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Sethupathy, P.

Publications and source records attributed to Sethupathy, P..

At least 19 recordsLinked to original sources

MHC-I downregulation is associated with immune-evasion in breast cancers with heterogeneous activation of epithelial-mesenchymal plasticity

Cancer cell-intrinsic epithelial-mesenchymal plasticity (EMP) drives carcinoma progression through increased intratumoral heterogeneity and an immunosuppressive tumor microenvironment (TME). Specifically, EMP activation in murine mammary carcinomas results in exclusion of CD8+ T-cells and recruitment of immunosuppressive M2-like macrophages, resulting in resistance to anti-CTLA-4 immune checkpoint blockade (ICB) therapy. Notably, mixed E-qM tumors in which only 10% of cancer cells have activated EMP are resistant anti-CTLA-4 immune checkpoint blockade (ICB) therapy. This suggests that minority fractions of qM cancer cells could, in theory, exert immunosuppressive effects on surrounding E cancer and immune cells to shield the entire heterogeneous tumor against immune attack. However, this has not yet been demonstrated. Here, we use spatial transcriptomic profiling to demonstrate that mixed E-qM tumors assemble immunosuppressive cellular niches. Furthermore, E cancer cells within mixed E-qM tumors experience long-term MHC-I downregulation leading to resistance to elimination by CD8+ T-cells. This is associated with the exclusion of T-cells from the tumor core and a dysfunctional phenotype in the remaining CD8+ T-cells. qM cancer cells recruit immunosuppressive M2-like macrophages to mixed tumors, which further downregulates E cancer cell-intrinsic MHC-I levels. Finally, we demonstrate that human triple-negative breast cancers with increased EMP activation exclude T-cells from the tumor core. Together, these findings reveal that qM cancer cells modulate the immune infiltrate of mixed tumors, promoting MHC-I downregulation and evasion of immunological control by CD8+ T-cells.

cancer biology↗

Spatial analysis of canine glioma reveals heterogeneous intratumoral niches characterized by distinct patterns of cell plasticity and immunosuppression

Glioma is a fatal tumor of the central nervous system (CNS) with limited therapeutic options. While immune checkpoint inhibitors (ICI) are gaining traction as a novel alternative treatment for human gliomas (HG), clinical trials have yielded mixed results. A proposed major barrier to ICI therapy in cancer is epithelial-mesenchymal plasticity (EMP)-driven intratumoral immunosuppression. However, studying this mechanism is difficult in ex vivo and murine models, which fail to adequately replicate cancer cell-immune cell interactions. Formalin-fixed, paraffin-embedded (FFPE) patient-derived canine glioma (CG) samples represent an attractive alternative approach. Although mechanistic perturbation is unachievable in archival FFPE tissues, CG is notoriously 'cold' immunologically and is actively being investigated as a comparative model of HG. Moreover, the immune microenvironment in CG advantageously develops under exposure to environmental conditions directly shared with humans. Yet, it remains unknown whether CGs harbor heterogeneous niches combining varied plastic phenotypes and immunosuppressive populations. Defining the immune landscape of CG in relation to EMP is therefore critical to determining its potential as a comparative model for immunotherapy response in HG. By combining standard histology, immunohistochemistry, and tissue microarray-based spatial transcriptomics, we analyzed 30 low-grade (LGr) and high-grade (HGr) FFPE CG cases (n=10 cases/subtype): astrocytomas (4xLGr, 6xHGr), oligodendrogliomas (1xLGr, 9xHGr), and undefined gliomas (3LxGr, 7xHGr). All CG subtypes harbored heterogeneous immunosuppressive niches associated with varied EMP phenotypes. Astrocytomas exhibited a more-mesenchymal identity correlated with recruitment of tumor-associated macrophages (TAMs). Oligodendrogliomas displayed a strong stem-like signature and uniquely upregulated a FOXP3/LAG3/SNAI1 triad, positioned directly at the interface of immunosuppression and EMP. Across all CG subtypes, a more-mesenchymal phenotype co-occurred with TAMs, and intratumoral T-cell dysfunction and exhaustion. We conclude that CG replicates the EMP-immunosuppression heterogeneity seen in humans. Importantly, our results emphasize the existence of subtype-specific mechanisms of EMP and immune evasion, underscoring the major relevance of subtyping for translational applications.

