Activation-induced cytidine deaminase causes recurrent splicing mutations in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p < 0.01). We validated this enrichment in two additional DLBCL cohorts (N > 2,000; p < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL.

Expression inactivation of SMARCA4 by microRNAs in lung tumors.

SMARCA4 is the catalytic subunit of the SWI/SNF chromatin-remodeling complex, which alters the interactions between DNA and histones and modifies the availability of the DNA for transcription. The latest deep sequencing of tumor genomes has reinforced the important and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer. However, although SWI/SNF complex plays a key role in gene expression, the regulation of this complex itself is poorly understood. Significantly, an understanding of the regulation of SMARCA4 expression has gained in importance due to recent proposals incorporating it in therapeutic strategies that use synthetic lethal interactions between SMARCA4-MAX and SMARCA4-SMARCA2. In this report, we found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3'UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functional relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the aforementioned synthetic lethal therapeutic strategies.

Benefits for cardiovascular system, bone density, and quality of life of a long-term hormone therapy in hysterectomized women: a 20-year follow-up study.

OBJECTIVE: The safety, consequences, and dosage of long-term hormone therapy (HT) for postmenopausal women remain unclear. Our aim was to analyze the effects of HT after 20 years of therapy in women after hysterectomy, focusing on the symptoms of menopause, blood pressure, lipid profiles, and bone density. METHODS: A prospective observational longitudinal study was designed. The initial transdermal estradiol dose was reduced in half (0.025 mg/d) at 60 years of age. Different parameters including demographic, cardiovascular, bone density, and metabolic variables, as well as quality of life characteristics, were analyzed using bivariate analyses. Multivariate generalized estimating equations for longitudinal data were fitted for differences over time and between doses (<60 vs ≥60 y) using the R package geepack. RESULTS: After 20 years of HT, the mean age of 56 studied hysterectomized women was 67.1 years. The mean Kupperman index score decreased from 26.7 to 12.0 ( P < 0.001). A trend with total and low-density lipoprotein cholesterol reduction and high-density lipoprotein cholesterol increase was observed over time. A decrease in very-low-density lipoprotein cholesterol ( P = 0.05) and an increase in T score vertebral densitometry ( P = 0.014) were detected after HT. No changes in health outcome were detected in women older than 60 years with the reduced dose of HT. Breast cancer was the reason for dropouts in 0.02% women. CONCLUSIONS: HT for up to 20 years after hysterectomy may be beneficial for bone and cardiovascular health and for the overall quality of life. Our data suggest the importance of evaluating the dose and the timing of HT.

BCL7A is silenced by hypermethylation to promote acute myeloid leukemia.

BACKGROUND: Recent massive sequencing studies have revealed that SWI/SNF complexes are among the most frequently altered functional entities in solid tumors. However, the role of SWI/SNF in acute myeloid leukemia is poorly understood. To date, SWI/SNF complexes are thought to be oncogenic in AML or, at least, necessary to support leukemogenesis. However, mutation patterns in SWI/SNF genes in AML are consistent with a tumor suppressor role. Here, we study the SWI/SNF subunit BCL7A, which has been found to be recurrently mutated in lymphomas, but whose role in acute myeloid malignancies is currently unknown. METHODS: Data mining and bioinformatic approaches were used to study the mutational status of BCL7A and the correlation between BCL7A expression and promoter hypermethylation. Methylation-specific PCR, bisulfite sequencing, and 5-aza-2'-deoxycytidine treatment assays were used to determine if BCL7A expression was silenced due to promoter hypermethylation. Cell competition assays after BCL7A expression restoration were used to assess the role of BCL7A in AML cell line models. Differential expression analysis was performed to determine pathways and genes altered after BCL7A expression restoration. To establish the role of BCL7A in tumor development in vivo, tumor growth was compared between BCL7A-expressing and non-expressing mouse xenografts using in vivo fluorescence imaging. RESULTS: BCL7A expression was inversely correlated with promoter methylation in three external cohorts: TCGA-LAML (N = 160), TARGET-AML (N = 188), and Glass et al. (2017) (N = 111). The AML-derived cell line NB4 silenced the BCL7A expression via promoter hypermethylation. Ectopic BCL7A expression in AML cells decreased their competitive ability compared to control cells. Additionally, restoration of BCL7A expression reduced tumor growth in an NB4 mouse xenograft model. Also, differential expression analysis found that BCL7A restoration altered cell cycle pathways and modified significantly the expression of genes like HMGCS1, H1-0, and IRF7 which can help to explain its tumor suppressor role in AML. CONCLUSIONS: BCL7A expression is silenced in AML by promoter methylation. In addition, restoration of BCL7A expression exerts tumor suppressor activity in AML cell lines and xenograft models.

