Implications of noncoding regulatory functions in the development of insulinomas.

Insulinomas are rare neuroendocrine tumors arising from pancreatic ß cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in ß cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of ß cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations.

A Single-Cell Atlas of the Murine Pancreatic Ductal Tree Identifies Novel Cell Populations With Potential Implications in Pancreas Regeneration and Exocrine Pathogenesis.

BACKGROUND & AIMS: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. METHODS: We used single cell RNA sequencing to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models, and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. RESULTS: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including a Wnt-responsive population, a ciliated population, and Flrt3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples. The expression of Wnt-responsive, interferon-responsive, and epithelial-to-mesenchymal transition population markers increases in chronic pancreatitis and tumor samples. CONCLUSIONS: In light of our discovery of previously unidentified ductal populations, we unmask potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis. Thus, novel lineage-tracing models are needed to investigate ductal-specific populations in vivo.

A single-cell atlas of the murine pancreatic ductal tree identifies novel cell populations with potential implications in pancreas regeneration and exocrine pathogenesis.

Background and aims: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. Methods: We used scRNA-seq to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. Results: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro , including Wnt-responsive-population, ciliated-population and FLRT3 + cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples, highlighting a putative role of WNT-responsive, IFN-responsive and EMT-populations in pancreatic exocrine pathogenesis as their expression increases in chronic pancreatitis and PanIN lesions. Conclusions: In light of our discovery of previously unidentified ductal populations, we unmask the potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis.

MacroH2As regulate enhancer-promoter contacts affecting enhancer activity and sensitivity to inflammatory cytokines.

MacroH2A histone variants have a function in gene regulation that is poorly understood at the molecular level. We report that macroH2A1.2 and macroH2A2 modulate the transcriptional ground state of cancer cells and how they respond to inflammatory cytokines. Removal of macroH2A1.2 and macroH2A2 in hepatoblastoma cells affects the contact frequency of promoters and distal enhancers coinciding with changes in enhancer activity or preceding them in response to the cytokine tumor necrosis factor alpha. Although macroH2As regulate genes in both directions, they globally facilitate the nuclear factor κB (NF-κB)-mediated response. In contrast, macroH2As suppress the response to the pro-inflammatory cytokine interferon gamma. MacroH2A2 has a stronger contribution to gene repression than macroH2A1.2. Taken together, our results suggest that macroH2As have a role in regulating the response of cancer cells to inflammatory signals on the level of chromatin structure. This is likely relevant for the interaction of cancer cells with immune cells of their microenvironment.

Cross-talk between IFN-γ and TWEAK through miR-149 amplifies skin inflammation in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease with disturbed interplay between immune cells and keratinocytes. A strong IFN-γ signature is characteristic for psoriasis skin, but the role of IFN-γ has been elusive. MicroRNAs are short RNAs regulating gene expression. OBJECTIVE: Our aim was to investigate the role of miR-149 in psoriasis and in the inflammatory responses of keratinocytes. METHODS: miR-149 expression was measured by quantitative RT-PCR in keratinocytes isolated from healthy skin and lesional and nonlesional psoriasis skin. Synthetic miR-149 was injected intradermally into the back skin of mice, and imiquimod was applied to induce psoriasis-like skin inflammation, which was then evaluated at the morphologic, histologic, and molecular levels. miR-149 was transiently overexpressed or inhibited in keratinocytes in combination with IFN-γ- and/or TNF-related weak inducer of apoptosis (TWEAK)-treatment. RESULTS: Here we report a microRNA-mediated mechanism by which IFN-γ primes keratinocytes to inflammatory stimuli. Treatment with IFN-γ results in a rapid and long-lasting suppression of miR-149 in keratinocytes. Depletion of miR-149 in keratinocytes leads to widespread transcriptomic changes and induction of inflammatory mediators with enrichment of the TWEAK pathway. We show that IFN-γ-mediated suppression of miR-149 leads to amplified inflammatory responses to TWEAK. TWEAK receptor (TWEAKR/Fn14) is identified as a novel direct target of miR-149. The in vivo relevance of this pathway is supported by decreased miR-149 expression in psoriasis keratinocytes, as well as by the protective effect of synthetic miR-149 in the imiquimod-induced mouse model of psoriasis. CONCLUSION: Our data define a new mechanism, in which IFN-γ primes keratinocytes for TWEAK-induced inflammatory responses through suppression of miR-149, promoting skin inflammation.

