Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness.

The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.

ßIII-tubulin overexpression is an independent predictor of prostate cancer progression tightly linked to ERG fusion status and PTEN deletion.

Evidence suggests that class III ß-tubulin (ßIII-tubulin) may represent a prognostic and predictive molecular marker in prostate cancer. ßIII-Tubulin expression was determined by IHC in 8179 prostate cancer specimens in a TMA format. Results were compared with tumor phenotype, biochemical recurrence, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) status, and deletions on PTEN, 3p13, 5q21, and 6q15. ßIII-Tubulin expression was detectable in 25.6% of 8179 interpretable cancers. High ßIII-tubulin expression was strongly associated with both TMPRSS2:ERG rearrangement and ERG expression (P < 0.0001 each). High ßIII-tubulin expression was tightly linked to high Gleason grade, advanced pT stage, and early prostate-specific antigen (PSA) recurrence in all cancers (P < 0.0001 each), but also in the subgroups of ERG-negative and ERG-positive cancers. When all tumors were analyzed, the prognostic role of ßIII-tubulin expression was independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features, such as biopsy specimen Gleason grade, preoperative PSA, cT stage, and ßIII-tubulin expression (P < 0.0001 each). ßIII-Tubulin expression was associated with PTEN (P < 0.0001) when all tumors were analyzed, but also in the subgroups of ERG-negative and ERG-positive cancers. ßIII-Tubulin expression is an independent prognostic parameter. The significant associations with key genomic alterations of prostate cancer, such as TMPRSS2:ERG fusions and PTEN deletions, suggest interactions with several pivotal pathways involved in prostate cancer.

High nuclear karyopherin α 2 expression is a strong and independent predictor of biochemical recurrence in prostate cancer patients treated by radical prostatectomy.

Increased levels of karyopherin α2 (KPNA2) expression have been described to be linked to poor prognosis in a variety of malignancies. This study was undertaken to evaluate the clinical impact of KPNA2 expression and its association with key genomic alterations in prostate cancers. A tissue microarray containing samples from 11 152 prostate cancers was analyzed for KPNA2 expression by immunohistochemistry. Results were compared with oncological follow-up data and genomic alterations such as TMPRSS2-ERG fusions and deletions of PTEN, 5q21, 6q15 or 3p13. KPNA2 expression was absent or weak in benign prostatic glands and was found to be in weak, moderate or strong intensities in 68.4% of 7964 interpretable prostate cancers. KPNA2 positivity was significantly linked to the presence of ERG rearrangement (P<0.0001). In ERG-negative and -positive prostate cancers, KPNA2 immunostaining was significantly associated with advanced pathological tumor stage (pT3b/pT4), high Gleason grade and early biochemical recurrence (P<0.0001 each). Multivariate analysis including all established prognostic criteria available after surgery revealed that the prognostic role of KPNA2 (P=0.001) was independent of high Gleason grade, advanced pathological tumor stage, high preoperative prostate-specific antigen level and positive surgical margin status (P<0.0001 each). The comparison of KPNA2 expression with deletions of PTEN, 5q21, 6q15 and 3p13 in ERG-positive and -negative cancers revealed a strong link to PTEN deletions in both subgroups (P<0.0001). In conclusion, the strong independent prognostic impact of KPNA2 expression raises the possibility that measurement of KPNA2 expression alone or in combination with other molecular parameters might possibly result in clinically useful information. The data also emphasize a critical role of the functionality of the nuclear import machinery for prostate cancer biology.

Recurrent deletion of 3p13 targets multiple tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion-positive prostate cancers.

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1-specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG(+) cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG(+) tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG(+) cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over-expression of FOXP1, RYBP and SHQ1 resulted in decreased colony-formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG(+) prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors.

Loss of pSer2448-mTOR expression is linked to adverse prognosis and tumor progression in ERG-fusion-positive cancers.

