Meox1 Expression Research Articles

Chromatin remodelling drives immune cell-fibroblast communication in heart failure.

Chronic inflammation and tissue fibrosis are common responses that worsen organ function, yet the molecular mechanisms governing their cross-talk are poorly understood. In diseased organs, stress-induced gene expression changes fuel maladaptive cell state transitions1 and pathological interaction between cellular compartments. Although chronic fibroblast activation worsens dysfunction in the lungs, liver, kidneys and heart, and exacerbates many cancers2, the stress-sensing mechanisms initiating transcriptional activation of fibroblasts are poorly understood. Here we show that conditional deletion of the transcriptional co-activator Brd4 in infiltrating Cx3cr1+ macrophages ameliorates heart failure in mice and significantly reduces fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1+ cells identified a large enhancer proximal to interleukin-1β (IL-1β, encoded by Il1b), and a series of CRISPR-based deletions revealed the precise stress-dependent regulatory element that controls Il1b expression. Secreted IL-1β activated a fibroblast RELA-dependent (also known as p65) enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL-1β neutralization improved cardiac function and tissue fibrosis in heart failure. Systemic IL-1β inhibition or targeted Il1b deletion in Cx3cr1+ cells prevented stress-induced Meox1 expression and fibroblast activation. The elucidation of BRD4-dependent cross-talk between a specific immune cell subset and fibroblasts through IL-1β reveals how inflammation drives profibrotic cell states and supports strategies that modulate this process in heart disease and other chronic inflammatory disorders featuring tissue remodelling.

Establishment of glioma prognosis nomogram based on the function of meox1 in promoting the progression of cancer

BackgroundGliomas stand out as highly predominant malignant nervous tumors and are linked to adverse treatment outcomes and short survival periods. Current treatment options are limited, emphasizing the need to identify effective therapeutic targets. The heterogeneity of tumors necessitates a personalized treatment approach with an effective grouping system. Meox1 has been implicated in promoting tumor progression in diverse cancers; nonetheless, its role in gliomas remains unelucidated. Material/methodsUtilized immunohistochemistry to assess the expression of Meox1 protein in glioma tissues. Proliferation and invasion assays were conducted on wild-type and meox1-overexpressed glioma cells using the CCK8 and Transwell assays, respectively. The expression levels of meox1 and its related genes in gliomas were obtained from Chinese Glioma Genome Atlas (CGGA), along with the corresponding patient survival periods. LASSO regression modeling was employed to construct a scoring system for patients with gliomas, categorizing them into high-/low-risk groups. Additionally, a nomogram for predicting the survival period of patients with glioma was developed using multivariate logistic analysis. ResultsWe attempted, for the first time, to demonstrate heightened expression of Meox1 in glioma tumor tissues, correlating with significantly increased invasion and proliferation abilities of glioma cells following meox1 overexpression. The scoring system effectively stratified patients with glioma into high-/low-risk groups, revealing differences in the survival period and immunotherapy efficacy between the two groups. The integration of this scoring system with other clinical indicators yielded a nomogram capable of effectively predicting the survival period of individuals with gliomas. ConclusionsOur study established a stratified investigation system based on the levels of meox1 and its related genes, providing a novel, cost-effective model for facilitating the prognosis prediction of individuals with glioma.

Unraveling the molecular mechanisms of lymph node metastasis in ovarian cancer: focus on MEOX1

