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Gastric cancer is the second leading cause of cancer-related death in the world. The exact molecular pathways in gastric cancer need for further study. We herein indicated miR-26a performed converse roles on oncogenicity in different gastric cancer cells. In gastric cancer cells MKN-28, miR-26a promoted cell proliferation, migration and invasion. However, in gastric cancer cells AGS, miR-26a reduced cell proliferation and metastasis. PTEN was identified as a direct target of miR-26a. In MKN-28 cells, PTEN was suppressed by miR-26a through 3'-UTR, and PTEN mediated miR-26a promoting oncogenicity including cell proliferation and metastasis. On the other hand, in AGS cells, the expression of PTEN was enhanced by miR-26a, and PTEN mediated miR-26a reducing oncogenicity. The mechanism in AGS cells may be the indirect regulation of PTEN by miR-26a overcame the direct targeting regulation. The model like MKN-28 cells was concordant with patients with a high level of miR-26a and a low level of PTEN and patients with a low level of miR-26a and a high level of PTEN which showed lower overall survival (OS); the model like AGS cells was concordant with patients with both high level of miR-26a and PTEN and both low level of miR-26a and PTEN which showed higher OS. These findings will facilitate a better understanding of the functions and mechanisms about miR-26a, miR-26a and PTEN are potential combined biomarkers in patients with gastric cancer.

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer. Cancer Res; 71(20); 6463–74. ©2011 AACR.

Regulation of hematopoietic stem cells (HSCs) by bone marrow (BM) niches has been extensively studied; however, whether and how HSC subpopulations are distinctively regulated by BM niches remain unclear. Here, we functionally distinguished reserve HSCs (rHSCs) from primed HSCs (pHSCs) based on their response to chemotherapy and examined how they are dichotomously regulated by BM niches. Both pHSCs and rHSCs supported long-term hematopoiesis in homeostasis; however, pHSCs were sensitive but rHSCs were resistant to chemotherapy. Surviving rHSCs restored the HSC pool and supported hematopoietic regeneration after chemotherapy. The rHSCs were preferentially maintained in the endosteal region that enriches N-cadherin+ (N-cad+) bone-lining cells in homeostasis and post-chemotherapy. N-cad+ cells were functional bone and marrow stromal progenitor cells (BMSPCs), giving rise to osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Finally, ablation of N-cad+ niche cells or deletion of SCF from N-cad+ niche cells impaired rHSC maintenance during homeostasis and regeneration.

In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.

We have previously identified an oncogenic role of artemin (ARTN), a member of glial cell derived neurotrophic factor family of ligands, in mammary carcinoma. We herein report that ARTN is an estrogen-inducible gene. Meta-analysis of gene expression data sets showed that ARTN expression is positively correlated to estrogen receptor (ER) status in human mammary carcinoma. Furthermore, in patients with ER-positive mammary carcinoma treated with tamoxifen, high ARTN expression is significantly correlated with decreased survival. Forced expression of ARTN in ER-positive human mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. ARTN-stimulated resistance to tamoxifen and fulvestrant is mediated by increased BCL-2 expression. Conversely, depletion of endogenous ARTN by small-interfering RNA or functional antagonism of ARTN by antibody enhanced the efficacy of antiestrogens. Tamoxifen decreased ARTN expression in tamoxifen-sensitive mammary carcinoma cells whereas ARTN expression was increased in tamoxifen-resistant cells and not affected by tamoxifen treatment. Antibody inhibition of ARTN in tamoxifen-resistant cells improved tamoxifen sensitivity. Functional antagonism of ARTN therefore warrants consideration as an adjuvant therapy to enhance antiestrogen efficacy in ER-positive mammary carcinoma.

Here, we provide evidence for a functional role of artemin (ARTN) in progression of endometrial carcinoma (EC). Increased ARTN protein expression was observed in EC compared with normal endometrial tissue, and ARTN protein expression in EC was significantly associated with higher tumor grade and invasiveness. Forced expression of ARTN in EC cells significantly increased total cell number as a result of enhanced cell cycle progression and cell survival. In addition, forced expression of ARTN significantly enhanced anchorage-independent growth and invasiveness of EC cells. Moreover, forced expression of ARTN increased tumor size in xenograft models and produced highly proliferative, poorly differentiated, and invasive tumors. The ARTN-stimulated increases in oncogenicity and invasion were mediated by increased expression and activity of AKT1. Small interfering RNA-mediated depletion or antibody inhibition of ARTN significantly reduced oncogenicity and invasion of EC cells. Thus, inhibition of ARTN may be considered as a potential therapeutic strategy to retard progression of EC.