A comprehensive analysis of coding and non-coding transcriptomic changes in cutaneous squamous cell carcinoma.

Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression. To identify mRNAs, lncRNAs and circRNAs, which can be involved in cSCC, RNA-seq was performed on nine cSCCs and seven healthy skin samples. Representative transcripts were validated by NanoString nCounter assays using an extended cohort, which also included samples from pre-cancerous skin lesions (actinic keratosis). 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs were identified to be differentially expressed in cSCC. Targets of 519 transcription factors were enriched among differentially expressed genes, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development (MYC, RELA, ETS1, TP63). Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched. In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were were identified to be dysregulated. A global downregulation of circRNAs was observed in cSCC, and novel skin-enriched circRNAs, circ_IFFO2 and circ_POF1B, were identified and validated. In conclusion, a reference set of coding and non-coding transcripts were identified in cSCC, which may become potential therapeutic targets or biomarkers.

Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells.

Chromosome segregation during mitosis is antagonistically regulated by the Aurora-B kinase and RepoMan (recruits PP1 onto mitotic chromatin at anaphase)-associated phosphatases PP1/PP2A. Aurora B is overexpressed in many cancers but, surprisingly, this only rarely causes lethal aneuploidy. Here we show that RepoMan abundance is regulated by the same mechanisms that control Aurora B, including FOXM1-regulated expression and proteasomal degradation following ubiquitination by APC/C-CDH1 or SCFFBXW7. The deregulation of these mechanisms can account for the balanced co-overexpression of Aurora B and RepoMan in many cancers, which limits chromosome segregation errors. In addition, Aurora B and RepoMan independently promote cancer cell proliferation by reducing checkpoint--induced cell-cycle arrest during interphase. The co-up-regulation of RepoMan and Aurora B in tumors is inversely correlated with patient survival, underscoring its potential importance for tumor progression. Finally, we demonstrate that high RepoMan levels sensitize cancer cells to Aurora-B inhibitors. Hence, the co-up-regulation of RepoMan and Aurora B is associated with tumor aggressiveness but also exposes a vulnerable target for therapeutic intervention.

Heterogeneous expression of EPCAM in human circulating tumour cells from patient-derived xenografts.

BACKGROUND: We aim to characterize the heterogeneous circulating tumour cells (CTCs) in peripheral blood, independently of physical or immunological purification, by using patient-derived xenografts (PDXs) models. CTC studies from blood generally rely on enrichment or purification. Conversely, we devised a method for the inclusive study of human cells from blood of PDX models, without pre-selection or enrichment. METHODS: A qRT-PCR assay was developed to detect human and cancer-related transcripts from CTCs in PDXs. We quantified the EPCAM and keratins CTC markers, in a PDX cohort of breast cancer. The murine beta actin gene was used for normalization. Spearman's rho coefficients were calculated for correlation. RESULTS: We demonstrated, for the first time, that we can quantify the content of CTCs and the expression of human CTC markers in PDX blood using human-specific qRT-PCR. Our method holds strong potential for the study of CTC heterogeneity and for the identification of novel CTC markers. CONCLUSIONS: The identification and the relative quantification of the diverse spectrum of CTCs in patients, irrespective of EPCAM or other currently used markers, will have a great impact on personalized medicine: unrestricted CTCs characterization will allow the early detection of metastases in cancer patients and the assessment of personalized therapies.

Genome-Wide Screen for MicroRNAs Reveals a Role for miR-203 in Melanoma Metastasis.

