Deciphering the roadmap of in vivo reprogramming toward pluripotency

Differentiated cells can be converted into pluripotent stem cells by expressing the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) in a process known as reprogramming. Here, using single-cell RNA sequencing of pancreas undergoing reprogramming, we identify markers along the trajectory from acinar cell identity to pluripotency. These markers allow direct in situ visualization of cells undergoing dedifferentiation and acquiring features of early and advanced intermediate reprogramming. We also find that a fraction of cells do not dedifferentiate upon OSKM expression and are characterized by stress markers of the REG3 and AP-1 families. Importantly, most markers of intermediate reprogramming in the pancreas are also observed in stomach, colon, and cultured fibroblasts expressing OSKM. Among them is LY6A, a protein characteristic of progenitor cells and generally upregulated during tissue repair. Our roadmap defines intermediate reprogramming states that could be functionally relevant for tissue regeneration and rejuvenation.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved

Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across Stages, Detection in Urine, and Lack of Association with Outcome

Background: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). Design, setting, and participants: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n = 174), or under surveillance after diagnosis of non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay. Outcome measurements and statistical analysis: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. Results and limitations: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p = 0.0002). There was no association between TERT mutations and mRNA expression (p = 0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Conclusions: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. (C) 2013 European Association of Urology. Published by Elsevier B. V. All rights reserved

Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across Stages, Detection in Urine, and Lack of Association with Outcome

Background: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). Design, setting, and participants: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n = 174), or under surveillance after diagnosis of non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay. Outcome measurements and statistical analysis: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. Results and limitations: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p = 0.0002). There was no association between TERT mutations and mRNA expression (p = 0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Conclusions: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. (C) 2013 European Association of Urology. Published by Elsevier B. V. All rights reserved

Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

PMCID: PMC3553106This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Is EGFR expression altered following postoperative chemotherapy for colorectal adenocarcinoma?

BACKGROUND: There is immunohistochemical evidence to suggest that expression of epidermal growth factor receptor (EGFR) in primary colorectal adenocarcinoma predicts its expression in recurrent disease. This study investigates whether postoperative chemotherapy affects the degree of concordance between EGFR statuses of the two tumors. METHODS: Thirty-three patients were identified from the files of Sunnybrook Health Sciences Center from July 1994 to June 2005. All patients had resection of their primary tumors and their distant recurrences. Eighteen patients received postoperative chemotherapy, 3 of which also received postoperative radiation therapy. Representative primary and recurrent tumor sections were stained using mouse anti-EGFR antibodies and only membranous staining of malignant cells was recorded. Results were reported as negative (no staining), 1+ (positivity in <50% of cells) or 2+ (positivity in >50% of cells). RESULTS: EGFR immunostaining in the 15 patients, who received no postoperative chemotherapy, was decreased in 3 recurrences, remained the same in 10 and increased in 2. In the group of 18 patients who received postoperative chemotherapy, EGFR immunostaining was decreased in 6 recurrences, remained the same in 9 and increased in 3 (p = 0.6598). In patients who received postoperative chemotherapy, the odds ratio for a recurrence to show lower levels of EGFR immunostaining compared to its originally resected primary was 4.75 (CI = 0.94 – 26.73). CONCLUSION: These preliminary data suggest that recurrences following postoperative chemotherapy are likely to have lower levels of EGFR expression compared to cases who receive no chemotherapy. Although the difference of immunostaining profiles between the two groups was not statistically significant, this observation might impact the management of these patients by targeted biologic therapies and its practical implications need further validation in larger series

Genetic Variation in the TP53 Pathway and Bladder Cancer Risk. A Comprehensive Analysis