We report that artemin, a member of the glial cell line-derived neurotrophic factor family of ligands, is oncogenic for human mammary carcinoma. Artemin is expressed in numerous human mammary carcinoma cell lines. Forced expression of artemin in mammary carcinoma cells results in increased anchorage-independent growth, increased colony formation in soft agar and in three-dimensional Matrigel, and also promotes a scattered cell phenotype with enhanced migration and invasion. Moreover, forced expression of artemin increases tumor size in xenograft models and leads to highly proliferative, poorly differentiated and invasive tumors. Expression data in Oncomine indicate that high artemin expression is significantly associated with residual disease after chemotherapy, metastasis, relapse and death. Artemin protein is detectable in 65% of mammary carcinoma and its expression correlates to decreased overall survival in the cohort of patients. Depletion of endogenous artemin with small interfering RNA, or antibody inhibition of artemin, decreases the oncogenicity and invasiveness of mammary carcinoma cells. Artemin is therefore oncogenic for human mammary carcinoma, and targeted therapeutic approaches to inhibit artemin function in mammary carcinoma warrant consideration.

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the “panjunctional sarcoplasmic reticulum” hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Multiple bone marrow stromal cell types have been identified as hematopoietic stem cell (HSC)-regulating niche cells. However, whether HSC progeny can serve directly as HSC niche cells has not previously been shown. Here we report a dichotomous role of megakaryocytes (MKs) in both maintaining HSC quiescence during homeostasis and promoting HSC regeneration after chemotherapeutic stress. We show that MKs are physically associated with HSCs in the bone marrow of mice and that MK ablation led to activation of quiescent HSCs and increased HSC proliferation. RNA sequencing (RNA-seq) analysis revealed that transforming growth factor β1 (encoded by Tgfb1) is expressed at higher levels in MKs as compared to other stromal niche cells. MK ablation led to reduced levels of biologically active TGF-β1 protein in the bone marrow and nuclear-localized phosphorylated SMAD2/3 (pSMAD2/3) in HSCs, suggesting that MKs maintain HSC quiescence through TGF-β-SMAD signaling. Indeed, TGF-β1 injection into mice in which MKs had been ablated restored HSC quiescence, and conditional deletion of Tgfb1 in MKs increased HSC activation and proliferation. These data demonstrate that TGF-β1 is a dominant signal emanating from MKs that maintains HSC quiescence. However, under conditions of chemotherapeutic challenge, MK ablation resulted in a severe defect in HSC expansion. In response to stress, fibroblast growth factor 1 (FGF1) signaling from MKs transiently dominates over TGF-β inhibitory signaling to stimulate HSC expansion. Overall, these observations demonstrate that MKs serve as HSC-derived niche cells to dynamically regulate HSC function.

Human growth hormone (hGH) plays critical roles in pubertal mammary gland growth, development and sexual maturation. Accumulated studies reported that autocrine hGH is an orthotopically expressed o...

The contemporary use of nanomedicines for cancer treatment has been largely limited to serving as carriers for existing therapeutic agents. Here, we provide definitive evidence that, the metallofullerenol nanomaterial Gd@C82(OH)22, while essentially not toxic to normal mammary epithelial cells, possesses intrinsic inhibitory activity against triple-negative breast cancer cells. Gd@C82(OH)22 blocks epithelial-to-mesenchymal transition with resultant efficient elimination of breast cancer stem cells (CSCs) resulting in abrogation of tumour initiation and metastasis. In normoxic conditions, Gd@C82(OH)22 mediates these effects by blocking TGF-β signalling. Moreover, under hypoxic conditions found in the tumour microenvironment, cellular uptake of Gd@C82(OH)22 is facilitated where it functions as a bi-potent inhibitor of HIF-1α and TGF-β activities, enhancing CSC elimination. These studies indicate that nanomaterials can be engineered to directly target CSCs. Thus, Gd-metallofullerenol is identified as a kind of non-toxic CSC specific inhibitors with significant therapeutic potential., A metallofullerenol nanomaterial, Gd@C82(OH)22, was shown to inhibit growth of several solid cancers in preclinical models and yet exhibit low toxicity. Herein the authors show that Gd@C82(OH)22 functions as an inhibitor of breast cancer stem cell function via blocking TGF-β and HIF-1α signalling, while sparing normal tissue.

