Engrailed 1 Research Articles

TGFβ-induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient-derived organoid model.

Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFβ signaling pathway was found associated with ACC, and the regulation of EN1 through TGFβ was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFβ-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFβ1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFβ1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFβ-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.

Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

Engrailed: Pathological and physiological effects of a multifunctional developmental gene.

Engrailed-1 (EN1) is a developmental gene that encodes En1, a highly conserved transcription factor involved in regionalization during early embryogenesis and in the later maintenance of normal neurons. After birth, EN1 still plays a role in the development and physiology of the body; for example, it exerts a protective effect on midbrain dopaminergic (mDA) neurons, and loss of EN1 causes mDA neurons in the ventral midbrain to gradually die approximately 6 weeks after birth, resulting in motor and nonmotor symptoms similar to those observed in Parkinson's disease. Notably, EN1 has been identified as a possible susceptibility gene for idiopathic Parkinson's disease in humans. EN1 is involved in the processes of wound-healing scar production and tissue and organ fibrosis. Additionally, EN1 can lead to tumorigenesis and thus provides a target for the treatment of some tumors. In this review, we summarize the effects of EN1 on embryonic organ development, describe the consequences of the deletion or overexpression of the EN1 gene, and discuss the pathways in which EN1 is involved. We hope to clarify the role of EN1 as a developmental gene and present potential therapeutic targets for diseases involving the EN1 gene.

Single-cell and bulk tissue sequencing unravels the heterogeneity of synovial microenvironment in arthrofibrosis

Arthrofibrosis (AF) is a debilitating complication that occurs after trauma or surgery, leading to functional impairment and surgical failures worldwide. This study aimed to uncover the underlying mechanism of AF. A total of 141 patients were enrolled, and synovial samples were collected from both patients and animal models at different time points. Single-cell RNA-sequencing (scRNA-seq) and bulk tissue RNA sequencing (bulk-seq) were employed to profile the distinct synovial microenvironment. This study revealed changes in cell proportions during AF pathogenesis and identified Engrailed-1 (EN1) as a key transcription factor strongly associated with disease severity and clinical prognosis. Additionally, the researchers discovered a specific type of synovial fibroblast called DKK3-SLF, which played a critical role in driving AF development. These findings shed light on the composition and heterogeneity of the synovial microenvironment in AF, offering potential avenues for identifying therapeutic targets and developing clinical treatments for AF and other fibrotic diseases.

Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development.

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

Placental stem cells-derived exosomes stimulate cutaneous wound regeneration via engrailed-1 inhibition.

Introduction: Skin wounds generally heal by scarring, a fibrotic process mediated by the Engrailed-1 (EN1) fibroblast lineage. Scar is detrimental to tissue structure and function, but perfect healing in clinical settings remains to be explored. Recent studies have shown that mesenchymal stem cell (MSC) transplantation can reduce scarring Methods: Here, we investigated the effects of placental MSCs (pMSCs) and exosomes derived from pMSCs (pMSC-exos) on wound healing using a full-thickness rat model. Results: The results showed that placental MSCs significantly accelerated the wound healing rate. Moreover, placental MSCs improved the quality of wound healing, including regenerating cutaneous appendages (hair follicles and sebaceous glands), decreasing collagen I and increasing collagen III, and improving collagen pattern (basket-wave-like) in the healed skin. placental MSCs treatment also increased the regeneration of blood vessels. Importantly, all these listed effects of placental MSCs were comparable to those of exosomes derived from pMSCs, but significantly stronger than those of adipose MSC-derived exosomes (aMSC-exos). Further studies showed that the effects of placental MSCs and exosomes derived from pMSCs on wound regeneration may be mainly achieved via the down-regulation of the Yes-associated protein signaling pathway, thus inhibiting the activation of EN1. Discussion: In summary, placental MSCs could effectively stimulate wound regeneration, and their effect could be achieved through their exosomes. This suggests that exosomes derived from pMSCs treatment could be used as a novel cell-free approach to induce wound regeneration in clinical settings.

EN1 Regulates Cell Growth and Proliferation in Human Glioma Cells via Hedgehog Signaling.

Glioblastoma is an aggressive cancer of the nervous system that accounts for the majority of brain cancer-related deaths. Through cross-species transcriptome studies, we found that Engrailed 1 (EN1) is highly expressed in serum-free cultured glioma cells as well as glioma tissues, and increased expression level predicts a worse prognosis. EN1 controls glioma cell proliferation, colony formation, migration, and tumorigenic capacity in vivo. It also influences sensitivity of glioma cells to γ-ray irradiation by regulating intracellular ROS levels. Mechanistically, EN1 influences Hedgehog signaling by regulating the level of Gli1 as well as primary cilia length and the primary cilia transport-related protein TULP3. In conclusion, we demonstrate that EN1 acts as an oncogenic regulator that contributes to glioblastoma pathogenesis and could serve as a diagnostic/prognostic marker and therapeutic target for glioblastoma.