BackgroundLymph node metastasis (LNM) is a major factor contributing to the high mortality rate of ovarian cancer, making the treatment of this disease challenging. However, the molecular mechanism underlying LNM in ovarian cancer is still not well understood, posing a significant obstacle to overcome.ResultsThrough data mining from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we have identified MEOX1 as a specific gene associated with LNM in ovarian cancer. The expression of MEOX1 was found to be relatively high in serous ovarian adenocarcinoma, and its higher expression were associated with increased tumor grade and poorer clinical prognosis for ovarian cancer patients. Bioinformatics analysis revealed that MEOX1 exhibited the highest mRNA levels among all cancer types in ovarian cancer tissues and cell lines. Furthermore, gene set enrichment analysis (GSEA) and pathway analysis demonstrated that MEOX1 was involved in various LNM-related biological activities, such as lymphangiogenesis, lymphatic vessel formation during metastasis, epithelial-mesenchymal transition (EMT), G2/M checkpoint, degradation of extracellular matrix, and collagen formation. Additionally, the expression of MEOX1 was positively correlated with the expression of numerous prolymphangiogenic factors in ovarian cancer. To validate our findings, we conducted experiments using clinical tissue specimens and cell lines, which confirmed that MEOX1 was highly expressed in high-grade serous ovarian cancer (HGSOC) tissues and various ovarian cancer cell lines (A2780, SKOV3, HO8910, and OVCAR5) compared to normal ovarian tissues and normal ovarian epithelial cell line IOSE-80, respectively. Notably, we observed a higher protein level of MEOX1 in tumor tissues of LNM-positive HGSOC compared to LNM-negative HGSOC. Moreover, our fundamental experiments demonstrated that suppression of MEOX1 led to inhibitory effects on ovarian cancer cell proliferation and EMT, while overexpression of MEOX1 enhanced the proliferation and EMT capacities of ovarian cancer cells.ConclusionsThe results of our study indicate that MEOX1 plays a role in the lymph node metastasis of ovarian cancer by regulating multiple biological activities, including the proliferation and EMT of ovarian cancer, lymphangiogenesis, and ECM remodeling. Our findings suggest that MEOX1 could serve as a potential biomarker for the diagnosis and treatment of ovarian cancer with LNM.

Analysis of immunoinfiltration and EndoMT based on TGF-β signaling pathway-related genes in acute myocardial infarction

Acute myocardial infarction (AMI), a critical manifestation of coronary heart disease, presents a complex and not entirely understood etiology. This study investigates the potential role of immune infiltration and endothelial-mesenchymal transition (EndoMT) in AMI pathogenesis. We conducted an analysis of the GSE24519 and MSigDB datasets to identify differentially expressed genes associated with the TGF-β signaling pathway (DE-TSRGs) and carried out a functional enrichment analysis. Additionally, we evaluated immune infiltration in AMI and its possible link to myocardial fibrosis. Key genes were identified using machine learning and LASSO logistic regression. The expression of MEOX1 in the ventricular muscles and endothelial cells of Sprague–Dawley rats was assessed through RT-qPCR, immunohistochemical and immunofluorescence assays, and the effect of MEOX1 overexpression on EndoMT was investigated. Our study identified five DE-TSRGs, among which MEOX1, SMURF1, and SPTBN1 exhibited the most significant associations with AMI. Notably, we detected substantial immune infiltration in AMI specimens, with a marked increase in neutrophils and macrophages. MEOX1 demonstrated consistent expression patterns in rat ventricular muscle tissue and endothelial cells, and its overexpression induced EndoMT. Our findings suggest that the TGF-β signaling pathway may contribute to AMI progression by activating the immune response. MEOX1, linked to the TGF-β signaling pathway, appears to facilitate myocardial fibrosis via EndoMT following AMI. These novel insights into the mechanisms of AMI pathogenesis could offer promising therapeutic targets for intervention.

Mesoderm/mesenchyme homeobox l may promote tumor progression in human hepatocellular carcinoma.

The clinical response rate for molecularly targeted medications is limited despite significant advancements in molecularly targeted therapy for hepatocellular carcinoma (HCC). Therefore, it is necessary to find new and robust therapeutic targets for the treatment of HCC. Recent research has shown that mesoderm/mesenchyme homeobox gene 1 (Meox1) is closely associated with cancer progression. The aim of this study was to evaluate the clinical relevance as well as biological function of Meox1 in HCC. Meox1 protein expression level was identified through immunohistochemistry (IHC) examination of pathological tissues from 25 HCC patients. The aim of the analysis was to investigate the relationship between clinicopathological traits and Meox1 expression. Biological function assays of Meox1 in HCC, including proliferation, colony formation, migration, and invasion, were performed with Huh7 and Hep3B cells. In this study, Meox1 expression in HCC tissues was significantly higher (p < 0.05) compared to paracancerous tissues. Especially in HCC tissues of patients with cirrhosis, the level of Meox1 expression was significantly elevated when compared to HCC tissues of patients without cirrhosis (p < 0.05). High Meox1 expression was significantly associated with tumor-node-metastasis (TNM) stage (p < 0.05) and the Barcelona Clinic Liver Cancer (BCLC) stage (p < 0.05). Moreover, Meox1 silencing suppressed the proliferation, colony formation, migration, and invasion of Huh7 and Hep3B cells. Our data reveal that Meox1 may play a crucial role in the development of HCC, and given the function of Meox1 in proliferation and metastasis, targeting Meox1 may offer a promising approach for combined and adjuvant therapeutics of HCC.