cancer biology↗

Neuroepithelial reprogramming and ERBB vulnerability in canine acanthomatous ameloblastoma

Canine acanthomatous ameloblastoma (CAA) is a locally invasive benign oral neoplasm that is difficult to distinguish from canine oral squamous cell carcinoma (COSCC) due to overlapping clinical, radiologic, and histologic features. Although both tumors exhibit MAPK pathway activation, their mutational landscapes are distinct. Furthermore, previous studies using bulk RNA sequencing (RNA-seq) have demonstrated pronounced differences in programs related to, among others, hypoxia, PI3K-AKT signaling, and cell proliferation. However, these bulk studies lacked the resolution to elucidate the cellular heterogeneity of CAA relative to COSCC. We therefore performed single-nucleus RNA-seq to define the cellular gene expression landscape of CAA, COSCC, and healthy gingiva. Across [~]205,000 nuclei, we identified major epithelial, immune, endothelial, and mesenchymal populations, as well as two epithelial subtypes uniquely enriched in CAA. The CAA-specific keratinocytes exhibited a neuronal-like expression program defined by synaptic regulators, KRAS-associated signaling pathways, and markedly elevated expression of PEG3, ERBB4, GABRB1, MAGI2, and CASK. These findings were validated by bulk RNA-seq, qPCR, and immunohistochemistry, which demonstrated strong nuclear localization of PEG3 exclusively in CAA epithelium. A kinase inhibitor screen independently identified ERBB4 as a candidate therapeutic vulnerability, and pharmacologic inhibition with neratinib was effective. Together, these findings reveal a previously unrecognized neuroepithelial cell state that defines CAA, distinguishes it from COSCC, and reveals unique diagnostic and therapeutic signaling dependencies. Given the molecular and histopathologic parallels between CAA and human ameloblastoma, these data further position CAA as a naturally occurring comparative model for studying ameloblastoma biology and therapeutic vulnerabilities. Significance StatementCanine acanthomatous ameloblastoma (CAA) is a common locally aggressive oral neoplasm often misdiagnosed as canine oral squamous cell carcinoma (COSCC) due to overlapping clinical, radiologic, and histologic features. Using single-nucleus RNA sequencing of CAA, COSCC, and healthy gingiva, we resolve the microenvironment of these tumors and detect two epithelial subpopulations unique to CAA. These CAA-specific keratinocytes exhibit a neuroepithelial-like signature characterized by synaptic regulators, KRAS-associated signaling, and elevated PEG3 and ERBB4 expression. Functional kinase inhibitor screening of patient derived microtumor slices independently converged on ERBB4 as an effective therapeutic target. Together, our study provides a molecular framework for distinguishing CAA from COSCC and establishes a comparative oncology model with translational relevance to human ameloblastoma and other ERBB-driven epithelial neoplasms.

cancer biology↗

The RNA-binding protein Imp1 promotes a Spdef transcriptional program and mucus fucosylation during necrotizing enterocolitis