Introduction: Germline variants in TP63 have been consistently associated with several tumors, including bladder cancer, indicating the importance of TP53 pathway in cancer genetic susceptibility. However, variants in other related genes, including TP53 rs1042522 (Arg72Pro), still present controversial results. We carried out an in depth assessment of associations between common germline variants in the TP53 pathway and bladder cancer risk. Material and Methods: We investigated 184 tagSNPs from 18 genes in 1,058 cases and 1,138 controls from the Spanish Bladder Cancer/EPICURO Study. Cases were newly-diagnosed bladder cancer patients during 1998–2001. Hospital controls were age-gender, and area matched to cases. SNPs were genotyped in blood DNA using Illumina Golden Gate and TaqMan assays. Cases were subphenotyped according to stage/grade and tumor p53 expression. We applied classical tests to assess individual SNP associations and the Least Absolute Shrinkage and Selection Operator (LASSO)-penalized logistic regression analysis to assess multiple SNPs simultaneously. Results: Based on classical analyses, SNPs in BAK1 (1), IGF1R (5), P53AIP1 (1), PMAIP1 (2), SERINPB5 (3), TP63 (3), and TP73 (1) showed significant associations at p-value#0.05. However, no evidence of association, either with overall risk or with specific disease subtypes, was observed after correction for multiple testing (p-value$0.8). LASSO selected the SNP rs6567355 in SERPINB5 with 83% of reproducibility. This SNP provided an OR = 1.21, 95%CI 1.05–1.38, p-value = 0.006, and a corrected p-value = 0.5 when controlling for over-estimation. Discussion: We found no strong evidence that common variants in the TP53 pathway are associated with bladder cancer susceptibility. Our study suggests that it is unlikely that TP53 Arg72Pro is implicated in the UCB in white Europeans. SERPINB5 and TP63 variation deserve further exploration in extended studies.This work was supported by the Fondo de Investigacion Sanitaria, Spain (grant numbers 00/0745, PI051436, PI061614, G03/174); Red Tematica de Investigacion Cooperativa en Cancer (grant number RD06/0020-RTICC), Spain; Marato TV3 (grant number 050830); European Commission (grant numbers EU-FP7-HEALTH-F2-2008-201663-UROMOL; US National Institutes of Health (grant number USA-NIH-RO1-CA089715); and the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute at the National Institutes of Health, USA; Consolider ONCOBIO (Ministerio de Economia y Competitividad, Madrid, Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Deciphering the complex interplay between pancreatic cancer, diabetes mellitus subtypes and obesity/BMI through causal inference and mediation analyses.

OBJECTIVES: To characterise the association between type 2 diabetes mellitus (T2DM) subtypes (new-onset T2DM (NODM) or long-standing T2DM (LSDM)) and pancreatic cancer (PC) risk, to explore the direction of causation through Mendelian randomisation (MR) analysis and to assess the mediation role of body mass index (BMI). DESIGN: Information about T2DM and related factors was collected from 2018 PC cases and 1540 controls from the PanGenEU (European Study into Digestive Illnesses and Genetics) study. A subset of PC cases and controls had glycated haemoglobin, C-peptide and genotype data. Multivariate logistic regression models were applied to derive ORs and 95% CIs. T2DM and PC-related single nucleotide polymorphism (SNP) were used as instrumental variables (IVs) in bidirectional MR analysis to test for two-way causal associations between PC, NODM and LSDM. Indirect and direct effects of the BMI-T2DM-PC association were further explored using mediation analysis. RESULTS: T2DM was associated with an increased PC risk when compared with non-T2DM (OR=2.50; 95% CI: 2.05 to 3.05), the risk being greater for NODM (OR=6.39; 95% CI: 4.18 to 9.78) and insulin users (OR=3.69; 95% CI: 2.80 to 4.86). The causal association between T2DM (57-SNP IV) and PC was not statistically significant (ORLSDM=1.08, 95% CI: 0.86 to 1.29, ORNODM=1.06, 95% CI: 0.95 to 1.17). In contrast, there was a causal association between PC (40-SNP IV) and NODM (OR=2.85; 95% CI: 2.04 to 3.98), although genetic pleiotropy was present (MR-Egger: p value=0.03). Potential mediating effects of BMI (125-SNPs as IV), particularly in terms of weight loss, were evidenced on the NODM-PC association (indirect effect for BMI in previous years=0.55). CONCLUSION: Findings of this study do not support a causal effect of LSDM on PC, but suggest that PC causes NODM. The interplay between obesity, PC and T2DM is complex

RNA Binding Protein CUGBP2/CELF2 Mediates Curcumin-Induced Mitotic Catastrophe of Pancreatic Cancer Cells

Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers

hnRNP I Inhibits Notch Signaling and Regulates Intestinal Epithelial Homeostasis in the Zebrafish

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins—including numerous keratins characterized only recently—are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family