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The aryl hydrocarbon receptor (AHR) was initially identified as a receptor that bound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental toxicants; however, there is increasing evidence that the AHR is an important new drug target for treating multiple diseases including breast cancer. Treatment of estrogen receptor (ER)-negative MDA-MB-231 and BT474 breast cancer cells with TCDD or the selective AHR modulator 6-methyl-1,3,-trichlorodibenzofuran (MCDF) inhibited breast cancer cell invasion in a Boyden chamber assay. These results were similar to those previously reported for the antimetastic microRNA-335 (miR-335). Both TCDD and MCDF induced miR-335 in MDA-MB-231 and BT474 cells and this was accompanied by downregulation of SOX4, a miR-335-regulated (inhibited) gene. The effects of TCDD and MCDF on miR-335 and SOX4 expression and interactions of miR-335 with the 3′-UTR target sequence in the SOX4 gene were all inhibited in cells transfected with an oligonucleotide (iAHR) that knocks down the AHR, thus confirming AHR-miR-335 interactions. MCDF (40 mg/kg/d) also inhibited lung metastasis of MDA-MB-231 cells in a tail vein injection model, showing that the AHR is a potential new target for treating patients with ER-negative breast cancer, a disease where treatment options and their effectiveness are limited. Mol Cancer Ther; 11(1); 108–18. ©2011 AACR.

Supplementary Figures 1-5 from Aryl Hydrocarbon Receptor Agonists Induce MicroRNA-335 Expression and Inhibit Lung Metastasis of Estrogen Receptor Negative Breast Cancer Cells

Tooth development starts with formation of the dental lamina, a localized thickening of the maxillary and mandibular epithelium, at specific location of oral cavity. How this dental lamina is specified within the continuous naïve maxillary and mandibular epithelium, remains as a critical and unresolved question. To identify potential genes and transcriptional regulatory networks that controlling dental lamina formation, we utilized single-cell Multiome-seq and Laser Microdissection coupled RNA-seq to profile gene expression and open chromatin of mandibular epithelium during tooth initiation. We comprehensively identified transcription factors (TFs) and signaling pathways that are differentially expressed among different domains (including dental lamina) along dorsal-ventral axis of mandibular epithelium. Specifically, we foundSox2andTfap2a/Tfap2bforming two complementary domains along the dorsal-ventral axis of mandibular epithelium. Furthermore, dental lamina specific or enriched TFs such asPitx1, Pitx2andZfp536are presented around the interface ofSox2andTfap2a/Tfap2b. We also identified correlation between signaling pathways and domain specific TFs. Through computational analysis of single-cell Multiome-seq dataset, we identified potential key/driver TFs and core transcriptional regulatory networks (TRNs) for lineages (oral, dental, skin) specification during tooth initiation. Our computational analysis also reveals a potential cross repression interactions between different groups of TFs (i.e.Pitx1/Pitx2andTfap2a/Tfap2b) that might be essential for dental lamina specification.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here, we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a β cell- and neural cell-enriched lncRNA TCL1 Upstream Neural Differentiation-Associated RNA (TUNAR, also known as TUNA, HI-LNC78, or LINC00617). We named this highly conserved 48-amino-acid micropeptide beta cell- and neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localizes at the endoplasmic reticulum (ER) in pancreatic β cells. Overexpression of BNLN lowered ER calcium levels, maintained ER homeostasis, and elevated glucose-stimulated insulin secretion in pancreatic β cells. We further assessed the BNLN expression in islets from mice fed a high-fat diet and a regular diet and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic β cell functions and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes., Graphical abstract, In this study, we identified one islet lncRNA, TUNAR, contains a sORF encoding for micropeptide BNLN. We further demonstrated that BNLN regulates insulin secretion in the pancreatic β cells in health and diseases. Our study provides important insights into the molecular mechanisms that underlie the fine-tuning of glucose-induced insulin secretion by translational function of lncRNA.