BackgroundIn the United States over 10% of all neonates are born premature (less than 37 weeks gestational age), and many face complications related to prematurity, including necrotizing enterocolitis (NEC). NEC is the most deadly gastrointestinal disease and the leading cause of death in preterm neonates, with up to 50% mortality. Since there is no cure for NEC, prevention is the best strategy. Enhancing our understanding of intestinal epithelial cell (IEC) responses to NEC damage will provide novel therapeutic targets to prevent NEC. Published evidence suggests that the RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) plays roles in intestinal development, barrier function, and intestinal repair. Notably, however, roles for IMP1 in NEC are not defined. Goblet cells produce protective mucus in the intestine, and their mature function is dependent on the transcription factor Spdef. Emerging evidence suggests that goblet cell mucus complexity impacts barrier function and inflammation susceptibility. This study aimed to define the role of IMP1 in NEC pathogenesis using neonatal human enteroids and a model of NEC-like intestinal injury in mice with IEC-specific Imp1 overexpression and loss. HypothesisIMP1 expression is protective in NEC. MethodsThis study used mice with intestinal epithelial Imp1 overexpression or loss and corresponding wild-type controls. At post-natal day 3, mice of both sexes were randomly assigned to control or NEC groups. NEC was induced with the well-established experimental NEC-like intestinal injury model that includes stress, formula feeding, and hypoxia. Imp1 effects on experimental NEC were assessed using RNA sequencing, western blotting, and immunostaining. ResultsInflammatory bacteria induced IMP1 expression in neonatal human enteroids. Mice with Imp1 overexpression incur worse intestinal damage during NEC. Pathway analysis of RNA sequencing data revealed a significant enrichment of the Spdef transcriptional network in Imp1IEC-OE during NEC. This included significant upregulation of Spdef target genes such as Agr2 (in NEC WT: 617.8 {+/-} 33.56 vs Imp1IEC-OE: 812.9 {+/-} 111.3, p=0.02) and Fut2 (in NEC WT: 219.4 {+/-} 34 vs Imp1IEC-OE: 396.6 {+/-} 62.9, p=0.05). In silico analysis predicted Imp1 binding to Spdef and mucus glycosylation mediator mRNAs. Although genotype did not affect goblet cell number, Imp1IEC-OE mice with NEC exhibited significant increases (p0.05) in Spdef protein, genes responsible for goblet cell function (Spink4, Klk1, Tspg1) and mucus glycosylation (Gcnt3, B3gnt7, Qsox1). Ultimately, Imp1 overexpression led to increased mucus fucosylation during NEC. ConclusionOur data indicate that during NEC, upregulation of Imp1 promotes goblet cell function via Spdef, including enhanced goblet cell maturation and mucus fucosylation. NEW AND NOTEWORTHYExpression of the RNA-binding protein Imp1 is enhanced in neonatal human enteroids in response to inflammatory bacteria. Imp1 regulates goblet cell function in response to early intestinal inflammation in a mouse model of necrotizing enterocolitis. Specifically, Imp1 promotes a Spdef transcriptional program by upregulating Spdef, resulting in elevated gene expression of Spdef target genes. Additionally, Imp1 increases the gene expression of (1,2)fucosyltransferase, Fut2, and subsequently, production of fucosylated mucus marked by UEA1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/702645v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@189f615org.highwire.dtl.DTLVardef@7aeeedorg.highwire.dtl.DTLVardef@dc258corg.highwire.dtl.DTLVardef@1252b98_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Single-cell multi-omic analysis of fibrolamellar carcinoma reveals rewired cell-to-cell communication patterns and unique vulnerabilities

Fibrolamellar carcinoma (FLC) is a rare malignancy disproportionately affecting adolescents and young adults with no standard of care. FLC is characterized by thick stroma, which has long suggested an important role of the tumor microenvironment. Over the past decade, several studies have revealed aberrant markers and pathways in FLC. However, a significant drawback of these efforts is that they were conducted on bulk tumor samples. Consequently, identities and roles of distinct cell types within the tumor milieu, and the patterns of intercellular communication, have yet to be explored. In this study we unveil cell-type specific gene signatures, transcription factor networks, and super-enhancers in FLC using a multi-omics strategy that leverages both single-nucleus ATAC-seq and single-nucleus RNA-seq. We also infer completely rewired cell-to-cell communication patterns in FLC including signaling mediated by SPP1-CD44, MIF-ACKR3, GDF15-TGFBR2, and FGF7-FGFR. Finally, we validate findings with loss-of-function studies in several models including patient tissue slices, identifying vulnerabilities that merit further investigation as candidate therapeutic targets in FLC.

cancer biology↗

Multi-omic study of genome-edited human colonoid models of colorectal cancer reveal genotype-specific patterns of microRNA regulation