Multi-omic alterations of the SWI/SNF complex define a clinical subgroup in lung adenocarcinoma.

SWI/SNF complexes are major targets of mutations in cancer. Here, we combined multiple "-omics" methods to assess SWI/SNF composition and aberrations in LUAD. Mutations in lung SWI/SNF subunits were highly recurrent in our LUAD cohort (41.4%), and over 70% of the mutations were predicted to have functional impact. Furthermore, SWI/SNF expression in LUAD suffered an overall repression that could not be explained exclusively by genetic alterations. Finally, SWI/SNF mutations were associated with poorer overall survival in TCGA-LUAD. We propose SWI/SNF-mutant LUAD as a separate clinical subgroup with practical implications.

PKP1 and MYC create a feedforward loop linking transcription and translation in squamous cell lung cancer.

PURPOSE: Plakophilin 1 (PKP1) is well-known as an important component of the desmosome, a cell structure specialized in spot-like cell-to-cell adhesion. Although desmosomes have generally been associated with tumor suppressor functions, we recently found that PKP1 is recurrently overexpressed in squamous cell lung cancer (SqCLC) to exert an oncogenic role by enhancing the translation of MYC (c-Myc), a major oncogene. In this study, we aim to further characterize the functional relationship between PKP1 and MYC. METHODS: To determine the functional relationship between PKP1 and MYC, we performed correlation analyses between PKP1 and MYC mRNA expression levels, gain/loss of function models, chromatin immunoprecipitation (ChIP) and promoter mutagenesis followed by luciferase assays. RESULTS: We found a significant correlation between the mRNA levels of MYC and PKP1 in SqCLC primary tumor samples. In addition, we found that MYC is a direct transcription factor of PKP1 and binds to specific sequences within its promoter. In agreement with this, we found that MYC knockdown reduced PKP1 protein expression in different SqCLC models, which may explain the PKP1-MYC correlation that we found. Conversely, we found that PKP1 knockdown reduced MYC protein expression, while PKP1 overexpression enhanced MYC expression in these models. CONCLUSIONS: Based on these results, we propose a feedforward functional relationship in which PKP1 enhances MYC translation in conjunction with the translation initiation complex by binding to the 5'-UTR of MYC mRNA, whereas MYC promotes PKP1 transcription by binding to its promoter. These results suggest that PKP1 may serve as a therapeutic target for SqCLC.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Acceleration of the DNA methylation clock among lynch syndrome-associated mutation carriers.

BACKGROUND: DNA methylation (DNAm) age metrics have been widely accepted as an epigenetic biomarker for biological aging and disease. The purpose of this study is to assess whether or not individuals carrying Lynch Syndrome-associated mutations are affected in their rate of biological aging, as measured by the epigenetic clock. METHODS: Genome-wide bisulfite DNA sequencing data were generated using DNA from CD4 + T-cells obtained from peripheral blood using 27 patient samples from Lynch syndrome families. Horvath's DNAm age model based on penalized linear regression was applied to estimate DNAm age from patient samples with distinct clinical and genetic characteristics to investigate cancer mutation-related aging effects. RESULTS: Both Lynch mutation carriers and controls exhibited high variability in their estimated DNAm age, but regression analysis showed steeper slope for the Lynch mutation carriers. Remarkably, six Lynch Syndrome-associated mutation carriers showed a strong correlation to the control group, and two sisters carrying Lynch Syndrome-associated mutations, with no significant difference in lifestyle and similar chronological age, were assigned very different DNAm age. CONCLUSIONS: Future studies will be required to explore, in larger patient populations, whether specific epigenetic age acceleration is predictive of time-to-cancer development, treatment response, and survival. Epigenetic clock DNAm metrics may be affected by the presence of cancer mutations in the germline, and thus show promise of potential clinical utility for stratified surveillance strategies based on the relative risk for imminent emergence of tumor lesions in otherwise healthy Lynch Syndrome-associated mutation carriers.