The balance between self-renewal and multilineage differentiation in hematopoietic stem cells (HSCs) is orchestrated by genetic regulatory networks, which are constituted by hundreds of thousands of cis-regulatory elements, such as promoters, enhancers, insulators, etc. Aberrant mutations in these cis-acting modules and their trans-acting factors have been frequently found in hematopoietic malignancies including leukemia. Although next-generation sequencing technologies have identified comprehensive maps of these modules, much remains unknown about their physiological functions and underlying mechanisms in HSC maintenance and leukemogenesis. Our transcriptome analysis in 17 murine hematopoietic cell types, including HSCs, committed progenitors and lineage cells, showed that most of HoxB cluster genes were predominantly enriched in the permanently reconstituting long-term (LT) HSCs. Interestingly, one of the two putative enhancers within HoxB cluster, identified by H3K27ac ChIP-Seq analysis, shared the same sequence as the retinoic acid responsive element DERARE, which was recently reported to regulate multiple HoxB gene expression in the central nervous system. To test whether DERARE is required for normal hematopoiesis, we utilized the DERARE knockout mouse and found that homozygous deletion of DERARE led to 2-fold reduction in both the frequency and absolute number of LT-HSCs. Functionally, limiting dilution, competitive repopulating unit assays showed a 2.5-fold decrease in functional HSCs of DERAREΔ mice compared to wildtype control. We further performed serial transplantation and observed a 4.3-fold reduction of repopulation rate after secondary transplantation of DERAREΔ HSCs, indicating long-term reconstitution capacity was impaired. Mechanistically, RNA-Seq in LT-HSCs from DERAREΔ mice exhibited significantly enriched apoptosis pathway in DERAREΔ compared with that from Wt control. Furthermore, DNA methylation analysis showed gradually gained methylation on DERARE during HSC differentiation, which is negatively correlated with HoxB cluster gene expression, suggesting DERARE might be a methylation-sensitive enhancer to control HoxB genes. Moreover, HoxB gene expression is markedly upregulated in Dnmt3a KO HSCs, indicating that Dnmt3a is responsible for the DERARE methylation in HSCs. Finally, the analysis of clinical data from acute myeloid leukemia 200 patients in the Cancer Genome Atlas project revealed that lowly methylated DERARE was significantly correlated with overexpression of HoxB genes, high cytogenetic risk, and poor prognosis, suggesting abnormal regulation of DERARE contributes to leukemogenesis. Collectively, our study demonstrates the essential roles of the cis-regulatory element DERARE in both maintenance of LT-HSCs and contribution to leukemogenesis through regulation of HoxB cluster genes. Disclosures No relevant conflicts of interest to declare.

Artemin (ARTN) has been reported to promote a TWIST1-dependent epithelial to mesenchymal transition of estrogen receptor negative mammary carcinoma (ER-MC) cells associated with metastasis and poor survival outcome. We therefore examined a potential role of ARTN in the promotion of the cancer stem cell (CSC)-like phenotype in mammary carcinoma cells. Acquired resistance of ER-MC cells to either ionizing radiation (IR) or paclitaxel was accompanied by increased ARTN expression. Small interfering RNA (siRNA)-mediated depletion of ARTN in either IR- or paclitaxel-resistant ER-MC cells restored cell sensitivity to IR or paclitaxel. Expression of ARTN was enriched in ER-MC cells grown in mammospheric compared with monolayer culture and was also enriched along with BMI1, TWIST1, and DVL1 in mammospheric and ALDH1+ populations. ARTN promoted mammospheric growth and self-renewal of ER-MC cells and increased the ALDH1+ population, whereas siRNA-mediated depletion of ARTN diminished these CSC-like cell behaviors. Furthermore, increased ARTN expression was significantly correlated with ALDH1 expression in a cohort of ER-MC patients. Forced expression of ARTN also dramatically enhanced tumor initiating capacity of ER-MC cells in xenograft models at low inoculum. ARTN promotion of the CSC-like cell phenotype was mediated by TWIST1 regulation of BCL-2 expression. ARTN also enhanced mammosphere formation and the ALDH1+ population in estrogen receptor-positive mammary carcinoma (ER+MC) cells. Increased expression of ARTN and the functional consequences thereof may be one common adaptive mechanism used by mammary carcinoma cells to promote cell survival and renewal in hostile tumor microenvironments.