Obesity is the major risk factor for nonalcoholic fatty liver disease (NAFLD), for which effective cures are lacking. Despite the notion that obesity is associated with aberrant levels and action of pituitary hormones that are essential for maintaining hepatic metabolic and inflammatory states, the intrinsic pituitary endocrine abnormalities and their systemic consequences are incompletely defined. By characterizing the impact of diet-induced obesity (DIO) on the pituitary whole tissue and single cell transcriptome, we demonstrated that obesity disrupts pituitary endoplasmic reticulum (ER) homeostasis by suppressing the inositol-requiring enzyme-α (IRE1α)-mediated adaptive unfolded protein response (UPR). We further showed that defective pituitary UPR by IRE1α-deletion in the anterior pituitary strikingly augmented obesity-associated systemic metabolic abnormalities, particularly the NAFLD-associated pathologies. Conversely, enhancing the adaptive UPR in the anterior pituitary, by genetic gain-of-function of spliced X-box binding protein 1 (sXBP1), ameliorated the systemic and hepatic metabolic defects observed in mice with pituitary IRE1α deletion. Intriguingly, disruption of the UPR in the pituitary resulted in impaired hepatic UPR, which was in part due to a defective thyroid hormone receptor (THR)-mediated activation of hepatic Xbp1. In contrast, activation of the hepatic THR signaling improved obesity-associated glucose intolerance and attenuated the impaired hepatic ER homeostasis in anterior pituitary-IRE1α deficient mice. Together, our study provides the first insight into disruption of endocrine signaling-mediated inter-organ UPR communication drives obesity-associated hepatic pathologies. Unraveling these connections might uncover new therapeutic targets for NAFLD and other obesity-associated diseases.

Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyzes of both primary hepatocytes and whole livers further showed that sXBP1 occupies the −743 to −523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction. Abbreviations: AAV8: adeno-associated virus serotype 8; ACTB: actin, beta; ANOVA: analysis of variance; ATF6: activating transcription factor-6; ATG: autophagy related; BECN1: beclin 1; BMI: body mass index; ChIP: chromatin immunoprecipitation; CLEAR: coordinated lysosomal expression and regulation; Cre: cre recombinase; DIO: diet-induced obesity; EBSS: Earle’s balanced salt solution; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HFD: high-fat diet; h: hours; HSCs: hepatic stellate cells; INS: insulin; L/A: ammonium chloride and leupeptin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; RD: regular diet; RFP: red fluorescent protein; SERPINA7/TBG: serpin family A member 7; SQSTM1/p62: sequestome 1; sXbp1 LOE: liver-specific overexpression of spliced Xbp1; TFEB: transcription factor EB; TG: thapsigargin; TN: tunicamycin; UPR: unfolded protein response; wks: weeks; WT: wild type; XBP1: X-box binding protein 1; xbp1 LKO: liver-specific Xbp1 knockout.

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre/Pitx2flox/flox (Pitx2cKO) embryos, and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, decreases progenitor cell proliferation, and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers, the initiation knot (IK) and enamel knot (EK). The loss of Pitx2 disrupted the patterning of these two signaling centers resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef-1 and Irx1.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNA (IncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a β-cell- and neural cell-enriched lncRNA TUNAR (also known as TUNA, HI-LNC78 or LINC00617). We named this highly conserved 48-amino-acid micropeptide Beta cell- and Neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localized at the endoplasmic reticulum in pancreatic β-cells. Overexpression of BNLN lowered ER calcium levels, increased cytosolic calcium levels, and maintained ER homeostasis in response to high glucose challenge. To determine the physiological and pathological roles of BNLN, we assessed the BNLN expression in islets from mice fed with a high-fat diet and a regular diet, and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN elevated glucose-stimulated insulin secretion in INS-1 cells. Lastly, BNLN overexpression enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic β-cell function and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.

Chemotherapy-induced diarrhea (CID), with clinical high incidence, adversely affects the efficacy of cancer treatment and patients’ quality of life. Our study demonstrates that the citrus flavonoid hesperetin (Hst) has a superior potential as a new agent to prevent and alleviate CID. In the animal model for irinotecan (CPT-11) induced CID, Hst could selectively inhibit intestinal carboxylesterase (CES2) and thus reduce the local conversion of CPT-11 to cytotoxic SN-38 which causes intestinal toxicity. Oral administration of Hst manifested an excellent anti-diarrhea efficacy, prohibiting 80% of severe and 100% of mild diarrhea in the CPT-11 administered tumor-bearing mice. In addition, a significant attenuation of intestinal inflammation contributed to the anti-diarrhea effect of Hst. Moreover, Hst was found to work synergistically with CPT-11 in tumor inhibition by suppressing the tumor's STAT3 activity and recruiting tumoricidal macrophages into the tumor microenvironment. The anti-intestinal inflammation and anti-STAT3 properties of Hst would contribute its broad benefits to the management of diarrhea caused by other chemo or targeted agents, and more importantly, enhance and reinforce the anti-tumor effects of these agents, to improve patient outcomes.