Identification of the novel FOXP3-dependent Treg cell transcription factor MEOX1 by high-dimensional analysis of human CD4+ T cells.

CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.

Spatio-temporal model of Meox1 expression control involvement of Sca-1-positive stem cells in neointima formation through the synergistic effect of Rho/CDC42 and SDF-1\u03b1/CXCR4

AimsNeointimal hyperplasia remains a major obstacle in vascular regeneration. Sca-1-positive progenitor cells residing within the vascular adventitia play a crucial role in the assemblage of vascular smooth muscle cell (VSMC) and the formation of the intimal lesion. However, the underlying mechanisms during vascular injury are still unknown.Methods and resultsAneointimal formation rat model was prepared by carotid artery injury using 2F-Forgaty. After vascular injury, Meox1 expressions time-dependently increased during the neointima formation, with its levels concurrently increasing in the adventitia, media, and neointima. Meox1 was highly expressed in the adventitia on the first day after vascular injury compared to the expression levels in the media. Conversely, by the 14th day post-injury, Meox1 was extensively expressed more in the media and neointima than the adventitia. Analogous to the change of Meox1 in injured artery, Sca-1+ progenitor cells increased in the adventitia wall in a time-dependent manner and reached peak levels on the 7th day after injury. More importantly, this effect was abolished by Meox1 knockdown with shRNA. The enhanced expression of SDF-1α after vascular injury was associated with the markedly enhanced expression levels of Sca1+ progenitor cell, and these levels were relatively synchronously increased within neointima by the 7th day after vascular injury. These special effects were abolished by the knockdown of Meox1 with shRNA and inhibition of CXCR4 by its inhibitor, AMD3100. Finally, Meox1 concurrently regulated SDF-1α expressions in VSMC via activating CDC42, and CDC42 inhibition abolished these effects by its inhibitor, ZCL278. Also, Meox1 was involved in activation of the CXCR4 expression of Sca-1+ progenitor cells by CDC42.ConclusionsSpatio-temporal model of Meox1 expression regulates theSca-1+progenitor cell migration during the formation of the neointima through the synergistic effect of Rho/CDC42 and SDF-1α/CXCR4.

Mesenchyme homeobox 1 mediated-promotion of osteoblastic differentiation is negatively regulated by mir-3064-5p

Human mesenchymal stem cells (hMSCs) are multipotent cells that can be differentiated into different cell types including osteoblasts. Herein we aimed to assess the regulation of transcription factor mesenchyme homeobox 1 (Meox1) in the osteogenic differentiation of hMSCs and to determine the microRNA which targets on Meox1. Total RNA was extracted from the isolated ligamentum flavum tissue samples and cultured hMSCs, and the expression of Meox1 was assessed by RT-PCR and Western blot assays. Cultured hMSCs were induced towards osteoblastic differentiation, and the osteoblast phenotype was determined by alkaline phosphatase activity and alizarin red staining. The microRNA targeting on the 3′-UTR of Meox1was predicted using bioinformatics tool, and the binding was validated by luciferase and RNA pulldown assays. The osteoblastic differentiation of hMSCs was checked with the knockdown of Meox1 and microRNA inhibitors. Higher expression of Meox1, and lower expression of miR-3064–5p in ossified ligamentum flavum (OLF) tissues were identified. In addition, increased expression along with the osteoblastic differentiation of hMSCs was found. Further research revealed that Meox was a direct target of miR-3064–5p, when the former promoted the differentiation of hMSCs into osteoblasts, the latter significantly suppressed the osteogenesis. The expression of Meox1 increased gradually with the osteoblastic differentiation of hMSCs, during which miR-3064–5p decreased. Meox1 is a direct target of miR-3064–5p, and they both play important roles in the osteogenesis. These findings provide potential target for the development of therapeutic drugs for skeletal system diseases.