A discontinuous pattern of LOH at chromosome 3p has been reported in 87% of primary breast cancers. Despite the identification of several tumor suppressor genes in this region, there has yet to be a detailed analysis of noncoding RNAs including miRNAs in this region. In this study, we identified 16 aberrant miRNAs in this region and determined several that are frequently lost or amplified in breast cancer. miR-128-2 was the most commonly deleted miRNA. Embedded in the intron of the ARPP21 gene at chromosome 3p22.3, miR-128-2 was frequently downregulated along with ARPP21 in breast cancer, where it was negatively associated with clinicopathologic characteristics and survival outcome. Forced expression of miR-128 impeded several oncogenic traits of mammary carcinoma cells, whereas depleting miR-128-2 expression was sufficient for oncogenic transformation and stem cell-like behaviors in immortalized nontumorigenic mammary epithelial cells, both in vitro and in vivo. miR-128-2 silencing enabled transforming capacity partly by derepressing a cohort of direct targets (BMI1, CSF1, KLF4, LIN28A, NANOG, and SNAIL), which together acted to stimulate the PI3K/AKT and STAT3 signaling pathways. We also found that miR-128-2 was directly downregulated by SNAIL and repressed by TGF-β signaling, adding 2 additional negative feedback loops to this network. In summary, we have identified a novel TGF-β/SNAIL/miR-128 axis that provides a new avenue to understand the basis for oncogenic transformation of mammary epithelial cells. Cancer Res; 72(22); 6036–50. ©2012 AACR.

The neurotrophic factor ARTEMIN (ARTN) has been reported to possess a role in mammary carcinoma progression and metastasis. Herein, we report that ARTN modulates endothelial cell behaviour and promotes angiogenesis in ER-mammary carcinoma (ER-MC). Human microvascular endothelial cells (HMEC-1) do not express ARTN but respond to exogenously added, and paracrine ARTN secreted by ER-MC cells. ARTN promoted endothelial cell proliferation, migration, invasion and 3D matrigel tube formation. Angiogenic behaviour promoted by ARTN secreted by ER-MC cells was mediated by AKT with resultant increased TWIST1 and subsequently VEGF-A expression. In a patient cohort of ER-MC, ARTN positively correlated with VEGF-A expression as measured by Spearman’s rank correlation analysis. In xenograft experiments, ER-MC cells with forced expression of ARTN produced tumors with increased VEGF-A expression and increased microvessel density (CD31 and CD34) compared to tumors formed by control cells. Functional inhibition of ARTN by siRNA decreased the angiogenic effects of ER-MC cells. Bevacizumab (a humanized monoclonal anti-VEGF-A antibody) partially inhibited the ARTN mediated angiogenic effects of ER-MC cells and combined inhibition of ARTN and VEGF-A by the same resulted in further significant decrease in the angiogenic effects of ER-MC cells. Thus, ARTN stimulates de novo tumor angiogenesis mediated in part by VEGF-A. ARTN therefore co-ordinately regulates multiple aspects of tumor growth and metastasis.