Targeting ribosomal G-quadruplexes with naphthalene-diimides as RNA polymerase I inhibitors for colorectal cancer treatment.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures commonly found in regulatory genomic regions. G4 targeting has emerged as a therapeutic approach in cancer. We have screened naphthalene-diimides (NDIs), a class of G4 ligands, in a cellular model of colorectal cancer (CRC). Here, we identify the leading compound T5 with a potent and selective inhibition of cell growth by high-affinity binding to G4s in ribosomal DNA, impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induces a rapid inhibition of Pol I transcription, nucleolus disruption, proteasome-dependent Pol I catalytic subunit A degradation and autophagy. Moreover, we attribute the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. Finally, we succinctly demonstrate that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Therefore, we report a mode of action for these NDIs involving ribosomal G4 targeting.

Correction: Preoperative chemoradiotherapy for rectal cancer: the sensitizer role of the association between miR-375 and c-Myc.

[This corrects the article DOI: 10.18632/oncotarget.19393.].

LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) are a heterogeneous class of non-coding RNAs whose biological roles are still poorly understood. LncRNAs serve as gene expression regulators, frequently interacting with epigenetic factors to shape the outcomes of crucial biological processes, and playing roles in different pathologies including cancer. Over the last years, growing scientific evidence supports the key role of some lncRNAs in tumor development and proposes them as valuable biomarkers for the clinic. In this study, we aimed to characterize lncRNAs whose expression is altered in tumor samples from patients with lung adenocarcinoma (LUAD) compared to adjacent normal tissue samples. On an RT-qPCR survey of 90 cancer-related lncRNAs, we found one lncRNA, DLG2-AS1, which was consistently downregulated in 70 LUAD patients. To gain insight into its biological function, DLG2-AS1 was cloned and successfully re-expressed in LUAD cancer cell lines. We determined that DLG2-AS1 is not a cis-regulatory element of its overlapping gene DLG2, as their transcription levels were not correlated, nor did DLG2-AS1 restoration modify the expression of DLG2 protein. Furthermore, after generating a receiver operating curve (ROC) and calculating the area under curve (AUC), we found that DLG2-AS1 expression showed high sensitivity and specificity (AUC = 0.726) for the classification of LUAD and normal samples, determining its value as a potential lung cancer biomarker.

The value of lncRNA FENDRR and FOXF1 as a prognostic factor for survival of lung adenocarcinoma.

It is increasingly evident that non-coding RNAs play a significant role in tumour development. However, we still have a limited knowledge of the clinical significance of long non-coding RNAs (lncRNAs) in lung cancer. The FENDRR is a long coding RNA (also named FOXF1-AS1) located in the vicinity of the protein-coding gene FOXF1 at 16q24.1 chromosomal region. The present study aimed to define the clinic pathological significance of the long-non-coding RNA FENDRR in lung adenocarcinomas. FENDRR expression measured by quantitative PCR was found significantly downregulated (p<0.001) in lung adenocarcinoma samples in comparison with their normal adjacent tissues (n=70). RNA in situ hybridization (RNA-FISH) corroborated independently the down-regulation of FENDRR. Interestingly, the expression of FENDRR correlated positively (p<0.001) with the expression of its protein-coding neighbor gene FOXF1. Additionally, FOXF1 expression was also found downregulated in adenocarcinomas compared to normal samples (p<0.001) and its expression was significantly correlated with overall survival alone (p=0.003) or in combination with FENDRR expression (p=0.01). In conclusion, our data support that FENDRR and FOXF1 expression is decreased in lung adenocarcinoma and should be considered as new potential diagnostic/prognosis biomarkers.

LncRNA-mRNA Co-Expression Analysis Identifies AL133346.1/CCN2 as Biomarkers in Pediatric B-Cell Acute Lymphoblastic Leukemia.