Mechanism of transcriptional regulation of Meox1 by transforming growth factor β (1) and its effect on cell migration of adult human dermal fibroblasts

Objective: To explore the transcriptional regulation mechanism of transforming growth factor β(1) (TGF-β(1)) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods: (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-β(1) stimulation group and blank control group. The cells in TGF-β(1) stimulation group were stimulated with 10 μL TGF-β(1) in the mass concentration of 1 mg/μL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 and P 0.05); the enrichment of Smad4 protein on the promoter of Meox1 in the cells of empty plasmid group was similar to that of Smad4 OE group (t=0.60, P>0.05). (5) Twenty-four hours after scratching, the scratch healing width of cells in siRNA-Meox1 group was narrower than that of negative interference group, while that of Meox1 OE group was wider than that of empty plasmid group. After 24 hours in culture, the number of migration cells in negative interference group was significantly higher than that in siRNA-Meox1 group (t=9.12, P<0.01), and that in empty plasmid group was significantly lower than that in Meox1 OE group (t=8.99, P<0.01). (6) The expression of Meox1 protein in the scar tissue was significantly higher than that in normal skin of patients with hypertrophic scars. Conclusions: TGF-β(1) transcriptionally regulates Meox1 expression via Smad2/3 in HDF-a cells, thus promoting cell migration.

MEOX1 Promotes Tumor Progression and Predicts Poor Prognosis in Human Non-Small-Cell Lung Cancer.

Background: MEOX1 is a homeobox transcriptional factor, and plays essential roles in regulating somite development. Our previous study indicated that MEOX1 is a critical molecular target in mesenchymal-like cancer cells in PTEN-deficient Trastuzumab resistant breast cancer. Despite the potential implication of MEOX1 for the cancer progression, no previous studies examined its level and clinical significance in lung cancer tissues. In this study, we aimed to detect the MEOX1 expression and correlate its level with clinical outcome in non-small-cell lung cancer patients (NSCLC).Methods: MEOX1 gene expression in lung cancer was examined by using the Oncomine database. MEOX1 protein levels were evaluated by IHC using the corresponding primary antibody on two different commercial lung cancer tissue arrays. siRNA knockdown was used to elucidate the function of MEOX1.Results: Analysis of the Oncomine datasets identified that an elevation of MEOX1 in gene amplification in lung cancer tissues in comparison to normal lung tissues. Immunohistochemistical analysis demonstrated that MEOX1 was localized predominantly in the nucleus, and positive rate was 67.3% (111/165) in NSCLC samples. Statistical analysis revealed high levels of MEOX1 significantly correlated with Lymph Node Metastasis and Stage. Kaplan-Meier survival analysis showed that high levels of MEOX1 were significantly associated with unfavorable survival in NSCLC patients, and MEOX1 nucleus staining had worse survival, than did patients with overall expression in lung squamous cell carcinoma patients. Multivariate Cox's regression analysis found that MEOX1 was an independent poor prognostic predictor for patients with NSCLC. Silencing of MEOX1 by specific SiRNA significantly inhibited H460 and H1299 cell proliferation and sphere formation in serum-free medium.Conclusions: Our results firstly indentified that high levels of MEOX1 especially nuclear staining was an independent prognostic factor for NSCLC, and it served a essential roles in the regulation of cell proliferation and colony formation in vitro. It may represent a potential target for the NSCLC treatment.

Retracted: Silencing of MEOX1 Gene Inhibits Proliferation and Promotes Apoptosis of LNCaP Cells in Prostate Cancer

Prostate cancer (PC), the most commonly diagnosed malignancy, is also the second leading cause of cancer-related deaths. Although tremendous efforts have been achieved to improve the early detection of PC, it is still urgent to find out novel markers and therapeutic targets for PC patients. Homeobox protein MOX-1 (MEOX1) is found to be ectopically expressed in human cancers. This study aims to test the hypothesis that MEOX1 gene silencing might suppress the proliferation and invasion of LNCaP cells in PC. Microarray analysis was conducted to screen PC-related differentially expressed gene. MEOX1 was silenced to detect the relationship between silenced MEOX1 and cell proliferation, migration, apoptosis, and invasion. Expression of MEOX1, Bcl-2, p53, and Bax in LNCaP cells was determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Cell proliferation, colony formation rate, cell migration, invasion, cell cycle, and apoptosis were detected respectively. MEOX1 gene was found to be expressed highly in PC tissues. After MEOX1 was silenced in LNCaP cells, the mRNA and protein expression of Bcl-2 decreased while the mRNA and protein expressions of p53 and Bax increased. Additionally, cell proliferation, migration, and invasion were reduced while cell apoptosis was increased by MEOX1, and MEOX1 gene silencing was revealed to inhibit cell proliferation and decrease tumorigenic ability of LNCaP cells in nude mice. Taken together, MEOX1 gene silencing could decrease the proliferation and increase apoptosis of LNCaP cells, and so it is expected to be a therapeutic target for PC.