Introduction ARTEMIN (ARTN) is an estrogen regulated growth factor, the expression of which promotes resistance to antiestrogen therapies and predicts poorer survival outcome of patients with estrogen receptor (ER) positive mammary carcinoma (ER+MC) treated with tamoxifen. ARTN is also expressed in ER negative mammary carcinoma (ER-MC). Herein, we determined the role of ARTN in ER-MC and defined the mechanism of action producing poor patient prognosis. Methods We modulated the expression of ARTN in two ER- (mesenchymal/claudin-low) mammary carcinoma cell lines (BT549 and MDA-MB-231) by forced expression or small interfering RNA (siRNA) mediated depletion. The effects of modulation of ARTN expression were examined by various in vitro measures of oncogenicity, including the expression of TWIST1 messenger RNA (mRNA) and protein. In vitro results were correlated to xenograft studies in immunodeficient mice. Co-expression of ARTN and TWIST1 and their association to poor survival outcome were examined in a cohort of patients with ER-MC. Pathway analysis was performed by pharmacological inhibition of phosphorylation of AKT (pAKT-Ser 473) or modulation of TWIST1 expression. Results ARTN expression resulted in ER-MC cells with enhanced mesenchymal characteristics, including increased invasion and a gene expression profile consistent with enhanced mesenchymal phenotype. ARTN stimulated ER-MC cell anchorage independent and 3D matrigel growth, endothelial cell adhesion and transmigration of ER-MC cells through an endothelial cell barrier. Forced expression of ARTN produced a larger, locally invasive tumour mass with tumour emboli that produced distant metastasis. ARTN regulated TWIST1 expression in ER-MC cells and ARTN expression was significantly correlated to TWIST1 expression in a panel of mammary carcinoma cell lines and in a cohort of patients with ER-MC. Low expression of both ARTN and TWIST1 predicted 100% relapse free and overall survival in patients with ER-MC, whereas high expression of both ARTN and TWIST1 was associated with a poor survival outcome. ARTN stimulated an increase in TWIST1 expression via increased AKT activity. siRNA mediated depletion of TWIST1 abrogated ARTN stimulated cellular behaviour associated with metastasis, and forced expression of TWIST1 abrogated the functional effects of ARTN depletion. Conclusions ARTN and TWIST1 synergize to produce a worse outcome in ER-MC and combined inhibition of ARTN and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) may therefore provide a novel therapeutic strategy in this subtype of mammary carcinoma.