Prevalence and clinical significance of mammalian target of rapamycin (mTOR) phosphorylation at the serine 2448 is disputed in prostate cancer. A tissue microarray containing 3,261 prostate cancers and 49 normal prostate samples with clinical follow-up data was analyzed for p(Ser2448)-mTOR expression by immunohistochemistry. Moderate to strong p(Ser2448)-mTOR staining was found in all (n = 49) normal prostate tissues, but was lost in 24% or weak in 29% cancers. Moderate and strong staining was found in 36 and 11% of tumors. Loss of p(Ser2448)-mTOR staining was significantly linked to advanced stage (p = 0.0027), high-grade (p = 0.0045), nodal positive cancers (p = 0.0483), early tumor recurrence (p < 0.0001, independently from stage and grade, p = 0.0016), lack of Ets-related gene (ERG) fusion (p < 0.0001), reduced androgen receptor expression (p < 0.0001 each) and increased cell proliferation (p = 0.0092) in all cancers and in the subset of ERG-fusion-positive cancers. Loss of p(Ser2448)-mTOR expression was linked to tumor metastasis (p = 0.0275) in ERG-fusion-positive cancers only. Molecular subset analysis using pre-existing phosphatase and tensin homolog (PTEN) deletion data revealed that loss of p(Ser2448) -mTOR expression is of prognostic relevance and defines a subpopulation of PTEN-deleted and ERG-fusion-positive cancers with a particular poor outcome. The results of our study strongly suggest that loss of p(Ser2448)-mTOR expression is a marker for activated AKT/mTOR signaling. Tumors with concomitant PTEN deletion and activated mTOR signaling indicated by loss of p(Ser2448)-mTOR expression characterize a small (4%) but clinically significant subset of prostate cancers that might optimally benefit from anti-mTOR therapies.

SPINK1 expression is tightly linked to 6q15- and 5q21-deleted ERG-fusion negative prostate cancers but unrelated to PSA recurrence.

BACKGROUND: The serine peptidase inhibitor, Kazal type 1 (SPINK1) has been suggested to define an aggressive molecular subtype of ERG-fusion negative prostate cancer. It was the aim of this study to further study the clinical relevance of SPINK1 expression and its relationship with other key genomic alterations of prostate cancer. METHODS: A tissue microarray containing more than 10,000 prostate cancers with clinical follow-up was used for immunohistochemical SPINK1 analysis. Data on ERG status as well as PTEN, 6q, 5q, and 3p deletions were available for comparison. RESULTS: SPINK1 expression was absent in benign prostate glands and detectable in 5.9% of 9,503 interpretable prostate cancers. Presence of SPINK1 expression was markedly more frequent in ERG negative (10.4%) than in ERG positive cancers (0.3%; P < 0.0001). However, SPINK1 expression was unrelated to tumor phenotype and biochemical recurrence in all cancers and in the subgroup of ERG negative cancers. Further subgroup analyses revealed, however, that--within ERG negative cancers--SPINK1 expression was significantly linked to deletions at 6q15 (P < 0.0001) and 5q21 (P = 0.0042). CONCLUSIONS: Our results exclude SPINK1 as a relevant prognostic prostate cancer biomarker. However, the data demonstrate that SPINK1 overexpression is tightly linked to the small subsets of 6q15- and 5q21-deleted ERG negative prostate cancers. These findings support the concept of molecularly defined subtypes of prostate cancers.

PML isoforms I and II participate in PML-dependent restriction of HSV-1 replication.