Weighted gene co-expression network analysis of the association between upregulated AMD1, EN1 and VGLL1 and the progression and poor prognosis of breast cancer.

Breast cancer is the most prevalent malignancy among females, but the molecular mechanisms involved in its pathogenesis and progression have remained to be fully elucidated. The aim of the present study was to identify novel potential therapeutic targets for breast cancer. The dataset GSE76275 was downloaded from the Gene Expression Omnibus database and weighted gene co-expression network analysis (WGCNA) was performed to identify hub genes. Furthermore, the dataset GSE25055, containing gene expression data and clinical information, was downloaded to validate the expression and survival association of these hub genes. In addition, the datasets GSE25065 and GSE42568 were used to validate the association between hub gene expression levels and clinical features. Immunohistochemistry (IHC), reverse transcription-quantitative PCR, as well as proliferation, migration, invasion and apoptosis assays, were used to verify gene expression and function. A total of 4,052 genes were selected for WGCNA and 18 modules were established; the red module was identified as the key module, as it had a strong positive correlation with the tumor grade. Survival analyses of hub genes [S-adenosylmethionine decarboxylase proenzyme (AMD1), homeobox protein engrailed-1 (EN1) and vestigial-like protein (VGLL1)] indicated that higher levels of gene expression were associated with poor prognosis of patients with breast cancer. This association was based on survival analysis of GSE25055 using the Kaplan-Meier plotter tool. Expression validation revealed that the upregulation of hub genes was associated with advanced tumor grade and malignant molecular subtype (basal-like). IHC results from the Human Protein Atlas also demonstrated that protein expression levels of the hub genes were higher in tumor tissues compared with those in adjacent normal tissues. Furthermore, the expression levels of AMD1, EN1 and VGLL1 were strongly correlated with each other. These results demonstrated that AMD1 is highly expressed in breast cancer tissues and cells and AMD1 knockdown decreased the proliferation and metastatic potential, while increasing apoptosis of breast cancer cells. These results suggested that AMD1, EN1 and VGLL1 are likely to contribute to breast cancer progression and unfavorable prognosis.

Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation.

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.

Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution

Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. Here, we show that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, we identified a human EN1 skin enhancer, hECE18. We showed that multiple epistatically interacting derived substitutions in the human ECE18 enhancer increased its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. Our study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.

Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use.

Human pluripotent stem cells show considerable promise for applications in regenerative medicine, including the development of cell replacement paradigms for the treatment of Parkinson's disease. Protocols have been developed to generate authentic midbrain dopamine (mDA) neurons capable of reversing dopamine-related deficits in animal models of Parkinson's disease. However, the generation of mDA neurons at clinical scale suitable for human application remains an important challenge. Here, we present an mDA neuron derivation protocol based on a two-step WNT signaling activation strategy that improves expression of midbrain markers, such as Engrailed-1 (EN1), while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. The resulting neurons exhibit molecular, biochemical, and electrophysiological properties of mDA neurons. Cryopreserved mDA neuron precursors can be successfully transplanted into 6-hydroxydopamine (6OHDA) lesioned rats to induce recovery of amphetamine-induced rotation behavior. The protocol presented here is the basis for clinical-grade mDA neuron production and preclinical safety and efficacy studies.

Electrochemically detecting DNA methylation in the EN1 gene promoter: implications for understanding ageing and disease.

There is a growing need for biomarkers which predict age-onset pathology. Although this is challenging, the methylome offers significant potential. Cancer is associated with the hypermethylation of many gene promoters, among which are developmental genes. Evolutionary theory suggests developmental genes arbitrate early-late life trade-offs, causing epimutations that increase disease vulnerability. Such genes could predict age-related disease. The aim of this work was to optimise an electrochemical procedure for the future investigation of a broad range of ageing-related pathologies. An electrochemical approach, which adopted three analytical techniques, was used to investigate DNA methylation in the engrailed-1 (EN1) gene promoter. Using synthetic single-stranded DNA, one technique was able to detect DNA at concentrations as low as 10 nM, with methylation status distinguishable at concentrations >25 nM. A negative correlation could be observed between % methylation of a heterogeneous solution and the key electrochemical parameter, charge transfer resistance (Rct; r = −0.982, P<0.01). The technique was applied to the breast cancer cell line Michigan Cancer Foundation-7 (MCF-7), where a similar correlation was observed (r = −0.965, P<0.01). These results suggest electrochemistry can effectively measure DNA methylation at low concentrations of DNA. This has implications for the future detection of age-related disease.