Introduction The development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies. Methods Gene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry. Results Gene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations. Conclusion Our study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. Disclosures No relevant conflicts of interest to declare.

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1−/− mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.

The role that noncoding regions of the genome play in the etiology of cleft palate is not well studied. A novel method of microRNA (miR) inhibition that allows for specific miR knockdown in vivo has been developed by our laboratory. To further understand the role of miRs in palatogenesis, we used a new mouse model to inhibit specific miRs within the miR-17-92 cluster. Transgenic mice expressing inhibitory complexes for miR-17 and miR-18 manifested a clefting phenotype that was distinct from that observed in mice carrying inhibitory complexes for miR-17, miR-18, miR-19, and miR-92. An in silico candidate gene analysis and bioinformatics review led us to identify TGFBR2 as a likely target of miR-17 and miR-19 family members. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TGFBR1 and TGFBR2 expression levels were elevated in the palates of these miR transgenic embryos at embryonic day 15.5. RT-PCR data also showed that the expression of mature miRs from the miR-17-92 cluster was significantly decreased in the transgenic embryos. Decreased expression of TGFB pathway signaling ligands was also observed. Experiments in cells showed that inhibition of miR-17 and miR-18 was sufficient to induce increases in expression of TGFB receptors, while a concomitant decrease in TGFB signaling ligands was not observed. RT-PCR of mature miR-17-92 in cells demonstrated the selectivity and specificity of inhibitory complexes. While this study builds on previous studies that have implicated miR-17-92 in the regulation of important molecular components of the TGFB signaling pathway, it is likely that interactions remain to be elucidated between miR-17-92 and as-of-yet unidentified molecules important for the control of palatogenesis. The differential regulation of palatogenesis by members of the miR-17-92 cluster indicates that several gene combinations regulate palate elevation and extension during development.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2(−/−) embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5’ upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance. Here, we show that a fluorescent reporter called SORE6 can identify SOX2/OCT4-overexpressing prostate cancer cells. Among tumor cells, the SORE6 reporter identified a small fraction with CSC hallmarks: rapid self-renewal, the capability to form tumors and metastasize, and resistance to chemotherapies. Transcriptome and biochemical analyses identified PI3K/AKT signaling as critical for maintaining the SORE6+ population. Moreover, a SORE6-driven herpes simplex virus thymidine kinase (TK) expression construct could selectively ablate SORE6+ cells in tumors, blocking tumor initiation and progression, and sensitizing tumors to chemotherapy. This study demonstrates a key role of SOX2/OCT4-associated prostate cancer stem cells in tumor development and therapeutic resistance, and identifies the SORE6 reporter system as a useful tool for characterizing CSCs functions in a native tumor microenvironment.

Accumulating evidence demonstrates that certain types of long-noncoding RNAs (lncRNA) encode very short open reading frames, called micropeptides. By regulating larger protein complexes, these micropeptides can regulate fundamental biological processes such as skeletal muscle development and cell migration. However, the ability of micropeptides to maintain cellular and metabolic homeostasis in islets is unknown. Using a bioinformatics approach, we comprehensively analyzed the protein-coding potential of lncRNAs that are expressed in the human pancreas, and identified more than 30 that likely encode micropeptides. We further characterized one conserved IncRNA that encodes a 65-amino-acid micropeptide, which we named beta cell glucose regulated micropeptide 1 (BGRM1). We found that BGRM1 expression was altered in islets from obese or diabetic humans and mice. Overexpression of BGRM1 elevated glucose-stimulated insulin production in INS-1 cells and in islets from humans and mice. BGRM1 promotes endoplasmic reticulum (ER) calcium efflux and enhances cytosolic calcium levels, which suggests it mediates insulin production is in part by modulating intracellular calcium homeostasis. This notion is further supported by our mass spectra analysis that reveals several BGRM1 binding partners involved in ER calcium homeostasis in beta cells. Notably, BGRM1 is localized in the ER and protects beta cells from ER stress. Taken together, our study provides the first evidence that lncRNA encoded micropeptides play a critical role in pancreatic beta cell function. These results provide a foundation on which to perform future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes. Disclosure M. Li: None. F. Shao: None. M.E. Sweat: None. Q. Qian: None. Y. Guo: None. S.B. Stephens: None. Y. Imai: None. L. Yang: None. H. Cao: None.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.