Combinations of oncogenic mutations drive inter-tumor heterogeneity in colorectal cancer (CRC), which promotes distinct phenotypes and affects therapeutic efficacy. We recently demonstrated that combinations of mutations in mouse small intestinal organoids lead to unique changes in microRNA (miRNA) expression profiles. However, it remains unknown how different mutational backgrounds shape miRNA profiles in the human colon. We leveraged human colonic organoid models, termed colonoids, with gene edits targeting genes commonly mutated in CRC to profile genotype-specific changes in miRNA expression. By small RNA-sequencing we characterized genotype-specific miRNA profiles. We identified one group of miRNAs, including mir-34a-5p and mir-10a-5p, that is strongly downregulated in APC/KRAS/TP53 mutant (AKP-mutant) colonoids. Using chromatin run-on sequencing, we showed that most miRNA alterations in AKP-mutant colonoids are concordant with transcriptional changes. Transcription factor (TF) motif enrichment analysis using transcriptional regulatory elements with increased activity in AKP-mutant colonoids revealed an enrichment of binding sites for multiple oncogenic TFs. Several of these harbor predicted binding sites for mir-10a-5p and/or mir-34a-5p, suggesting these miRNAs may play a role in regulating transcriptional programs in AKP-mutant contexts. Ultimately, our study offers a glimpse into regulatory mechanisms that drive inter-tumor heterogeneity, and we highlight candidate therapeutic targets for the advancement of precision medicine.

cancer biology↗

Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution

The gut plays a key role in regulating metabolic health. Dietary factors disrupt intestinal physiology and contribute to obesity and diabetes, whereas bariatric procedures such as vertical sleeve gastrectomy (VSG) cause gut adaptations that induce robust metabolic improvements. However, our understanding of these adaptations at the cellular and molecular levels remains limited. In a validated murine model, we leverage single-cell transcriptomics to determine how VSG impacts different cell lineages of the small intestinal epithelium. We define cell type-specific genes and pathways that VSG rescues from high-fat diet perturbation and characterize additional rescue-independent changes brought about by VSG. We show that Paneth cells have increased expression of the gut peptide Reg3g after VSG. We also find that VSG restores pathways pertaining to mitochondrial respiration and cellular metabolism, especially within crypt-based cells. Overall, our study provides unprecedented molecular resolution of VSGs therapeutic effects on the gut epithelium.

genomics↗

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene DNAJB1-PRKACA (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long non-coding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and in vivo models of disease. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, an indicator of mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease. HighlightsFibrolamellar carcinoma (FLC) is a lethal liver cancer lacking effective therapeutic options. Ma et al. demonstrate that primate-specific RNA LINC00473 is enriched in tumor epithelial cells and functions to promote FLC growth and dysregulate cellular energetics, unveiling an important mechanism downstream of the fusion oncogene, DNAJB1-PRKACA, in FLC pathogenesis. In BriefO_LILINC00473 is consistently elevated in primary FLC tumor tissue from different patient cohorts and in multiple disease models. C_LIO_LIDP fusion, the signature oncoprotein of FLC, drives LINC00473 expression. C_LIO_LILINC00473 promotes FLC growth via anti-apoptotic function. C_LIO_LILINC00473 modulates FLC energetics by promoting glycolysis and altering mitochondrial fitness. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/543290v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@20e0f2org.highwire.dtl.DTLVardef@904fbborg.highwire.dtl.DTLVardef@62af84org.highwire.dtl.DTLVardef@f114be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

CDK7 is a Novel Therapeutic Vulnerability in Fibrolamellar Carcinoma

Fibrolamellar carcinoma (FLC) is a rare and lethal cancer that afflicts young individuals. The tumor arises in the background of a healthy liver, and patients typically present with advanced cancer at the time of diagnosis. Unfortunately, for these patients with advanced or recurrent cancer, no proven systemic therapies exist resulting in only 30-45% of patients surviving to 5 years. Investigations into the molecular underpinning of FLC have revealed a unique gene fusion between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA), leading to the formation of an oncoprotein (DNAJ-PKAc) that retains kinase activity and is a proven tumor-causing event in FLC. To uncover potential therapeutic targets, we engineered an FLC cell line by introducing the DNAJB1-PRKACA oncogene rearrangement into human hepatocellular cells using CRISPR/Cas9. We identified aberrant cell cycle progression, and follow-up molecular analysis revealed evidence of enhanced cyclin dependent kinase 7 (CDK7) activation in the DNAJB1-PRKACA expressing FLC cells. These findings were confirmed in human samples of FLC. In turn, targeting CDK7 with selective inhibitors demonstrated efficacy in several patient-derived models of FLC, with minimal toxicity to normal liver. Collectively, this work uncovers a novel candidate therapeutic vulnerability in FLC.