Opposing Functions of BRD4 Isoforms in Breast Cancer

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

Abstract B25: Engrailed-1 promotes pancreatic cancer progression via antagonizing COMPASS activity

Abstract Pancreatic ductal adenocarcinoma (PDA) is the most deadly disease in human malignancies. Using pancreatic organoid models, we have shown that enhancer reprogramming allows pancreatic cancer cells to acquire aggressive characteristics during disease progression. Recently we found that organoid cultures (mM) derived from metastatic lesions of KPC mice (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) can survive in the reduced condition, unlike the paired primary tumor-derived organoid cultures (mT). We implicate Engrailed-1 (EN1), a neurodevelopmental transcription factor (TF), in this process as a prosurvival factor. We show that EN1 is indispensable for organoid survival in the reduced condition, highly expressed in mM compared to mT organoid cultures, and associated with poor prognosis. Gain- and loss-of-function experiments further show that EN1 is responsible for the aggressiveness of pancreatic cancer cells in vitro (anchorage-independent growth, migration, and invasion) and in vivo. On the other hand, subcutaneous tumors derived from EN1 expressing mT organoids exhibit squamous-like differentiation compared to control. In line with this, squamous-type PDA patients tend to express EN1 compared to other subtypes of PDA. Furthermore, EN1 perturbation renders the changes of squamous-PDA identity and KDM6A deficiency signatures. Mechanistically, EN1 can physically interact with KDM6A and repress H3K4me3 occupancy in a subset of genes without affecting H3K27me3 and H3K27ac occupancies, suggesting that the regulation of COMPASS (Complex Proteins Associated with Set1) activity by EN1 might be critical in this process. Finally, by combining EN1 with FOXA1, previously shown to be responsible for enhancer activation in PDA organoids, we were able to fully reprogram pancreatic cancer cells into highly aggressive pancreatic cancer cells. In sum, we report that EN1 confers the aggressiveness of pancreatic cancer cells via regulation of COMPASS activity and contributes to squamous-type identity and PDA progression. Citation Format: Chang-il Hwang, Jae-Seok Roe, Eun Jung Lee, Claudia Tonelli, Tim D.D. Somervile, Melissa Yao, Joseph P. Milazzo, Hervé Tiriac, Youngkyu Park, Christopher Vakoc, David Tuveson. Engrailed-1 promotes pancreatic cancer progression via antagonizing COMPASS activity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B25.

EN1 Is a Transcriptional Dependency in Triple-Negative Breast Cancer Associated with Brain Metastasis.

To define transcriptional dependencies of triple-negative breast cancer (TNBC), we identified transcription factors highly and specifically expressed in primary TNBCs and tested their requirement for cell growth in a panel of breast cancer cell lines. We found that EN1 (engrailed 1) is overexpressed in TNBCs and its downregulation preferentially and significantly reduced viability and tumorigenicity in TNBC cell lines. By integrating gene expression changes after EN1 downregulation with EN1 chromatin binding patterns, we identified genes involved in WNT and Hedgehog signaling, neurogenesis, and axonal guidance as direct EN1 transcriptional targets. Quantitative proteomic analyses of EN1-bound chromatin complexes revealed association with transcriptional repressors and coactivators including TLE3, TRIM24, TRIM28, and TRIM33. High expression of EN1 correlated with short overall survival and increased risk of developing brain metastases in patients with TNBC. Thus, EN1 is a prognostic marker and a potential therapeutic target in TNBC. SIGNIFICANCE: These findings show that the EN1 transcription factor regulates neurogenesis-related genes and is associated with brain metastasis in triple-negative breast cancer.

Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1-iPeps and RGD peptides

Triple negative breast cancers (TNBC) are aggressive malignancies for which chemotherapy is the only treatment option. Many TNBC acquire chemotherapy resistance, notably docetaxel, which has been associated with the overexpression of transcription factors (TFs), such as ENGRAILED1 (EN1). Here, we have developed a tumor delivery system for docetaxel-PGMA-PAA-nanoparticles and interference peptides designed to specifically inhibit EN1 (EN1-iPeps). To promote tumor specific targeting, we functionalized these nanoparticles with EN1-iPeps engineered with RGD sequences. We found that these peptides reduce cell viability and induce apoptosis in TNBC cells with negligible effects on normal cells (EN1−). Moreover, EN1-RGD-iPeps-mediated nanoparticle internalization into breast cancer cells was via integrins and intravenous injection of this nanoformulation increased tumor accumulation. Furthermore, docetaxel nanoparticles functionalized with EN1-RGD-iPeps significantly reduced TNBC growth both in vitro and in vivo without showing toxicity. Our results suggest that this targeted nanoformulation represents a new and safe therapeutic approach for chemoresistant TNBCs.