Leukemia is often maintained by a relatively rare subset of tumor-initiating cells, which may be resistant to traditional chemotherapy. Targeting these leukemic stem cells is expected to eliminate or reduce acquired resistance to chemotherapy and disease recurrence, but this goal has been elusive. It's also not clear whether eliminating the 'root' of cancer is sufficient or whether targeting both 'root' and 'branch' (i.e. LSCs and their bulk progeny) is necessary. To test this, we established and characterized a mouse model with clearly defined populations of rare leukemic stem cells (LSCs), abundant leukemic blast cell progeny, and normal hematopoietic stem/progenitor cells (HSPCs). This was achieved by simultaneously activating the Wnt/β-catenin and PI3K/Akt signaling pathways in HSPCs to drive stem cell self-renewal-resulting in successive expansion of HSPCs, LSCs, and leukemic blasts. Functional analysis showed a 1,300-fold increase in leukemia-initiating activity of sorted LSCs compared to bulk blast cells or sorted HSPCs. Since Akt activates β-catenin by phosphorylation at serine 552 and thus represents a molecular link mediating the synergistic, self-renewal promoting activity of these pathways, we sought to specifically target pS552-β-catenin. Unexpectedly, high-throughput screening (HTS) and subsequent validation assays found that doxorubicin (DXR) inhibits pS552-β-catenin with minimal effects on total β-catenin. DXR exhibits the broadest spectrum of anti-cancer activity known and has been employed as a standard chemotherapeutic agent for decades, but severe side-effects limit its use. We found that while standard chemotherapeutic treatment reduced the bulk leukemic blast cells as expected, it also induced pS552-β-catenin uniquely in LSCs and stimulated LSC expansion. Using long-circulating nanoparticles (NanoDXR) to deliver very low, sustained doses of DXR, we repurposed DXR as a targeted therapy for pS552-β-catenin inhibition rather than a broadly cytotoxic agent. In vivo, this treatment reduced pS552-β-catenin levels in LSCs, prevented LSC expansion, essentially eliminated LSC tumorigenic activity in transplant recipients, and was accompanied by recovery of HSPCs and substantially increased median survival from 44.5 days to 139 days. Our data reveals a dynamic relationship between rare LSCs and bulk leukemic blast cells in their response to cytotoxic chemotherapy and show how oncogenic self-renewal in chemoresistant LSCs can be targeted while sparing normal HSPCs. In distinguishing the unique properties of LSCs and their progeny, we find that both populations must be differentially targeted at both the 'root' and 'branch' of leukemia and can be effectively reduced or eliminated while allowing for recovery of normal HSPCs. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) sustain lifelong production of multiple blood cell types through a finely-tuned balance between stem cell maintenance and activation to prevent bone marrow exhaustion or overgrowth. The highly conserved Hox family of homeodomain containing transcription factors have been identified as key regulators and contributors in both normal hematopoiesis and leukemogenesis. Most previous work has focused on individual Hox genes; however, it remains largely unknown whether and how multiple Hox genes in a cluster are regulated and function in hematopoiesis. We initiated a study to perform systematic, high-throughput transcriptome analysis in the following 17 cell types from the bone marrow (BM) of C57BL/6J mice: 4 hematopoietic stem and progenitor cells (CD49blo long-term (LT)-HSC, CD49bhi intermediate-term (IT)-HSC, short-term (ST)-HSC, and MPP); and 4 committed progenitors (CLP, CMP, GMP and MEP); and 9 mature lineage cells (B cell, T cell, NK cell, dendritic cell, monocyte, macrophage, granulocyte, megakaryocyte and nucleated erythrocyte). Intriguingly, as part of a unique fingerprint observed in the most primitive CD49blo LT-HSCs, we detected expression from the Hoxb cluster. Further analysis on all the four Hox clusters revealed that most of the genes from the Hoxb cluster, and not from the other Hox clusters, were predominantly expressed in the CD49blo LT-HSCs. This suggests that they might function as a cluster to maintain CD49blo LT-HSCs. A previous study has shown that one cis -regulatory retinoic acid responsive element (RARE), is conserved among vertebrate species and regulates multiple Hoxb gene expression in central nervous system development. Thus, we asked whether RARE is essential for maintenance of primitive CD49blo LT-HSCs by regulation of Hoxb cluster. To test this hypothesis, we utilized a RAREΔ knockout mouse model and assayed for HSC numbers in BM. We observed that homozygous deletion of RARE led to 2-fold reduction in both the frequency and absolute number of CD49blo LT-HSCs. Functionally, we first conducted limiting dilution, competitive repopulating unit (CRU) assays by transplanting 2.5×104, 7.5×104 or 2×105 of BM cells from RAREΔ mutants and their control littermates, together with 2×105 recipient BM cells derived from the Ptprc mutant strain, into lethally irradiated recipient mice. Our data showed a 2.5-fold decrease in functional HSCs in RAREΔ HSCs (1/20,326) compared to control (1/50,839). To further evaluate the long-term effect of RARE on HSCs, we performed serial BM transplantation and observed a 12.9-fold reduction of reconstitution ability after secondary transplantation. These data indicate that deletion of RARE compromised HSC long-term reconstitution capacity. Collectively, our work provides evidence showing that RARE is essential for maintenance of the primitive HSCs by regulation of Hoxb cluster genes. Disclosures No relevant conflicts of interest to declare.

