and pro-caspase-8 to initiate apoptosis. 1,16 Hence, ubiquitin conjugation has a crucial role in allowing cells to decide between cell death and survival in the TNF signalling cascade. Although a number of ubiquitin ligases, that presumably modify components of the TNFR1 complex, have been identified, so far only two de-ubiquitinating proteases have functionally been found in the TNF signalling pathway that impact on cell death, A20 and CYLD (cylindromatosis tumour suppressor). 21-24 A20 is a ...

Dynamic ubiquitination impacts on the degradation of proteins by the proteasome as well as on their effects as signalling factors. Of the many cellular responses that are regulated by changes in ubiquitination, apoptosis has garnered special attention. We have found that USP2a and USP2c, two isoforms of the ubiquitin-specific protease USP2, cause cell death upon ectopic expression. We show that both USP2 isoforms can control the ubiquitination status of many proteins but from a panel of potential targets only the protein level of RIP1 was increased by these enzymes. This effect is responsible for the activity of USP2a and USP2c to cause cell death. Both enzymes likewise de-ubiquitinate TRAF2, a ubiquitin-ligase in the TNFR1 complex. Whilst this and the similar sub-cellular localisations of both enzyme isoforms indicate a substantial overlap of activities, inactivation by RNAi revealed that only the knock-down of USP2c resulted in apoptosis, whilst targeting USP2a did not have any consequence on the cells' survival. Consequently, we focussed our studies on USP2a and found that TRAF2 inhibits USP2a's effect on K48- but not on K63-linked ubiquitin chains. Hence, the ratio between USP2a and TRAF2 protein levels determines the cells' sensitivity to cell death.

This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

ORCTL3, an organic cation/anion transporter expressed in various tissue types, was isolated in a genome-wide cDNA screen as a gene with a tumor-specific apoptosis activity. When overexpressed it elicits an apoptosis response in many transformed cells, while normal cells remain unaffected. It can be activated for apoptosis induction by individual tumorigenic mutations in renal cells. This effect is independent of the tumor cells' proliferation status and mediated by an incomplete ER stress response, characterized by the accumulation of the endoplasmic reticulum-stress marker ATF4, but not BiP. Recent studies show that for its apoptosis induction activity ORCTL3 targets the enzyme stearoyl-CoA desaturase-1 (SCD-1) that is involved in the fatty acid metabolism. This is evidenced by the inhibition of apoptosis induced through ORCTL3 when the SCD-1 product oleic acid is exogenously supplemented or when SCD-1 is co-transfected in the transformed cells. ORCTL3's activity to specifically target tumor cells is caused by the transmembrane domains 3 and 4 of the mouse, but not the human, gene. In an in vivo model ORCTL3 shows a significant shrinkage in the size of xenograft tumors when injected with an adenoviral carrier carrying the mouse ORCTL3 gene. An ex vivo study using human renal cancer cells confirmed the promising tumor-specific apoptosis effect of ORCTL3. Since ORCTL3 targets fatty acid metabolism in transformed cells and induces an ER stress specifically in these cells, it reveals a novel therapeutic interference option for tumor cells.

ORCTL3 is a member of a group of genes, the so-called anticancer genes, that cause tumour-specific cell death. We show that this activity is triggered in isogenic renal cells upon their transformation independently of the cells’ proliferation status. For its cell death effect ORCTL3 targets the enzyme stearoyl-CoA desaturase-1 (SCD1) in fatty acid metabolism. This is caused by transmembrane domains 3 and 4, which are more efficacious in vitro than a low molecular weight drug against SCD1, and critically depend on their expression level. SCD1 is found upregulated upon renal cell transformation indicating that its activity, while not impacting proliferation, represents a critical bottleneck for tumourigenesis. An adenovirus expressing ORCTL3 leads to growth inhibition of renal tumours in vivo and to substantial destruction of patients’ kidney tumour cells ex vivo. Our results indicate fatty acid metabolism as a target for tumour-specific apoptosis in renal tumours and suggest ORCTL3 as a means to accomplish this.

BackgroundWith the ever-increasing information emerging from the various sequencing and gene annotation projects, there is an urgent need to elucidate the cellular functions of the newly discovered genes. The genetically regulated cell suicide of apoptosis is especially suitable for such endeavours as it is governed by a vast number of factors.Methodology/Principal FindingsWe have set up a high-throughput screen in 96-well microtiter plates for genes that induce apoptosis upon their individual transfection into human cells. Upon screening approximately 100,000 cDNA clones we determined 74 genes that initiate this cellular suicide programme. A thorough bioinformatics analysis of these genes revealed that 91% are novel apoptosis regulators. Careful sequence analysis and functional annotation showed that the apoptosis factors exhibit a distinct functional distribution that distinguishes the cell death process from other signalling pathways. While only a minority of classic signal transducers were determined, a substantial number of the genes fall into the transporter- and enzyme-category. The apoptosis factors are distributed throughout all cellular organelles and many signalling circuits, but one distinct signalling pathway connects at least some of the isolated genes. Comparisons with microarray data suggest that several genes are dysregulated in specific types of cancers and degenerative diseases.Conclusions/SignificanceMany unknown genes for cell death were revealed through our screen, supporting the enormous complexity of cell death regulation. Our results will serve as a repository for other researchers working with genomics data related to apoptosis or for those seeking to reveal novel signalling pathways for cell suicide.

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