Engrailed 1 overexpression as a potential prognostic marker in quintuple-negative breast cancer

ABSTRACTTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further categorized as either basal-like (BLBC) or quintuple-negative breast cancer (QNBC). In the present study, we aimed to identify novel molecular therapeutic targets for TNBC by analyzing the mRNA expression of TNBC-related genes in publicly available microarray data sets. We found that Engrailed 1 (EN1) was significantly overexpressed in TNBC. Using breast cancer cell lines, we found that EN1 was more highly expressed in TNBC than in other breast cancer subtypes. EN1 expression was analyzed in 199 TNBC paraffin-embedded tissue samples by immunohistochemistry. EN1 protein expression was positively associated with reduced overall survival (OS) rate in patients with QNBC, but not those with BLBC. The importance of EN1 expression in QNBC cell viability and tumorigenicity was evaluated using the QNBC cell lines, HCC38 and HCC1395. Based on our data, EN1 may promote the proliferation, migration, and multinucleation of QNBC cells, likely via the transcriptional activation of HDAC8, UTP11L, and ZIC3. We also demonstrated that actinomycin D effectively inhibits EN1 activity in QNBC cells. The results of the present study suggest that EN1 activity is highly clinically relevant to the survival prognosis of patients with QNBC and EN1 is a promising potential therapeutic target for future QNBC treatment.

APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors.

Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71-80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms.

In-depth characterization of the salivary adenoid cystic carcinoma transcriptome with emphasis on dominant cell type.

Adenoid cystic carcinoma (ACC), 1 of the most common salivary gland malignancies, arises from the intercalated ducts, which are composed of inner ductal epithelial cells and outer myoepithelial cells. The objective of this study was to determine the genomic subtypes of ACC with emphasis on dominant cell type to identify potential specific biomarkers for each subtype and to improve the understanding of this disease. A whole-genome expression study was performed based on 42 primary salivary ACCs and 5 normal salivary glands. RNA from these specimens was subjected to expression profiling with RNA sequencing, and results were analyzed to identify transcripts in epithelial-dominant ACC (E-ACC), myoepithelial-dominant ACC (M-ACC), and all ACC that were expressed differentially compared with the transcripts in normal salivary tissue. In total, the authors identified 430 differentially expressed transcripts that were unique to E-ACC, 392 that were unique to M-ACC, and 424 that were common to both M-ACC and E-ACC. The sets of E-ACC-specific and M-ACC-specific transcripts were sufficiently large to define and differentiate E-ACC from M-ACC. Ingenuity pathway analysis identified known cancer-related genes for 60% of the E-ACC transcripts, 69% of the M-ACC transcripts, and 68% of the transcripts that were common in both E-ACC and M-ACC. Three sets of highly expressed candidate genes-distal-less homeobox 6 (DLX6) for E-ACC; protein keratin 16 (KRT16), SRY box 11 (SOX11), and v-myb avian myeloblastosis viral oncogene homolog (MYB) for M-ACC; and engrailed 1 (EN1) and statherin (STATH), which are common to both E-ACC and M-ACC)-were further validated at the protein level. The current results enabled the authors to identify novel potential therapeutic targets and biomarkers in E-ACC and M-ACC individually, with the implication that EN1, DLX6, and OTX1 (orthodenticle homeobox 1) are potential drivers of these cancers. Cancer 2016;122:1513-22. © 2016 American Cancer Society.

The Engrailed-1 Gene Stimulates Brown Adipogenesis.

As a thermogenic organ, brown adipose tissue (BAT) has received a great attention in treating obesity and related diseases. It has been reported that brown adipocyte was derived from engrailed-1 (EN1) positive central dermomyotome. However, functions of EN1 in brown adipogenesis are largely unknown. Here we demonstrated that EN1 overexpression increased while EN1 knockdown decreased lipid accumulation and the expressions of key adipogenic genes including PPARγ2 and C/EBPα and mitochondrial OXPHOS as well as BAT specific marker UCP1. Taken together, our findings clearly indicate that EN1 is a positive regulator of brown adipogenesis.