Melanoma is one of the deadliest human cancers with limited therapeutic options. MicroRNAs are a class of short noncoding RNAs regulating gene expression at the post-transcriptional level. To identify important miRNAs in melanoma, we compared the miRNome of primary and metastatic melanomas in The Cancer Genome Atlas dataset and found lower miR-203 abundance in metastatic melanoma. Lower level of miR-203 was associated with poor overall survival in metastatic disease. We found that the methylation levels of several CpGs in the MIR203 promoter negatively correlated with miR-203 expression and that treatment with the demethylating agent 5-aza-2-deoxycytidine induced miR-203 expression, which was associated with demethylation of the promoter CpGs, in melanoma cell lines. In vitro, there was a decreased expression of miR-203 in melanoma cell lines in comparison with primary melanocytes. Ectopic overexpression of miR-203 suppressed cell motility, colony formation, and sphere formation as well as the angiogenesis-inducing capacity of melanoma cells. In vivo, miR-203 inhibited xenograft tumor growth and reduced lymph node and lung metastasis. SLUG was shown as a target of miR-203, and knockdown of SLUG recapitulated the effects of miR-203, whereas its restoration was able to reverse the miR-203-mediated suppression of cell motility. These results establish a role for miR-203 as a tumor suppressor in melanoma which suppresses both early and late steps of metastasis. Hence, restoration of miR-203 has therapeutic potential in melanoma.

Profiling of the Predicted Circular RNAs in Ductal In Situ and Invasive Breast Cancer: A Pilot Study.

The recent advantage obtained by next generation sequencing allows a depth investigation of a new "old" kind of noncoding transcript, the circular RNAs. Circular RNAs are nontranslated RNAs, typically nonpolyadenylated, with a resistance to exonucleases that gives them the ability to be more stable than the common linear RNA isoforms. We used a bioinformatic detection tool (CIRCexplorer) to research predictive circRNAs from the next generation sequenced data of five samples of ductal in situ carcinoma (DCIS) and matched adjacent invasive ductal carcinoma (IDC). Furthermore, we also investigated the circular RNAs expressed in MCF7, an invasive breast ductal carcinoma cell line. We described the genomic context of the predicted circular RNAs and we address the hypothetical possible functional roles. This study showed a perspective of a panel of predictive circRNAs identified and the function that circRNAs could exert.

Transancestral fine-mapping of four type 2 diabetes susceptibility loci highlights potential causal regulatory mechanisms.

To gain insight into potential regulatory mechanisms through which the effects of variants at four established type 2 diabetes (T2D) susceptibility loci (CDKAL1, CDKN2A-B, IGF2BP2 and KCNQ1) are mediated, we undertook transancestral fine-mapping in 22 086 cases and 42 539 controls of East Asian, European, South Asian, African American and Mexican American descent. Through high-density imputation and conditional analyses, we identified seven distinct association signals at these four loci, each with allelic effects on T2D susceptibility that were homogenous across ancestry groups. By leveraging differences in the structure of linkage disequilibrium between diverse populations, and increased sample size, we localised the variants most likely to drive each distinct association signal. We demonstrated that integration of these genetic fine-mapping data with genomic annotation can highlight potential causal regulatory elements in T2D-relevant tissues. These analyses provide insight into the mechanisms through which T2D association signals are mediated, and suggest future routes to understanding the biology of specific disease susceptibility loci.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Quantification of CpG island methylation in progressive breast lesions from normal to invasive carcinoma.

Epigenetic silencing of specific genes is associated with cancer progression. CpG islands are present at higher frequency in promoter regions, their methylation leading to gene underexpression. Pyrosequencing provides sequencing analysis of genetic markers, e.g., single nucleotide polymorphisms and DNA methylation. We investigated methylation levels of a spectrum of neoplastic breast lesions, ranging from hyperplasia to invasive carcinoma obtained from the same patient. Assays were designed to analyze promoter regions of RASSF1A, GSTP1, RARbeta, and E-cadherin. Methylation increased from normal to hyperplasia, the increase being significantly higher in invasive and in situ tumors for RASSF1A (p=0.00006 and p=0.009, respectively).

Rehabilitation of adaptive immunity and regeneration of beta cells.

Type 1 Diabetes (T1D) is an autoimmune disease resulting from the destruction of pancreatic insulin-producing beta cells that most frequently occurs in genetically predisposed children. Recent observations illustrating the regenerative capability of the endocrine pancreas in addition to advances in stem cell and gene therapy technologies enable the exploration of alternatives to allogeneic islet transplantation. Living-cell-mediated approaches can abrogate autoimmunity and the consequent destruction of beta cells without the need for immunosuppressive drugs. Such approaches can be used as a foundation for new protocols that more easily translate to the clinical setting. The twin goals of controlling autoimmune disease and promoting stable regeneration of insulin-producing beta cells should be considered the cornerstones of the successful development of a cure for this chronic disease.