Mesenchyme homeobox 1 mediates transforming growth factor-β (TGF-β)–induced smooth muscle cell differentiation from mouse mesenchymal progenitors

Differentiation of smooth muscle cells (SMCs) is critical for proper vasculogenesis and angiogenesis. However, the molecular mechanisms controlling SMC differentiation are not completely understood. During embryogenesis, the transcription factor mesenchyme homeobox 1 (Meox1) is expressed in the early developing somite, which is one of the origins of SMCs. In the present study, we identified Meox1 as a positive regulator of SMC differentiation. We found that transforming growth factor-β (TGF-β) induces Meox1 expression in the initial phase of SMC differentiation of pluripotent murine C3H10T1/2 cells. shRNA-mediated Meox1 knockdown suppressed TGF-β-induced expression of SMC early markers, whereas Meox1 overexpression increased expression of these markers. Mechanistically, Meox1 promoted SMAD family member 3 (Smad3) nuclear retention during the early stage of TGF-β stimulation because Meox1 inhibited protein phosphatase Mg2+/Mn2+-dependent 1A (PPM1A) and thereby prevented PPM1A-mediated Smad3 dephosphorylation. Meox1 appears to promote PPM1A degradation, leading to sustained Smad3 phosphorylation, thus allowing Smad3 to stimulate SMC gene transcription. In vivo, Meox1 knockdown in mouse embryos impaired SMC marker expression in the descending aorta of neonatal mice, indicating that Meox1 is essential for SMC differentiation during embryonic development. In summary, the transcriptional regulator Meox1 controls TGF-β-induced SMC differentiation from mesenchymal progenitor cells by preventing PPM1A-mediated Smad3 dephosphorylation, thereby supporting SMC gene expression.

Abstract 2509: Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem cells

Abstract Many mechanisms have been attributed to de novo and acquired trastuzumab drug resistance in patients with HER2-enriched breast cancer; common mediated resistance is inactivation of the tumor suppressor gene PTEN seen in approximately 40% of patients. Evidence show the HER2 gene plays a significant role in regulating breast cancer stem cells through PTEN deletion; increasing evidence show these cancer stem cells may be responsible for drug resistance, cancer recurrence and metastasis. Studies demonstrate how PTEN deletion in HER2-overexpressing breast cancer activates an IL6 inflammatory feedback loop expanding the cancer stem cell population displaying EMT properties, conferring trastuzumab drug resistance and metastasis. While new therapeutic approaches for targeting HER2-enriched tumors with PTEN deletion are in progress, further understanding of molecular systems involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance is important for improving therapeutic options. Our lab conducted RNA sequencing to further explore functional mechanisms behind this aforementioned molecular system and found the upregulation of mesenchyme homeobox 1 (Meox1) as a potential transcriptional factor regulator. Meox1 is a non-cluster homeobox transcription factor expressed during embryonic development, critical in somite morphogenesis. Recent evidence has linked Meox1 as a critical PBX1 cofactor in ovarian cancer. As of yet, the role of Meox1 has never been studied in the context of breast cancer in correlation with cancer stem cells. Therefore specifically, the purpose of this study is to elucidate the molecular role of Meox1 in HER2-amplified PTEN-deleted trastuzumab-resistant breast cancer in order to provide functional and mechanistic insight involving EMT cancer stem cell expansion as well as of de novo and acquired trastuzumab drug resistance. RT-qPCR analyses of Meox1 expression using different breast cancer cell lines show correlation between increase in Meox1 expression with PTEN deletion. In vitro studies show siRNA knockdown of Meox1 decreases cancer stem cell properties of both colony and mammosphere formation in the BT474 HER2-enriched breast cell line that is PTEN-deleted and long term trastuzumab treated. Furthermore, lentiviral stable overexpression of Meox1 in normal immortalized MCF10A breast cancer cell line shows an increase in mammosphere formation compared to control infections. These preliminary results suggest Meox1 may be a critical transcription factor responsible for cancer stem cell progression in PTEN-deleted trastuzumab-resistant breast cancer. As such, further functional and mechanistic investigation of this homeobox transcription factor may provide additional understanding for de novo and acquired drug resistance and disease recurrence, potentially offering advanced therapeutic options. Citation Format: Mari Gasparyan, Joseph P. Burnett, Lichao Sun, Duxin Sun. Determining the functional and mechanistic role of homeobox transcription factor Meox1 in breast cancer and breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2509.