Background Treatment failures in cancers, including multiple myeloma (MM), are most likely due to the persistence of a minor population of tumor-initiating cells (TICs), which are noncycling or slowly cycling and very drug resistant. Methods Gene expression profiling and real-time quantitative reverse transcription polymerase chain reaction were employed to define genes differentially expressed between the side-population cells, which contain the TICs, and the main population of MM cells derived from 11 MM patient samples. Self-renewal potential was analyzed by clonogenicity and drug resistance of CD24+ MM cells. Flow cytometry (n = 60) and immunofluorescence (n = 66) were applied on MM patient samples to determine CD24 expression. Therapeutic effects of CD24 antibodies were tested in xenograft MM mouse models containing three to six mice per group. Results CD24 was highly expressed in the side-population cells, and CD24+ MM cells exhibited high expression of induced pluripotent or embryonic stem cell genes. CD24+ MM cells showed increased clonogenicity, drug resistance, and tumorigenicity. Only 10 CD24+ MM cells were required to develop plasmacytomas in mice (n = three of five mice after 27 days). The frequency of CD24+ MM cells was highly variable in primary MM samples, but the average of CD24+ MM cells was 8.3% after chemotherapy and in complete-remission MM samples with persistent minimal residual disease compared with 1.0% CD24+ MM cells in newly diagnosed MM samples (n = 26). MM patients with a high initial percentage of CD24+ MM cells had inferior progression-free survival (hazard ratio [HR] = 3.81, 95% confidence interval [CI] = 5.66 to 18.34, P < .001) and overall survival (HR = 3.87, 95% CI = 16.61 to 34.39, P = .002). A CD24 antibody inhibited MM cell growth and prevented tumor progression in vivo. Conclusion Our studies demonstrate that CD24+ MM cells maintain the TIC features of self-renewal and drug resistance and provide a target for myeloma therapy.

Background & Aims The lysosome is an acidic organelle that is important for maintaining cellular and metabolic homeostasis in hepatocytes. Lysosomal dysfunction and chronic inflammation coexist, and both contribute to obesity-associated hepatic insulin resistance. However, in the context of obesity, the interplay between inflammatory signals and hepatic lysosomal function remains largely unknown. Inducible nitric oxide synthase (iNOS) is a hallmark for inflammation, and is activated in obesity. The aim of this study is to understand the molecular link between iNOS-mediated lysosomal nitric oxide (NO) production, hepatic lysosomal function, and autophagy in the context of obesity-associated insulin resistance. Methods The role of iNOS in hepatic autophagy, as related to insulin and glucose homeostasis were studied in mice with diet-induced obesity (DIO). The effects and mechanisms of iNOS-mediated lysosomal NO production on lysosomal function and hepatic autophagy were studied in primary hepatocytes as well as in a mouse model of DIO. Results We demonstrate that obesity promotes iNOS localization to the lysosome and decreases levels of lysosomal arginine, resulting in an accumulation of NO in hepatic lysosomes. This lysosomal NO production is attenuated by treatment with a NO scavenger, while co-overexpression of mTOR and a lysosomal arginine transporter (SLC38A9) enhances lysosomal NO production and suppresses autophagy. In addition, we show that deletion of iNOS ameliorates lysosomal nitrosative stress in the livers of DIO mice, promotes lysosomal biogenesis by activating transcription factor EB (TFEB), and enhances lysosomal function and autophagy. Lastly, deletion of iNOS in mice with DIO improves hepatic insulin sensitivity, which is diminished by suppression of TFEB or autophagy related 7 (Atg7). Conclusions Our studies suggest that lysosomal iNOS-mediated NO signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.