Intrinsic antiviral resistance mediated by constitutively expressed cellular proteins is one arm of defence against virus infection. Promyelocytic leukaemia nuclear bodies (PML-NBs, also known as ND10) contribute to host restriction of herpes simplex virus type 1 (HSV-1) replication via mechanisms that are counteracted by viral regulatory protein ICP0. ND10 assembly is dependent on PML, which comprises several different isoforms, and depletion of all PML isoforms decreases cellular resistance to ICP0-null mutant HSV-1. We report that individual expression of PML isoforms I and II partially reverses the increase in ICP0-null mutant HSV-1 plaque formation that occurs in PML-depleted cells. This activity of PML isoform I is dependent on SUMO modification, its SUMO interaction motif (SIM), and each element of its TRIM domain. Detailed analysis revealed that the punctate foci formed by individual PML isoforms differ subtly from normal ND10 in terms of composition and/or Sp100 modification. Surprisingly, deletion of the SIM motif from PML isoform I resulted in increased colocalisation with other major ND10 components in cells lacking endogenous PML. Our observations suggest that complete functionality of PML is dependent on isoform-specific C-terminal sequences acting in concert.

Cysteine-rich secretory protein 3 overexpression is linked to a subset of PTEN-deleted ERG fusion-positive prostate cancers with early biochemical recurrence.

The aim of this study was to determine whether cysteine-rich secretory protein 3 (CRISP3) expression is linked to clinically or molecularly relevant subgroups of prostate cancer. A tissue microarray representing samples from >10,000 prostate cancers from radical prostatectomy specimens with clinical follow-up data were analyzed for CRISP3 expression by immunohistochemistry. CRISP3 expression was also compared with key genomic alterations of prostate cancer. CRISP3 staining was found as weak in 15%, moderate in 8.5%, and strong in 7.2% of prostate cancers, whereas no expression was detected in normal prostate. Strong CRISP3 expression was linked to advanced tumor stage, high Gleason score, and positive surgical margin status (P<0.0001 each). There was a marked accumulation of high CRISP3 expression in PTEN-deleted ERG-positive tumors (P<0.0001). A total of, 21.7% of ERG-positive and PTEN-deleted cancers had strong CRISP3 expression, but only 10.4% of ERG-positive cancers without PTEN deletion (P<0.0001). The rate of high CRISP3 expression was 2.5% in ERG-negative cancers (P=0.0001; vs ERG-positive cancers). Accordingly, CRISP3 overexpression was associated with early prostate-specific antigen recurrence in all tumors (P=0.0013) as well as in ERG-negative (P=0.004) and ERG-positive cancers (P=0.0318). CRISP3 expression did not retain prognostic significance in models also involving PTEN deletions. Strong CRISP3 expression is associated with unfavorable tumor phenotype and early recurrence in prostate cancers. The tight link of strong CRISP3 expression to the ERG fusion-positive prostate cancers with PTEN deletions provides further evidence for the existence of molecularly distinct subgroups of prostate cancers.

Genomic deletion of MAP3K7 at 6q12-22 is associated with early PSA recurrence in prostate cancer and absence of TMPRSS2:ERG fusions.

6q12-22 is the second most commonly deleted genomic region in prostate cancer. Mapping studies have described a minimally deleted area at 6q15, containing MAP3K7/TAK1, which was recently shown to have tumor suppressive properties. To determine prevalence and clinical significance of MAP3K7 alterations in prostate cancer, a tissue microarray containing 4699 prostate cancer samples was analyzed by fluorescence in situ hybridization. Heterozygous MAP3K7 deletions were found in 18.48% of 2289 interpretable prostate cancers. MAP3K7 deletions were significantly associated with advanced tumor stage (P<0.0001), high Gleason grade (P<0.0001), lymph node metastasis (P<0.0108) and early biochemical recurrence (P<0.0001). MAP3K7 alterations were typically limited to the loss of one allele as homozygous deletions were virtually absent and sequencing analyses revealed no evidence for MAP3K7 mutations in 15 deleted and in 14 non-deleted cancers. There was a striking inverse association of MAP3K7 deletions and TMPRSS2:ERG fusion status with 26.7% 6q deletions in 1125 ERG-negative and 11.1% 6q deletions in 1198 ERG-positive cancers (P<0.0001). However, the strong prognostic role of 6q deletions was retained in both ERG-positive and ERG-negative cancers (P<0.0001 each). In summary, our study identifies MAP3K7 deletion as a prominent feature in ERG-negative prostate cancer with strong association to tumor aggressiveness. MAP3K7 alterations are typically limited to one allele of the gene. Together with the demonstrated tumor suppressive function in cell line experiments and lacking evidence for inactivation through hypermethylation, these results indicate MAP3K7 as a gene for which haploinsufficency is substantially tumorigenic.

