A splicing-dependent transcriptional checkpoint associated with prespliceosome formation

There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit’s heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription

Aromatase inhibitor-induced modulation of breast density: clinical and genetic effects

Background: Change in breast density may predict outcome of women receiving adjuvant hormone therapy for breast cancer. We performed a prospective clinical trial to evaluate the impact of inherited variants in genes involved in oestrogen metabolism and signalling on change in mammographic percent density (MPD) with aromatase inhibitor (AI) therapy. Methods: Postmenopausal women with breast cancer who were initiating adjuvant AI therapy were enrolled onto a multicentre, randomised clinical trial of exemestane vs letrozole, designed to identify associations between AI-induced change in MPD and single-nucleotide polymorphisms in candidate genes. Subjects underwent unilateral craniocaudal mammography before and following 24 months of treatment. Results: Of the 503 enrolled subjects, 259 had both paired mammograms at baseline and following 24 months of treatment and evaluable DNA. We observed a statistically significant decrease in mean MPD from 17.1 to 15.1% (P<0.001), more pronounced in women with baseline MPD ⩾20%. No AI-specific difference in change in MPD was identified. No significant associations between change in MPD and inherited genetic variants were observed. Conclusion: Subjects with higher baseline MPD had a greater average decrease in MPD with AI therapy. There does not appear to be a substantial effect of inherited variants in biologically selected candidate genes

Humoral response to a 13-valent pneumococcal conjugate vaccine in kidney transplant recipients

Background: Vaccination against S. pneumoniae is recommended by national guidelines. Moderate immunogenicity of the 13-valent pneumococcal conjugate vaccine (PCV13) has been reported in adult kidney transplant recipients (KTR). This study further defines the immunogenicity of PCV13 in this cohort. Methods: 49 KTR were immunized with PCV13. A validated opsonophagocytic killing assay (OPA), a global anti-pneumococcal capsular polysaccharide (anti-PCP) IgG, IgG2, IgM and IgA ELISA, and - for selected patients - a serotype specific anti-PCP WHO reference ELISA were performed pre-vaccination and at month 1 and 12 post-vaccination. Results: Geometric mean OPA titers increased significantly for 13/13 serotypes at month 1 and for 10/13 serotypes at month 12 post-vaccination. Vaccine response defined as an OPA titer ≥1:8 was reached in 9/13 serotypes (median). 53% reached the vaccine response criteria at month 1 and 45% at month 12. At month 1 after vaccination, the median OPA titer in an age-group matched healthy reference population was 5- to 10-fold higher than in KTR. OPA titers correlated strongly with results to the global and serotype specific anti-PCP IgG ELISA. Lower OPA titers significantly (p < 0.05) correlated with albuminuria, an interval between vaccination and transplantation <12 months, age and treatment with mycophenolate mofetil. Global IgG, IgG2, IgM and IgA, as well as serotype specific anti-PCP antibody concentrations (12/13 serotypes) increased significantly at month 1 and 12 post-vaccination. Conclusions: Kidney transplant recipients show a significant humoral response after vaccination with PCV13. Functional antibody response exists, but is not as vigorous as in healthy adults

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies. © 2012 Coronnello et al

Correlation of Fc Receptor Polymorphisms with Pneumococcal Antibodies in Vaccinated Kidney Transplant Recipients