Transforming growth factor-β (TGF-β) signaling plays fundamental roles in the development and homeostasis of somatic cells. Dysregulated TGF-β signaling contributes to cancer progression and relapse to therapies by inducing epithelial-to-mesenchymal transition (EMT), enriching cancer stem cells, and promoting immunosuppression. Although many TGF-β-regulated genes have been identified, only a few datasets were obtained by next-generation sequencing. In this study, we performed RNA-sequencing analysis of MCF10A cells and identified 1166 genes that were upregulated and 861 genes that were downregulated by TGF-β. Gene set enrichment analysis revealed that focal adhesion and metabolic pathways were the top enriched pathways of the up- and downregulated genes, respectively. Genes in these pathways also possess significant predictive value for renal cancers. Moreover, we confirmed that TGF-β induced expression of MICAL1 and 2, and the histone demethylase, KDM7A, and revealed their regulatory roles on TGF-β-induced cell migration. We also show a critical effect of KDM7A in regulating the acetylation of H3K27 on TGF-β-induced genes. In sum, this study identified novel effectors that mediate the pro-migratory role of TGF-β signaling, paving the way for future studies that investigate the function of MICAL family members in cancer and the novel epigenetic mechanisms downstream TGF-β signaling.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.

Many cancers, including multiple myeloma (MM), retain more cytosolic iron to promote tumor cell growth and drug resistance. Higher cytosolic iron promotes oxidative damage due to its interaction with reactive oxygen species generated by mitochondria. The variation of mitochondrial biogenesis in different stages of MM disease was evaluated using gene expression profiles in a large clinical dataset. Sixteen of 18mitochondrial biogenesis related gene sets, including mitochondrial biogenesis signature and oxidative phosphorylation, were increased in myeloma cells compared with normal plasma cells and high expression was associated with an inferior patient outcome. Relapsed and drug resistant myeloma samples had higher expression of mitochondrial biogenesis signatures than newly diagnosed patient samples. The expression of mitochondrial biogenesis genes was regulated by the cellular iron content, which showed a synergistic effect in patient outcome in MM. Pharmacological ascorbic acid induced myeloma cell death by inhibition of mitochondria oxidative phosphorylation in an in vivo model. Here, we identify that dysregulated mitochondrial biogenesis and iron homeostasis play a major role in myeloma progression and patient outcome and that pharmacological ascorbic acid, through cellular iron content and mitochondrial oxidative species, should be considered as a novel treatment in myeloma including drug-resistant and relapsed patients.

Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature miRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT+ progenitors and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT+ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT+ progenitor expansion during organogenesis.

Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.

Heterogeneity of genomic instabilities among individual patients is believed to be a major cause of drug response heterogeneity. Cancer patients who are sensitive to anti-cancer drugs are often re-examined to understand the unknown mechanism of action (MoA) of given drugs. For example, a non-small cell lung cancer (NSCLC) patient was reported to be responsive to Dasatinib treatment and remained cancer-free four years later. Though follow-up genomic analysis showed the patient bears an inactivating BRAF [1]mutation in the tumor, the MoA remains unclear. There are two challenges in uncovering the MoA. First, Dasatinib is a kinase inhibitor, which often has many protein targets. Second, the downstream MoA signaling pathways regulated by these targets are too complicated to delineate. Currently, there is no computational tool that can effectively address both challenges. To fill this gap, we developed a computational tool DrugMoaMiner (Drug MoA Miner) that can be used to identify the comprehensive set of kinase inhibitor targets; delineate the underlying drug MoA; and predict personalized sensitivity to a given drug based on an individual's gene expression profiles. We applied the DrugMoaMiner to lung cancer cell lines to uncover the potential MoA signaling network of Dasatinib sensitivity; our result is in agreement with previous protein data analysis. Moreover, we can predict Dasatinib response of an independent set of NSCLC cell lines using the MoA signaling network uncovered.

