Systematic Analysis of FASTK Gene Family Alterations in Cancer.

The FASTK family of proteins have been recently reported to play a key role in the post-transcriptional regulation of mitochondrial gene expression, including mRNA stability and translation. Accumulated studies have provided evidence that the expression of some FASTK genes is altered in certain types of cancer, in agreement with the central role of mitochondria in cancer development. Here, we obtained a pan-cancer overview of the genomic and transcriptomic alterations of FASTK genes. FASTK, FASTKD1, FASTKD3 and FASTKD5 showed the highest rates of genetic alterations. FASTK and FASTKD3 alterations consisted mainly of amplifications that were seen in more than 8% of ovarian and lung cancers, respectively. FASTKD1 and FASTKD5 were the most frequently mutated FASTK genes, and the mutations were identified in 5-7% of uterine cancers, as well as in 4% of melanomas. Our results also showed that the mRNA levels of all FASTK members were strongly upregulated in esophageal, stomach, liver and lung cancers. Finally, the protein-protein interaction network for FASTK proteins uncovers the interaction of FASTK, FASTKD2, FASTKD4 and FASTKD5 with cancer signaling pathways. These results serve as a starting point for future research into the potential of the FASTK family members as diagnostic and therapeutic targets for certain types of cancer.

Role of POLE and POLD1 in familial cancer.

PURPOSE: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. METHODS: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. RESULTS: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. CONCLUSIONS: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.

Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae.

BACKGROUND: Specific histone modifications play important roles in chromatin functions; i.e., activation or repression of gene transcription. This participation must occur as a dynamic process. Nevertheless, most of the histone modification maps reported to date provide only static pictures that link certain modifications with active or silenced states. This study, however, focuses on the global histone modification variation that occurs in response to the transcriptional reprogramming produced by a physiological perturbation in yeast. RESULTS: We did a genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after saline stress. The most striking change was rapid acetylation loss in lysines 9 and 14 of H3 and in lysine 8 of H4, associated with gene repression. The genes activated by saline stress increased the acetylation levels at these same sites, but this acetylation process was quantitatively minor if compared to that of the deacetylation of repressed genes. The changes in the tri-methylation of lysines 4, 36 and 79 of H3 and the di-methylation of lysine 79 of H3 were slighter than those of acetylation. Furthermore, we produced new genome-wide maps for seven histone modifications, and we analyzed, for the first time in S. cerevisiae, the genome-wide profile of acetylation of lysine 8 of H4. CONCLUSIONS: This research reveals that the short-term changes observed in the post-stress methylation of histones are much more moderate than those of acetylation, and that the dynamics of the acetylation state of histones during activation or repression of transcription is a much quicker process than methylation.

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1.

BACKGROUND: Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. METHODS: A training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered. RESULTS: Gene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign. CONCLUSIONS: Our recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

Brain radiotoxicity-related 15CAcBRT gene expression signature predicts survival prognosis of glioblastoma patients.

BACKGROUND: Glioblastoma is the most common and devastating primary brain cancer. Radiotherapy is standard of care; however, it is associated with brain radiation toxicity (BRT). This study used a multi-omics approach to determine whether BRT-related genes (RGs) harbor survival prognostic value and whether their encoded proteins represent novel therapeutic targets for glioblastoma. METHODS: RGs were identified through analysis of single-nucleotide variants associated with BRT (R-SNVs). Functional relationships between RGs were established using Protein-Protein Interaction networks. The influence of RGs and their functional groups on glioblastoma prognosis was evaluated using clinical samples from the Glioblastoma Bio-Discovery Portal database and validated using the Chinese Glioma Genome Atlas dataset. The identification of clusters of radiotoxic and putative pathogenic variants in proteins encoded by RGs was achieved by computational 3D structural analysis. RESULTS: We identified the BRT-related 15CAcBRT molecular signature with prognostic value in glioblastoma, by analysis of the COMT and APOE protein functional groups. Its external validation confirmed clinical relevance independent of age, MGMT promoter methylation status, and IDH mutation status. Interestingly, the genes IL6, APOE, and MAOB documented significant gene expression levels alteration, useful for drug repositioning. Biological networks associated with 15CAcBRT signature involved pathways relevant to cancer and neurodegenerative diseases. Analysis of 3D clusters of radiotoxic and putative pathogenic variants in proteins coded by RGs unveiled potential novel therapeutic targets in neuro-oncology. CONCLUSIONS: 15CAcBRT is a BRT-related molecular signature with prognostic significance for glioblastoma patients and represents a hub for drug repositioning and development of novel therapies.

Solving the enigma of POLD1 p.V295M as a potential cause of increased cancer risk.

Germline variants that affect the proofreading activity of polymerases epsilon (POLE) and delta (POLD1) predispose to colorectal adenomas and carcinomas, among other cancers. All cancer-associated pathogenic variants reported to date consist of non-disruptive genetic changes affecting the sequence that codifies the exonuclease domain (ED). Generally, disruptive (frameshift, stop-gain) POLE and POLD1 variants and missense variants outside the ED do not predispose to cancer. However, this statement may not be true for some, very specific variants that would indirectly affect the proofreading activity of the corresponding polymerase. We evaluated, by using multiple approaches, the possibility that POLD1 c.883G>A; p.(Val295Met), -a variant located 9 amino acids upstream the ED and present in ~0.25% of hereditary cancer patients-, affects POLD1 proofreading activity. Our findings show cumulative evidence that support no alteration of the proofreading activity and lack of association with cancer. The variant is classified as likely benign according to the ACMG/AMP guidelines.

A computational and structural analysis of germline and somatic variants affecting the DDR mechanism, and their impact on human diseases.