Abstract 218: MEOX-1 as a novel cancer stem cell target for treatment of trastuzumab-resistant Her2+ breast cancers

Abstract Introduction: Previous studies showed that 60-80% of Her2+ breast cancers develop Trastuzumab-resistance after one year treatment and more than 50% of Her2+ Trastuzumab-resistant breast cancers have PTEN deletion and increased populations of cancer stem cells (CSCs). The purpose of this study is to explore novel targets to regulate cancer stem cells and to test treatment option for Her2+ Trastuzumab-resistant breast cancer. Method: Trastuzumab resistance of Her2+ breast cancer cells (BT474) was induced by shRNA knockdown of PTEN and long term treatment of Trastuzumab (LTT) in comparison with Trastuzumab-sensitive BT474. The whole transcriptome RNA sequencing was performed and the differentially expressed genes were evaluated using dChip analyzer and the bioinformatics tool DAVID. Colony formation in soft agar and mammosphere formation were used to evaluate the effect of candidate genes on breast CSCs. Immunohistochemistry (IHC) was used to assess the protein expression levels in xenograft animal tissues and human breast cancer tissues. MTS assay was used to evaluate the effect of drug treatment to inhibit proliferation of BT474 and LTT. A xenograft model was used to determine whether drug efficacy to inhibit tumor formation. The secondary implantation was used to monitor cancer stem cells growth in vivo after treatment. Result: In comparison of BT474 and LTT under treatment of Sulforaphane (2-10 uM, 8-24 hr), RNA-Seq analysis revealed that 51 genes, including 23 up-regulated and 28 down-regulated by Sulforaphane treatment. DAVID functional clustering analysis revealed that 6 significantly altered genes were associated with 9 distinct functional categories. Among them, MEOX-1 was identified as top candidate to regulate Trastuzumab-resistance phenotype. siRNA knockdown of MEOX-1 in LTT cells inhibited mammosphere formation by 60.4% and colony formation by 86.7%. Further, in the IHC of 68 primary breast cancer samples, expression of MEOX-1 was positive in 34 primary breast cancer tissues (34/68, 50%), especially in triple negative breast cancer tissues (P&amp;lt;0.05). Sulforaphane inhibited the proliferation of LTT cells by 20% to 80%, while it showed no effect on BT474 cells in vitro. Also Sulforaphane decreased MEOX-1 mRNA level by 6-fold LTT cells. Daily injection with 50 mg/kg Sulforaphane reduced the growth of mouse xenografts by &amp;gt;50% in SCID xenograft tumors (P&amp;lt;0.05) and abrogated tumor growth after the reimplantation of primary tumor cells into the secondary mice (P&amp;lt;0.05). Immunostainng showed that MEOX1 protein level in nucleus, and Sulforaphane significantly reduced MEOX-1 expression in primary xenograft tumors. Conclusion: MEOX1 is a novel target to regulate cancer stem cells in Her2+ Trastuzumab-resistant breast cancer cells. Drug treatment to down-regulate Meox1 provides treatment option for Trastuzumab-resistant breast cancer by eliminating breast cancer stem cells. Citation Format: Lichao Sun, Joseph P. Burnett, Mari Gasparyan, Hasan Korkaya, Hui Jiang, Yajing Liu, Jamie Connarn, Max Wicha, Duxin Sun. MEOX-1 as a novel cancer stem cell target for treatment of trastuzumab-resistant Her2+ breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 218. doi:10.1158/1538-7445.AM2014-218

Abstract 76: Meox1 is a Cell-cycle Oscillator Required for Mitotic Transition and Proliferation in Cardiac Fibroblasts