Genomic deletion of PTEN is associated with tumor progression and early PSA recurrence in ERG fusion-positive and fusion-negative prostate cancer.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.

Reduced CD147 expression is linked to ERG fusion-positive prostate cancers but lacks substantial impact on PSA recurrence in patients treated by radical prostatectomy.

The extracellular matrix metalloproteinase inducer CD147 has been suggested as a prognostic marker in prostate cancer. CD147 expression was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence, ERG status and deletions on PTEN, 3p13, 6q15 and 5q21. CD147 expression was strong in benign prostatic glands and often reduced in prostate cancers. CD147 immunostaining was found in 71.7% of 7628 interpretable cases. CD147 staining was considered strong in 34.6%, moderate in 24.3% and weak in 12.8% of cancers while 28.3% did not show any CD147 reactivity. Reduced CD147 staining was strongly associated with both TMPRSS2-ERG-rearrangement and ERG expression (p<0.0001 each). Within the subgroups of ERG positive and negative cancers, deletions of PTEN, 3p13, 6q15 and 5q21 were unrelated to the CD147 expression status. Decreased CD147 expression was significantly linked to high preoperative PSA values, high Gleason grade, advanced tumor stage (p<0.0001 each), and positive lymph node involvement (p=0.0026) in all cancers. There was a marginal, but statistically significant, association of reduced CD147 expression with early biochemical recurrence (p=0.0296). The significant reduction of CD147 expression in ERG positive prostate cancer provides further evidence for marked biological differences between "fusion type" and "non-fusion type" prostate cancer. Despite a weak association with PSA recurrence, CD147 cannot be considered a relevant prognostic biomarker.

Intratumoral heterogeneity of KRAS mutation is rare in non-small-cell lung cancer.

BACKGROUND: Several lines of evidence indicate that mutational activation of KRAS is an early event in the carcinogenesis of non-small cell lung cancer (NSCLC). Nonetheless, previous studies report high frequencies of divergent KRAS mutational status between primary NSCLC and corresponding metastases. This suggests heterogeneity of the primary tumor in respect to its KRAS status. We therefore aimed to examine the frequency and the extent of such intratumoral heterogeneity. METHODS: 40 NSCLC were examined for intratumoral heterogeneity of KRAS mutation (20 adenocarcinomas, 10 squamous cell carcinomas and 10 large cell carcinomas). Three to eight different tumor areas were analyzed for KRAS mutation and up to four corresponding lymph node metastases were included for analysis in nineteen cases. A combination of different methods for screening of heterogeneity and its validation were used including direct sequencing, laser-capture microdissection for tumor cell enrichment and the very sensitive ARMS/S method. RESULTS: Mutations of KRAS were found in 13/30 adenocarcinomas and large cell carcinomas. No mutations were detected in 10 squamous cell carcinomas. Four cases showed heterogeneous KRAS results by direct sequencing. More sensitive methods for KRAS mutation analysis revealed false negative results due to admixture of non-neoplastic cells in all of these samples. Intratumoral heterogeneity of KRAS mutational status was therefore confirmed in none of the analyzed cases. In addition, identical KRAS mutations were present in the primary tumor and the corresponding lymph node metastases in 19 cases examined. CONCLUSIONS: Intratumoral heterogeneity of KRAS mutational status is rare in NSCLC but highly sensitive tools are required to reliably identify these mutations. This finding is in line with the hypothesis that oncogenic activation of KRAS is an early event and a bona fide "driver mutation" in NSCLC. Furthermore, future therapies targeting KRAS will not be limited by intratumoral heterogeneity.

The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation.

BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.