Several polymorphisms within Fc receptors (FCR) have been described, some of which correlate with allograft function. In the current study, we determined three Fcγ receptor and five Fcα receptor dimorphisms in 47 kidney transplant recipients who had been vaccinated against Streptococcus pneumoniae. We analyzed if FCR genotypes correlated with pneumococcal antibodies and their serotype-specific opsonophagocytic function, tested prior to and at months 1 and 12 post-vaccination. In parallel, we assessed antibodies against HLA and MICA and determined kidney function. We observed that IgG2 antibodies against pneumococci at months 1 and 12 after vaccination and IgA antibodies at month 1 differed significantly between the carriers of the three genotypes of FCGR3A rs396991 (V158F, p = 0.02; 0.04 and 0.009, respectively). Moreover, the genotype of FCGR3A correlated with serotype-specific opsonophagocytic function, reaching statistical significance (p < 0.05) at month 1 for 9/13 serotypes and at month 12 for 6/13 serotypes. Heterozygotes for FCGR3A had the lowest antibody response after pneumococcal vaccination. On the contrary, heterozygotes tended to have more antibodies against HLA class I and impaired kidney function. Taken together, our current data indicate that heterozygosity for FCGR3A may be unfavorable in kidney transplant recipients

High rates of loss of heterozygosity on chromosome 19p13 in human breast cancer

We have recently discovered that the nuclear matrix protein SAFB is an oestrogen receptor corepressor. Since it has become clear that many steroid receptor cofactors play important roles in breast tumorigenesis, we investigated whether SAFB could also be involved in breast cancer. To address this question, the gene locus was examined for structural alterations in breast cancer tissue. Laser capture microdissection was used for isolating DNA from paired primary breast tumour and normal tissue specimens, and the loss of heterozygosity (LOH) at chromosome 19p13.2–3 was determined by use of microsatellite markers. LOH was detected at the marker D19S216, which colocalizes with the SAFB locus, in specimens from 29 (78.4%) of 37 informative patients. The peak LOH rate occurred at D19S216 near the SAFB locus, with LOH frequencies ranging from 21.6% to 47.2% at other markers. The finding of a very high LOH rate at the marker D19S216 strongly indicates the presence of a breast tumour-suppressor gene locus. While preliminary findings of mutations in SAFB suggest that this indeed may be a promising candidate, other potential candidate genes are located at this locus. © 2001 Cancer Research Campaign http://www.bjcancer.co

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis

Background: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood–brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets.Methods: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis.Results: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis.Conclusion: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease

CYP2D6 Genotype is Not Associated with Survival in Breast Cancer Patients Treated with Tamoxifen: Results from a Population-based Study

Purpose: A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. Methods: Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). Results: CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00-2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). Conclusions: This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice

Association between manganese superoxide dismutase promoter gene polymorphism and breast cancer survival

BACKGROUND: Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk and survival in breast cancer. This base pair transition (-9 T > C) leads to a valine to alanine amino acid change in the mitochondrial targeting sequence. A polymorphism has also been identified in the proximal region of the promoter (-102 C>T) that alters the recognition sequence of the AP-2 transcription factor, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the -102 C>T polymorphism with overall and relapse-free breast cancer survival in a hospital-based case-only study. MATERIALS AND METHODS: The relationship between the MnSOD -102 C>T polymorphism and survival was examined in a cohort of 291 women who received chemotherapy and/or radiotherapy for incident breast cancer. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. Patient survival was evaluated according to the MnSOD genotype using Kaplan–Meier survival functions. Hazard ratios were calculated from adjusted Cox proportional hazards modeling. All statistical tests were two-sided. RESULTS: In an evaluation of all women, there was a borderline significant reduction in recurrence-free survival with either one or both variant alleles (CT + TT) when compared with patients with wild-type alleles (CC) (odds ratio, 0.65; 95% confidence interval, 0.42–1.01). When the analysis was restricted to patients receiving radiation therapy, there was a significant reduction in relapse-free survival in women who were heterozygous for the MnSOD -102 genotype (relative risk, 0.40; 95% confidence interval, 0.18–0.86). Similarly, when the homozygous and heterozygous variant genotypes were combined, there remained a significant reduction in relapse-free survival in this group (hazard ratio, 0.42; 95% confidence interval, 0.20–0.87). CONCLUSION: The MnSOD -102 variant allele appears to be associated with an improved recurrence-free survival in all patients, and more dramatically in subjects who received adjuvant radiation therapy