Background: Tissue fibrosis plays important roles in the pathogenesis of chronic diseases, including heart failure. The mechanism underlying interstitial fibroblast proliferation is a promising analytical target for therapeutic applications. Here we developed quantitative epigenome profiling to identify a critical regulator in interstitial cell populations that emerges during the progression of heart failure. Methods and Results: We subjected pressure-overloaded hearts of mice to trimethylated histone H3 lysine 4 (H3K4me3) ChIP-sequence and RNA-sequence. Expression analysis followed by quantitative H3K4me3 profiling identified 45 fibrosis-related genes with significant H3K4me3 enrichment in failing hearts, including Meox1 transcription factor. Meox1 emerged in the interstitial fibrotic region in failing heart, and intriguingly Meox1 was expressed in the limited population of cardiac fibroblasts both in vivo and in vitro. Meox1-positive fibroblasts were increased in response to a paracrine signal from cardiomyocytes, and knockdown of Meox1 completely inhibited the reactive proliferation of cardiac fibroblasts stimulated by conditioned medium from cardiomyocytes. Gene expression profiling combined with siRNAs clarified that Meox1 depletion resulted in down regulation in the mitosis-related genes including Aurora B kinase. Indeed, Meox1 depletion decreased the cells under mitosis, but conversely increased the proportion of DNA synthesizing cells, thereby inhibited mitotic transition. The cell-cycle synchronization analysis and promoter analysis using live-cell imaging clarified that Meox1 oscillated throughout the cell-cycle and specifically emerged in G2/M phase. Finally, we revealed that Meox1 heterogenously expressed in the interstitial fibrotic are of human ventricular heart tissues from patients with end-stage heart failure. Notably, Meox1 expression was significantly correlated with the fibrosis-related genes in diseased ventricular heart tissues (n=15), suggesting the pathological relevance in clinical settings. Conclusion: Our findings identify a novel cell-cycle regulator and propose that Meox1 is a potential target for therapies aimed at preventing tissue fibrosis.

The Ski-Zeb2-Meox2 pathway provides a novel mechanism for regulation of the cardiac myofibroblast phenotype

Cardiac fibrosis is linked to fibroblast-to-myofibroblast phenoconversion and proliferation but the mechanisms underlying this are poorly understood. Ski is a negative regulator of TGF-β-Smad signaling in myofibroblasts, and might redirect the myofibroblast phenotype back to fibroblasts. Meox2 could alter TGF-β-mediated cellular processes and is repressed by Zeb2. Here, we investigated whether Ski diminishes the myofibroblast phenotype by de-repressing Meox2 expression and function through repression of Zeb2 expression. We show that expression of Meox1 and Meox2 mRNA and Meox2 protein is reduced during phenoconversion of fibroblasts to myofibroblasts. Overexpression of Meox2 shifts the myofibroblasts into fibroblasts, whereas the Meox2 DNA-binding mutant has no effect on myofibroblast phenotype. Overexpression of Ski partially restores Meox2 mRNA expression levels to those in cardiac fibroblasts. Expression of Zeb2 increased during phenoconversion and Ski overexpression reduces Zeb2 expression in first-passage myofibroblasts. Furthermore, expression of Meox2 is decreased in scar following myocardial infarction, whereas Zeb2 protein expression increases in the infarct scar. Thus Ski modulates the cardiac myofibroblast phenotype and function through suppression of Zeb2 by upregulating the expression of Meox2. This cascade might regulate cardiac myofibroblast phenotype and presents therapeutic options for treatment of cardiac fibrosis.

Cross talk between hedgehog and bone morphogenetic proteins occurs during cardiomyogenesis in P19 cells

Hedgehog (Hh) signaling plays a role in heart morphogenesis and can initiate cardiomyogenesis in P19 cells. To determine if Hh signaling is essential for P19 cell cardiomyogenesis, we determined which Hh factors are expressed and the effect of Hh signal transduction inhibitors. Here, we find that the Hh gene family and their downstream mediators are expressed during cardiomyogenesis but an active Hh signaling pathway is not essential. However, loss of Hh signaling resulted in a delay of BMP-4, GATA-4, Gli2, and Meox1 expression during cardiomyogenesis. By using Noggin-overexpressing P19 cells, we determined that Hh signaling was not active during Noggin-mediated inhibition of cardiomyogenesis. Thus, there is cross talk between the Hh and BMP signaling pathways and the Hh pathway appears important for timely cardiomyogenesis.