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology., Author summary The basic question of how human faces develop, undergo morphogenesis and grow after birth to define our final characteristic shape has been studied from the earliest days of comparative vertebrate developmental research. While many studies have shown the factors and mechanisms that contribute to the cells and tissues of the face during embryology, fewer studies have determined mechanisms that promote face growth after birth and into childhood. In our quest to understand developmental mechanisms of facial growth we used murine gene expression and bioinformatics analyses combined with human 3D facial variations and genome-wide association studies to identify genes and variants controlling post-natal face growth. Bioinformatics analyses of mouse craniofacial gene expression identified FoxO6 as a transcription factor expressed at late stages of face development. Ablation of FoxO6 in the mouse resulted in specific anterior growth of the mouse face. The increased FOXO6 expression activated Hippo signaling to reduce face growth. These data indicate that changes in FOXO6 expression control face growth during early childhood.

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

We created a cDNA microarray representing approximately 3,500 pig genes for functional genomic studies. The array elements were selected from 6,494 cDNA clones identified in a large-scale expressed sequence tag (EST) project. These cDNA clones came from normalized and subtracted porcine adipose tissue cDNA libraries. Sequence similarity searches of the 3,426 ESTs represented on the array using BLASTN identified 2,790 (81.4%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. We used the gene microarray to profile transcripts expressed by adipose tissue of fatty Chinese Xiang pig (XP) and muscley Large White (LW). Microarray analysis of RNA extracted from adipose tissue of fatty XP and muscley LW identified 81 genes that were differently expressed two fold or more. Transcriptional differences of four of these genes, adipocyte fatty acid binding protein (aP2), stearyl-CoA desaturase (SCD), sterol regulatory element binding transcription factor 1 (SREBF1) and lipoprotein lipase (LPL) were confirmed using SYBR Green quantitative RT-PCR technology. Our results showed that high expression of SCD and SREBF1 may be one of the reasons that larger fat deposits are observed in the XP. In addition, our findings also illustrate the potential power of microarrays for understanding the molecular mechanisms of porcine development, disease resistance, nutrition, fertility and production traits.

T-box transcription factor TBX1 is the major candidate gene for 22q11.2 deletion syndrome (22q11.2DS, DiGeorge syndrome/Velo-cardio-facial syndrome), whose phenotypes include craniofacial malformations such as dental defects and cleft palate. In this study, Tbx1 was conditionally deleted or over-expressed in the oral and dental epithelium to establish its role in odontogenesis and craniofacial developmental. Tbx1 lineage tracing experiments demonstrated a specific region of Tbx1-positive cells in the labial cervical loop (LaCL, stem cell niche). We found that Tbx1 conditional knockout (Tbx1(cKO)) mice featured microdontia, which coincides with decreased stem cell proliferation in the LaCL of Tbx1(cKO) mice. In contrast, Tbx1 over-expression increased dental epithelial progenitor cells in the LaCL. Furthermore, microRNA-96 (miR-96) repressed Tbx1 expression and Tbx1 repressed miR-96 expression, suggesting that miR-96 and Tbx1 work in a regulatory loop to maintain the correct levels of Tbx1. Cleft palate was observed in both conditional knockout and over-expression mice, consistent with the craniofacial/tooth defects associated with TBX1 deletion and the gene duplication that leads to 22q11.2DS. The biochemical analyses of TBX1 human mutations demonstrate functional differences in their transcriptional regulation of miR-96 and co-regulation of PITX2 activity. TBX1 interacts with PITX2 to negatively regulate PITX2 transcriptional activity and the TBX1 N-terminus is required for its repressive activity. Overall, our results indicate that Tbx1 regulates the proliferation of dental progenitor cells and craniofacial development through miR-96-5p and PITX2. Together, these data suggest a new molecular mechanism controlling pathogenesis of dental anomalies in human 22q11.2DS.

Patients with Axenfeld–Rieger Syndrome (ARS) present various dental abnormalities, including hypodontia, and enamel hypoplasia. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in early tooth development. However, because Pitx2 is also a major regulator of later stages of tooth development, especially during amelogenesis, it is unclear how mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel )e xpression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is controlled by chromatin-associated factor Hmgn2, and that Hmgn2 prevents Pitx2 from efficiently binding to and activating the Amel promoter. Consistent with a physiological significance to this interaction, we show that K14-Hmgn2 transgenic mice display a severe loss of Amel expression on the labial side of the lower incisors, as well as enamel hypoplasia—consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carryPITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in a novel mouse model of ARS.