Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Familial clustering of a rare syndrome

Ectrodactyly, ectodermal dysplasia and cleft palate syndrome is a rare autosomal dominant multiple congenital anomaly syndrome with variable expressivity and reduced penetration. The cardinal features are cleft palate/lip, lobster hand deformity, sparse hypopigmented hair, dry scaly skin, and lacrimal and urogenital anomalies. A neonate presented to us with typical features, his mother and other two siblings were also affected

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated β-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect β-catenin, demonstrating USP14 but not UCHL5 has a specific effect on β-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides β-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function

Ubiquitinated proteins accumulated in the cells expressing USP14 C114A.

Ubiquitinated proteins accumulated in the cells expressing USP14 C114A.</p

UCHL5 C88A causes an accumulation of ubiquitinated proteins but does not affect β-catenin degradation.

(A) Immunoblot showing the effects UCHL5 WT and C88A in HEK293T cells. Note the lack of band smearing of β-catenin but the presence of band laddering of UCHL5 and the increase of conjugated ubiquitin in the C88A lane. (B) Quantification from (A). n = 3–6, * P P < 0.01, error bars represent SEM. (C) Immunoblot showing USP14 C114A does not induce UCHL5 band laddering in cells. (D) β-catenin IP followed by K48 IB showing the lack of ubiquitinated β-catenin accumulation in the cells expressing UCHL5 C88A. (E) K48 IP followed by K48 and β-catenin IB showing although the accumulated ubiquitinated proteins were immunoprecipitated but ubiquitinated β-catenin was not accumulated in the cells expressing UCHL5 C88A. (F) K48-linked ubiquitinated proteins were immunoprecipitated from the cells expressing the indicated proteins. A control IP was done with IgG from the cells expressing UCHL5 C88A. The IP samples were subjected to Coomassie blue staining. The gel pieces above the antibody heavy chain were cut for mass spectrometry analysis.</p

Expression of USP14 C114A has no effect on TDP-43, tau or α-synuclein but leads to an accumulation of ubiquitinated proteins.

(A) Immunoblot showing the effects of a control plasmid, USP14 WT or C114A co-expression with myc-TDP-43 WT, myc-TDP-43 M337V, tau or α-synuclein in HEK293T cells. * Non-specific band. (B) USP14 WT or C114A has no effect on the protein level of TDP-43, tau and α-synuclein. Quantification of protein levels from A. n = 3–6, error bars represent SEM. USP14 C114A causes an accumulation of polyubiquitinated proteins. Quantification of conjugated ubiquitin (conj. ubiquitin) from A. n = 4 for endogenous and WT TDP-43, n = 3 for M337V TDP-43, n = 3 for tau, n = 6 for α-syn, error bars represent SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.</p

Confirmation that ubiquitinated PSMC1 and PSMD4 accumulate in USP14 C114A expressing cells.

(A) Immunoblot showing effects of USP14 WT and C114A in HEK293T cells. Note the increased ubiquitin conjugates, the band smearing of β-catenin, and the band laddering of PSMC1 and PSMD4 in C114A samples. (B) Immunoblot showing effects of UCHL5 WT and C88A in HEK293T cells. Note the increased ubiquitin conjugates and the band laddering of UCHL5 in C88A samples.</p