cancer biology↗

A Genetic Locus Mediating Attentional Processing

Attention is required for most higher-order cognitive functions. Prior studies have revealed functional roles for the prefrontal cortex and its extended circuits to enabling attention, but the underlying molecular processes and their impacts on cellular and circuit function remain poorly understood. To develop insights, we here took an unbiased forward genetics approach to identify single genes of large effect on attention. We studied 200 genetically diverse mice on measures of pre-attentive processing and through genetic mapping identified a small locus on chromosome 13 (95%CI: 92.22-94.09 Mb) driving substantial variation (19%) in this trait. Further characterization of the locus revealed a causative gene, Homer1, encoding a synaptic protein, where down-regulation of its short isoforms in prefrontal cortex (PFC) during early postnatal development led to improvements in multiple measures of attention in the adult. Subsequent mechanistic studies revealed that prefrontal Homer1 down-regulation is associated with GABAergic receptor up-regulation in those same cells. This enhanced inhibitory influence, together with dynamic neuromodulatory coupling, led to strikingly low PFC activity at baseline periods of the task but targeted elevations at cue onset, predicting short-latency correct choices. Notably high-Homer1, low-attentional performers, exhibited uniformly elevated PFC activity throughout the task. We thus identify a single gene of large effect on attention - Homer1 - and find that it improves prefrontal inhibitory tone and signal-to-noise (SNR) to enhance attentional performance. A therapeutic strategy focused on reducing prefrontal activity and increasing SNR, rather than uniformly elevating PFC activity, may complement the use of stimulants to improve attention.

neuroscience↗

Breast adipose tissue-derived extracellular vesicles from women with obesity stimulate mitochondrial-induced dysregulated tumor cell metabolism

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. Mitochondrial complex I inhibitor, metformin, reverses O-EV-induced cell proliferation. Several miRNAs, miR-155-5p, miR-10a-3p, and miR-30a-3p, which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing the metabolic reprogramming of ER+ breast cancer cells.

cancer biology↗

Aberrant miR-29 is a predictive feature of severe phenotypes in pediatric Crohn's disease

Crohns disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-sequencing of patient-matched non-inflamed colon and ileum biopsies from treatment-naive pediatric patients with CD (n=169) and a control cohort (n=108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we also found that pediatric CD patients with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 up-regulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

genomics↗

miR-29 is an important driver of aging-related phenotypes

Aging is a consequence of complex molecular changes, but the roles of individual microRNAs (miRNAs) in aging remain unclear. One of the few miRNAs that are upregulated during both normal and premature aging is miR-29. We confirmed this finding in our study in both mouse and monkey models. Follow-up analysis of the transcriptomic changes during normal aging revealed that miR-29 is among the top miRNAs predicted to drive the aging-related gene expression changes. We also showed that partial loss of miR-29 extends the lifespan of Zmpste24-/- mice, an established model of progeria, which indicates that miR-29 is functionally important in this accelerated aging model. To examine whether miR-29 upregulation alone is sufficient to promote aging-related phenotypes in vivo, we generated mice in which miR-29 can be conditionally overexpressed (miR-29TG). We found that miR-29 overexpression in mice is sufficient to drive aging-related phenotypes including alopecia, kyphosis, osteoporosis, senescence, and leads to early lethality. Transcriptomic analysis of both young miR-29TG and old WT mice revealed shared downregulation of genes enriched in extracellular matrix and fatty acid metabolism, and shared upregulation of genes in pathways linked to inflammation. Together, these results highlight the functional importance of miR-29 in controlling a gene expression program that drives agingrelated phenotypes.

physiology↗

Integrative genome-scale analyses reveal post-transcriptional signatures of early human smallintestinal development in a directed differentiation organoid model

MicroRNAs (miRNAs) are important post-transcriptional gene regulators in organ development. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Our analyses showed that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/499825v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ebf48eorg.highwire.dtl.DTLVardef@4a0d9org.highwire.dtl.DTLVardef@1dc3be9org.highwire.dtl.DTLVardef@1e14207_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

In situ polyadenylation enables spatial mapping of the total transcriptome

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We apply this Spatial Total RNA-Sequencing (STRS) approach to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration, and highlight host transcriptional responses associated with local viral RNA abundance. In situ polyadenylation requires the addition of only a single step to a widely used protocol for spatial RNA-sequencing, and thus could be broadly and quickly adopted. Spatial RNA-sequencing of the total transcriptome will enable new insights into spatial gene regulation and biology.