Balanced regulation is essential for the long-term preservation of stem cells while providing for ongoing tissue maintenance. We and others have previously shown that these dichotomous functions are accomplished through the co-existence of at least two stem cell populations—reserve and primed stem cells. This balance has been shown previously to be regulated by protein-coding genes; however, the potential roles of noncoding RNAs (ncRNAs) and their relationships with protein-coding genes in regulating hematopoietic stem cells (HSCs) remain largely unknown. To systematically identify ncRNAs involved in the murine hematopoiesis, we used RNA sequencing and identified unique, differentially expressed (fingerprint) ncRNAs representing reserve HSCs, primed HSCs, and more active stem/progenitor cells. These were also compared with committed progenitors and all major mature hematopoietic lineages. Intriguingly, all of the fingerprint ncRNAs uniquely expressed in reserve HSCs were derived from the imprinted Dlk1-Gtl2 locus, which spans a 780kb region on the mouse chromosome 12qF1 and is precisely controlled by the Intergenic Germ line-derived Differentially Methylated Region (IG-DMR). The Gtl2 locus contains a large cluster of snoRNAs (23 snoRNAs) and the largest cluster of mammalian miRNAs (57 miRNAs) as part of a single transcript of long length ncRNA downstream of Gtl2. To determine the role of Dlk1-Gtl2 locus in hematopoiesis, we utilized the IG-DMR knockout mouse model and carried out phenotypic and functional assays in E15.0 fetal liver HSCs since the embryos loss of maternal IG-DMR are lethal after E16. We observed that deletion of the maternal IG-DMR (ΔmIG-DMR), but not the paternal one, leads to 2-fold reduction in CD93+ fetal liver HSC number and 4-fold decrease of reconstitution ability after tertiary transplantation. Further, we employed RNA-seq using fetal liver HSCs from wt and ΔmIG-DMR and found that several pathways involved in growth control, mitochondrial function and energy metabolism, such as mTOR, PI3K/Akt and Wnt, are significantly enhanced in ΔmIG-DMR HSCs. We also carried out small RNA-seq in both adult HSCs and fetal liver HSCs and identified 13 HSC-specific miRNAs, which are predominantly expressed in reserve HSCs and predicted to target multiple proteins in PI3K/Akt/mTOR pathway. Mechanistically, maternal IG-DMR deletion leads to down-regulation of Gtl2-derived 13 miRNAs and hyperactivation of PI3K/Akt/mTOR pathway, which further enhances mitochondrial activity and biogenesis, increases oxidative phosphorylation (OXPHOS) mediated ATP production and ROS levels, and eventually causes HSC exhaustion. Moreover, either pharmacological inhibition of the mTOR activity by rapamycin or overexpression of Gtl2-derived miRNAs could partially, if not all, rescues the defective HSC phenotype and bioenergetic activities caused by mIG-DMR deletion. Collectively, our work provides a global landscape of murine hematopoietic lncRNAs and demonstrates that Dlk1-Gtl2 locus is critical in maintaining primitive HSCs with a fundamentally epigenetic regulation of mitochondrial function, energy metabolism via repression of PI3K/Akt/mTOR pathway. Disclosures No relevant conflicts of interest to declare.

A population of reserve HSCs has been reported to be in a deep-quiescent (or dormant) state and function as a back-up population of HSCs to support life-time hematopoiesis (Li and Clevers, 2010; Wilson et al., 2008). Currently, characterization of reserve HSCs is mainly based on cell cycle quiescence, however, the metabolic state in reserve HSCs is yet to be defined. Here, we show that reserve HSCs maintain not only a quiescent state but also an overall low metabolic activity whereas the primed HSCs maintain still a quiescent state but primed at metabolic state. First, we used CD49b(Benveniste et al., 2010) to further separate conventional long-term (LT) HSCs (CD34-Flk2-Lineage-Sca-1+c-Kit+)(Yang et al., 2005) into CD49blo and CD49bhi subpopulations and confirmed their enrichment with previously identified dormant (Scl-H2B-GFP label retaining cells, LRCs), thus we termed CD49blo and CD49bhi subpopulations as candidates of reserve and primed HSCs. We then determined the cell cycle frequencies of reserve, primed, and ST-HSCs (Cd34+Flk2-LSK) respectively as once in 3 months, 3-4 weeks, and 3-4 days. Functionally, Reserve HSCs had on average 3.5-fold higher functional capacity compared with primed HSCs. RNA-seq analysis revealed that reserve HSC predominantly expressed a list of imprinting genes that associate with growth-restriction functions; primed HSCs expressed relatively-high number of genes involving in mitochondria fusion, organization, and function, while ST-HSCs expressed genes reflecting an active cycling state. Conducting metabolic assays, we found that reserve HSCs not only maintain quiescence but also maintain overall low metabolic activity in both glycolysis and mitochondrial capacity. In contrast, primed HSCs are in a quiescent state, but with their metabolic state primed as evidenced by an increased glycolytic activity and mitochondrial potential for subsequent active proliferating state in ST-HSCs. Intriguingly, our data suggests that the functionality of reserve HSCs correlates to metabolic state rather than cell cycle. Disclosures No relevant conflicts of interest to declare.