Pediatric acute B-cell lymphoblastic leukemia (B-ALL) constitutes a heterogeneous and aggressive neoplasia in which new targeted therapies are required. Long non-coding RNAs have recently emerged as promising disease-specific biomarkers for the clinic. Here, we identified pediatric B-ALL-specific lncRNAs and associated mRNAs by comparing the transcriptomic signatures of tumoral and non-tumoral samples. We identified 48 lncRNAs that were differentially expressed between pediatric B-ALL and healthy bone marrow samples. The most relevant lncRNA/mRNA pair was AL133346.1/CCN2 (previously known as RP11-69I8.3/CTGF), whose expression was positively correlated and increased in B-ALL samples. Their differential expression pattern and their strong correlation were validated in external B-ALL datasets (Therapeutically Applicable Research to Generate Effective Treatments, Cancer Cell Line Encyclopedia). Survival curve analysis demonstrated that patients with "high" expression levels of CCN2 had higher overall survival than those with "low" levels (p = 0.042), and this gene might be an independent prognostic biomarker in pediatric B-ALL. These findings provide one of the first detailed descriptions of lncRNA expression profiles in pediatric B-ALL and indicate that these potential biomarkers could help in the classification of leukemia subtypes and that CCN2 expression could predict the survival outcome of pediatric B-cell acute lymphoblastic leukemia patients.

Frequent mutations in the amino-terminal domain of BCL7A impair its tumor suppressor role in DLBCL.

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.

Comprehensive Analysis of SWI/SNF Inactivation in Lung Adenocarcinoma Cell Models.

Mammalian SWI/SNF (SWitch/Sucrose Non-Fermentable) complexes are ATP-dependent chromatin remodelers whose subunits have emerged among the most frequently mutated genes in cancer. Studying SWI/SNF function in cancer cell line models has unveiled vulnerabilities in SWI/SNF-mutant tumors that can lead to the discovery of new therapeutic drugs. However, choosing an appropriate cancer cell line model for SWI/SNF functional studies can be challenging because SWI/SNF subunits are frequently altered in cancer by various mechanisms, including genetic alterations and post-transcriptional mechanisms. In this work, we combined genomic, transcriptomic, and proteomic approaches to study the mutational status and the expression levels of the SWI/SNF subunits in a panel of 38 lung adenocarcinoma (LUAD) cell lines. We found that the SWI/SNF complex was mutated in more than 76% of our LUAD cell lines and there was a high variability in the expression of the different SWI/SNF subunits. These results underline the importance of the SWI/SNF complex as a tumor suppressor in LUAD and the difficulties in defining altered and unaltered cell models for the SWI/SNF complex. These findings will assist researchers in choosing the most suitable cellular models for their studies of SWI/SNF to bring all of its potential to the development of novel therapeutic applications.

Preoperative chemoradiotherapy for rectal cancer: the sensitizer role of the association between miR-375 and c-Myc.

Administration of chemoradiation before tumor resection has revolutionized the management of locally advanced rectal cancer, but many patients have proven resistant to this preoperative therapy. Our group recently reported a negative correlation between c-Myc gene expression and this resistance. In the present study, integrated analysis of miRNA and mRNA expression profiles was conducted in 45 pre-treatment rectal tumors in order to analyze the expressions of miRNAs and c-Myc and their relationship with clinicopathological factors and patient survival. Twelve miRNAs were found to be differentially expressed by responders and non-responders to the chemoradiation. Functional classification revealed an association between the differentially expressed miRNAs and c-Myc. Quantitative real-time PCR results showed that miRNA-148 and miRNA-375 levels were both significantly lower in responders than in non-responders. Notably, a significant negative correlation was found between miRNA-375 expression and c-Myc expression. According to these findings, miRNA-375 and its targeted c-Myc may be useful as a predictive biomarker of the response to neoadjuvant treatment in patients with locally advanced rectal cancer.

Allelic expression and quantitative RT-PCR study of TAp73 and DeltaNp73 in non-Hodgkin's lymphomas.

p73 shares structural and functional homology to p53. p73 generates different proteins using alternative promoters and splicing which have different biological characteristics. We investigated the pattern (monoallelic or biallelic) of expression of TAp73 and DeltaNp73 in normal lymphocytes and lymphomas using two p73 polymorphisms. We found monoallelic expression of TAp73 in normal lymphocytes and tumors, and a selective expression of AT allele in all cases. Moreover, the quantitative expression analysis revealed DeltaNp73 over-expression in both B- and T-cell lymphomas comparing with normal lymphoid cells, suggesting a role in tumorigenesis. Finally, we have confirmed that although DeltaNp73 over-expression could be an alternative mechanism of p53 inactivation, both alterations may appear together.