genomics↗

Human pancreatic islet microRNAs implicated in diabetes and related traits by large-scale genetic analysis

Genetic studies have identified [≥]240 loci associated with risk of type 2 diabetes (T2D), yet most of these loci lie in non-coding regions, masking the underlying molecular mechanisms. Recent studies investigating mRNA expression in human pancreatic islets have yielded important insights into the molecular drivers of normal islet function and T2D pathophysiology. However, similar studies investigating microRNA (miRNA) expression remain limited. Here, we present data from 63 individuals, representing the largest sequencing-based analysis of miRNA expression in human islets to date. We characterize the genetic regulation of miRNA expression by decomposing the expression of highly heritable miRNAs into cis- and trans-acting genetic components and mapping cis-acting loci associated with miRNA expression (miRNA-eQTLs). We find (i) 81 heritable miRNAs, primarily regulated by trans-acting genetic effects, and (ii) 5 miRNA-eQTLs. We also use several different strategies to identify T2D-associated miRNAs. First, we colocalize miRNA-eQTLs with genetic loci associated with T2D and multiple glycemic traits, identifying one miRNA, miR-1908, that shares genetic signals for blood glucose and glycated hemoglobin (HbA1c). Next, we intersect miRNA seed regions and predicted target sites with credible set SNPs associated with T2D and glycemic traits and find 32 miRNAs that may have altered binding and function due to disrupted seed regions. Finally, we perform differential expression analysis and identify 13 miRNAs associated with T2D status--including miR-187-3p, miR-21-5p, miR-668, and miR-199b-5p--and 4 miRNAs associated with a polygenic score for HbA1c levels--miR-216a, miR-25, miR-30a-3p, and miR-30a-5p.

genetics↗

Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles

Somatic mutations drive colorectal cancer (CRC) by disrupting gene regulatory mechanisms. Distinct combinations of mutations can result in unique changes to regulatory mechanisms leading to variability in the efficacy of therapeutics. MicroRNAs are important regulators of gene expression, and their activity can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect microRNAs. Here, using genetically-modified mouse intestinal organoid (enteroid) models, we identify 12 different modules of microRNA expression patterns across distinct combinations of mutations common in CRC. We also show that miR-24-3p is aberrantly upregulated in genetically-modified mouse enteroids irrespective of mutational context. Furthermore, we identify an enrichment of miR-24-3p predicted targets in downregulated gene lists from various mutational contexts compared to WT. In follow-up experiments, we demonstrate that miR-24-3p promotes CRC cell survival in multiple cell contexts. Our novel characterization of genotype-specific patterns of miRNA expression offer insight into the mechanisms that drive inter-tumor heterogeneity and highlight candidate microRNA therapeutic targets for the advancement of precision medicine for CRC.

cancer biology↗

Chemical, molecular, and single cell analysis reveal chondroitin sulfate proteoglycan aberrancy in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and non-malignant liver tissue (n=27) to identify changes in glycosaminoglycan (GAG) biosynthesis pathways and found that genes associated with production of chondroitin sulfate, but not other GAGs, are significantly increased by 8-fold. We then implemented a novel LC-MS/MS based method to quantify the abundance of different types of GAGs in patient tumors (n=16) and found that chondroitin sulfate is significantly more abundant in FLC tumors by 6-fold. Upon further analysis of GAG-associated proteins we found that versican (VCAN) expression is significantly up-regulated at the mRNA and protein levels, the latter of which was validated by immunohistochemistry. Finally, we performed single-cell assay for transposon-accessible chromatin-sequencing on FLC tumors (n=3), which revealed for the first time the different cell types in FLC tumors and also showed that VCAN is likely produced not only from FLC tumor epithelial cells but also activated stellate cells. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells. SignificanceThis study leverages a multi-disciplinary approach, including state-of-the-art chemical analyses and cutting-edge single-cell genomic technologies, to identify for the first time a marked chondroitin sulfate aberrancy in fibrolamellar carcinoma (FLC) that could open novel therapeutic avenues in the future.

cancer biology↗