DNA-Damage Response (DDR) proteins are crucial for maintaining the integrity of the genome by identifying and repairing errors in DNA. Variants affecting their function can have severe consequences since failure to repair damaged DNA can result in cells turning cancerous. Here, we compare germline and somatic variants in DDR genes, specifically looking at their locations in the corresponding three-dimensional (3D) structures, Pfam domains, and protein-protein interaction interfaces. We show that somatic variants in metastatic cases are more likely to be found in Pfam domains and protein interaction interfaces than are pathogenic germline variants or variants of unknown significance (VUS). We also show that there are hotspots in the structures of ATM and BRCA2 proteins where pathogenic germline, and recurrent somatic variants from primary and metastatic tumours, cluster together in 3D. Moreover, in the ATM, BRCA1 and BRCA2 genes from prostate cancer patients, the distributions of germline benign, pathogenic, VUS, and recurrent somatic variants differ across Pfam domains. Together, these results provide a better characterisation of the most recurrent affected regions in DDRs and could help in the understanding of individual susceptibility to tumour development.

Geleophysic dysplasia: novel missense variants and insights into ADAMTSL2 intracellular trafficking.

Geleophysic dysplasia (GPHYSD1, MIM231050; GPHYSD2, MIM614185; GPHYSD3, MIM617809) is an autosomal disorder characterized by short-limb dwarfism, brachydactyly, cardiac valvular disease, and laryngotracheal stenosis. Mutations in ADAMTSL2, FBN1, and LTBP3 genes are responsible for this condition. We found that three previously described cases of GPHYSD diagnosed clinically were homozygote or compound heterozygotes for five ADAMTSL2 variants, four of which not being previously reported. By electron microscopy, skin fibroblasts available in one case homozygote for an ADAMTSL2 variant showed a defective intracellular localization of mutant ADAMTSL2 protein that did not accumulate within lysosome-like intra-cytoplasmic inclusions. Moreover, this mutant ADAMTSL2 protein was less secreted in medium and resulted in increased SMAD2 phosphorylation in transfected HEK293 cells.

PROREPAIR-B: A Prospective Cohort Study of the Impact of Germline DNA Repair Mutations on the Outcomes of Patients With Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Germline mutations in DNA damage repair (DDR) genes are identified in a significant proportion of patients with metastatic prostate cancer, but the clinical implications of these genes remain unclear. This prospective multicenter cohort study evaluated the prevalence and effect of germline DDR (gDDR) mutations on metastatic castration-resistance prostate cancer (mCRPC) outcomes. PATIENTS AND METHODS: Unselected patients were enrolled at diagnosis of mCRPC and were screened for gDDR mutations in 107 genes. The primary aim was to assess the impact of ATM/BRCA1/BRCA2/ PALB2 germline mutations on cause-specific survival (CSS) from diagnosis of mCRPC. Secondary aims included the association of gDDR subgroups with response outcomes for mCRPC treatments. Combined progression-free survival from the first systemic therapy (PFS) until progression on the second systemic therapy (PFS2) was also explored. RESULTS: We identified 68 carriers (16.2%) of 419 eligible patients, including 14 with BRCA2, eight with ATM, four with BRCA1, and none with PALB2 mutations. The study did not reach its primary end point, because the difference in CSS between ATM/BRCA1/BRCA2/PALB2 carriers and noncarriers was not statistically significant (23.3 v 33.2 months; P = .264). CSS was halved in germline BRCA2 (g BRCA2) carriers (17.4 v 33.2 months; P = .027), and g BRCA2 mutations were identified as an independent prognostic factor for CCS (hazard ratio [HR], 2.11; P = .033). Significant interactions between g BRCA2 status and treatment type (androgen signaling inhibitor v taxane therapy) were observed (CSS adjusted P = .014; PFS2 adjusted P = .005). CSS (24.0 v 17.0 months) and PFS2 (18.9 v 8.6 months) were greater in g BRCA2 carriers treated in first line with abiraterone or enzalutamide compared with taxanes. Clinical outcomes did not differ by treatment type in noncarriers. CONCLUSION: g BRCA2 mutations have a deleterious impact on mCRPC outcomes that may be affected by the first line of treatment used. Determination of g BRCA2 status may be of assistance for the selection of the initial treatment in mCRPC. Nonetheless, confirmatory studies are required before these results can support a change in clinical practice.

Hesperetin induces melanin production in adult human epidermal melanocytes.

One of the major sources of flavonoids for humans are citrus fruits, hesperidin being the predominant flavonoid. Hesperetin (HSP), the aglycon of hesperidin, has been reported to provide health benefits such as antioxidant, anti-inflammatory and anticarcinogenic effects. However, the effect of HSP on skin pigmentation is not clear. Some authors have found that HSP induces melanogenesis in murine B16-F10 melanoma cells, which, if extrapolated to in vivo conditions, might protect skin against photodamage. Since the effect of HSP on normal melanocytes could be different to that observed on melanoma cells, the described effect of HSP on murine melanoma cells has been compared to the effect obtained using normal human melanocytes. HSP concentrations of 25 and 50 µM induced melanin synthesis and tyrosinase activity in human melanocytes in a concentration-dependent manner. Compared to control melanocytes, 25 µM HSP increased melanin production and tyrosinase activity 1.4-fold (p < 0.01) and 1.1-fold (p < 0.01), respectively, and the corresponding increases in the case of 50 µM HSP were 1.9-fold (p < 0.001) and 1.3-fold (p < 0.001). Therefore, HSP could be considered a valuable photoprotective substance if its capacity to increase melanin production in human melanocyte cultures could be reproduced on human skin.

Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex.

Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5'-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.