Introduction Estrogen signaling is pivotal in the progression of estrogen receptor positive breast cancer primarily by the regulation of cell survival and proliferation. Micro (mi)RNAs have been demonstrated to be regulated by estrogen to mediate estrogenic effects. Herein, we determined the role of estrogen regulated miR-26 and its underlying molecular mechanisms associated with estrogen receptor (ER)+ breast cancer proliferation. Methods The expression of miR-26a and miR-26b was evaluated by real-time quantitative (RT)-PCR. The expression of miR-26a or miR-26b was modulated in ER+ breast cancer cells (MCF-7 and T47D) and tumor cell growth in vitro and an in vivo xenograft model was determined. Bioinformatics analyses were utilized to screen for estrogen responsive genes, which were also predicted to be targeted by miR-26. Luciferase reporter assays were performed to confirm miR-26 regulation of the 3' UTR of target genes. The levels of miR-26 target genes (CHD1, GREB1 and KPNA2) were evaluated by western blotting and immunohistochemistry. Results Estrogen reduced the expression of miR-26a and miR-26b in ER+ breast cancer cells. Forced expression of miR-26a or miR-26b significantly inhibited the estrogen stimulated growth of ER+ breast cancer cells and tumor growth in xenograft models, whereas miR-26a/b depletion increased the growth of ER+ breast cancer cells in the absence of estrogen treatment. Screening of estrogen responsive genes, which were also predicted to be targeted by miR-26, identified GREB1 and nine other genes (AGPAT5, AMMECR1, CHD1, ERLIN1, HSPA8, KPNA2, MREG, NARG1, and PLOD2). Further verification has identified nine genes (AGPAT5, CHD1, ERLIN1, GREB1, HSPA8, KPNA2, MREG, NARG1 and PLOD2) which were directly targeted by miR-26 via their 3′ UTR. Functional screening suggested only three estrogen regulated miR-26 target genes (CHD1, GREB1 and KPNA2) were involved in the regulation of estrogen promoted cell proliferation. Depletion of either CHD1, GREB1 or KPNA2 significantly abrogated the enhanced growth of ER+ breast cancer cells due to miR-26 depletion. We further demonstrated that estrogen stimulated c-MYC expression was both sufficient and necessary for the diminished expression of miR-26a and miR-26b. Conclusions We have identified a novel estrogen/MYC/miR-26 axis that mediates estrogen stimulated cell growth via CHD1, GREB1 and KPNA2.

We have previously reported that artemin (ARTN) stimulates the oncogenicity and invasiveness of endometrial carcinoma cells. Herein, we demonstrate that ARTN modulates the sensitivity of endometrial carcinoma cells to agents used to treat late-stage endometrial carcinoma. Forced expression of ARTN in endometrial carcinoma cells decreased sensitivity to doxorubicin and paclitaxel. Accordingly, depletion of ARTN by small interfering RNA or functional inhibition of ARTN with antibodies significantly increased sensitivity of endometrial carcinoma cells to doxorubicin and paclitaxel. Forced expression of ARTN in endometrial carcinoma cells abrogated doxorubicin-induced G2-M arrest and paclitaxel-induced apoptosis. ARTN increased CD24 expression in endometrial carcinoma cells by transcriptional up-regulation, and CD24 was partially correlated to ARTN expression in endometrial carcinoma. Forced expression of CD24 in endometrial carcinoma cells stimulated cell proliferation and oncogenicity, enhanced cell invasion, and decreased sensitivity to doxorubicin and paclitaxel. Depletion of CD24 in endometrial carcinoma cells abrogated ARTN-stimulated resistance to doxorubicin and paclitaxel. ARTN-stimulated resistance to doxorubicin and paclitaxel in endometrial carcinoma cells is therefore mediated by the specific regulation of CD24. Functional inhibition of ARTN may therefore be considered as an adjuvant therapeutic approach to improve the response of endometrial carcinoma to specific chemotherapeutic agents.