CHD1 loss negatively influences metastasis-free survival in R0-resected prostate cancer patients and promotes spontaneous metastasis in vivo.

The outcome of prostate cancer (PCa) patients is highly variable and depends on whether or not distant metastases occur. Multiple chromosomal deletions have been linked to early tumor marker PSA recurrence (biochemical relapse, BCR) after radical prostatectomy (RP), but their potential role for distant metastasis formation is largely unknown. Here, we specifically analyzed whether deletion of the tumor suppressor CHD1 (5q21) influences the post-surgical risk of distant metastasis and whether CHD1 loss directly contributes to metastasis formation in vivo. By considering >6800 patients we found that the CHD1 deletion negatively influences metastasis-free survival in R0 patients (HR: 2.32; 95% CI: 1.61, 3.33; p < 0.001) independent of preoperative PSA, pT stage, pN status, Gleason Score, and BCR. Moreover, CHD1 deletion predicts shortened BCR-free survival in pT2 patients and cancer-specific survival in all patients. In vivo, CHD1 loss increases spontaneous pulmonary metastasis formation in two distinct PCa models coupled with a higher number of multicellular colonies as compared to single-cell metastases. Transcriptome analyses revealed down-regulation of the PCa-specific metastasis suppressor and TGFß signaling regulator PMEPA1 after CHD1 depletion in both tested PCa models. CHD1 loss increases the risk of postoperative metastasis in R0-resected PCa patients and promotes spontaneous metastasis formation in vivo.

Proteasome-dependent degradation of Daxx by the viral E1B-55K protein in human adenovirus-infected cells.

The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin alpha3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level.

TMPRSS2-ERG -specific transcriptional modulation is associated with prostate cancer biomarkers and TGF-ß signaling.

BACKGROUND: TMPRSS2-ERG gene fusions occur in about 50% of all prostate cancer cases and represent promising markers for molecular subtyping. Although TMPRSS2-ERG fusion seems to be a critical event in prostate cancer, the precise functional role in cancer development and progression is still unclear. METHODS: We studied large-scale gene expression profiles in 47 prostate tumor tissue samples and in 48 normal prostate tissue samples taken from the non-suspect area of clinical low-risk tumors using Affymetrix GeneChip Exon 1.0 ST microarrays. RESULTS: Comparison of gene expression levels among TMPRSS2-ERG fusion-positive and negative tumors as well as benign samples demonstrated a distinct transcriptional program induced by the gene fusion event. Well-known biomarkers for prostate cancer detection like CRISP3 were found to be associated with the gene fusion status. WNT and TGF-ß/BMP signaling pathways were significantly associated with genes upregulated in TMPRSS2-ERG fusion-positive tumors. CONCLUSIONS: The TMPRSS2-ERG gene fusion results in the modulation of transcriptional patterns and cellular pathways with potential consequences for prostate cancer progression. Well-known biomarkers for prostate cancer detection were found to be associated with the gene fusion. Our results suggest that the fusion status should be considered in retrospective and future studies to assess biomarkers for prostate cancer detection, progression and targeted therapy.

Selection and counterselection of the rtI233V adefovir resistance mutation during antiviral therapy.

Recently, we reported on three patients with chronic hepatitis B virus (HBV) infection for whom adefovir (ADF) therapy virologically failed, most likely due to a preexisting rtI233V HBV polymerase mutation. Here, we describe two further patients with chronic HBV infection who were found to develop the rtI233V mutation after initiation of ADF therapy. These patients represent the first cases known so far in which the rtI233V ADF resistance mutation evolved under persistent HBV replication during HBV therapy with ADF. Interestingly, one of the previously described patients, who was initially successfully switched from ADF to tenofovir (TDF) and became virologically suppressed subsequently, experienced a moderate but remarkable rebound of HBV viremia after switching from TDF to entecavir, due to the emergence of renal toxicity. Thus, we provide evidence for the selection and counterselection of the rtI233V ADF resistance mutation during antiviral therapy.

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100.

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.

Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry.

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.