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Journal of Experimental & Clinical Cancer Research
Abnormally elevated USP37 expression in breast cancer stem cells regulates stemness, epithelial-mesenchymal transition and cisplatin sensitivity
Tao Qin1, 2,
Bai Li1, 2,
Xiaoyue Feng1, 2,
Shujun Fan1, 2,
Lei Liu3,
Dandan Liu1, 2,
Jun Mao1, 2,
Ying Lu4,
Jinfeng Yang5,
Xiaotang Yu1,
Qingqing Zhang1,
Jun Zhang6,
Bo Song1, 2,
Man Li7Email author and
Lianhong Li1, 2Email author
Journal of Experimental & Clinical Cancer Research201837:287
Recent studies have indicated that deubiquitinating enzymes (DUBs) are related to the stem-cell pathway network and chemo-resistance in cancer. Ubiquitin-specific peptidase 37 (USP37), a novel DUB, was identified to be a potential factor associated with tumor progression. However, the biological functions of USP37 in breast cancer remain unclear.
The distribution of USP37 expression in breast cancer and the correlation between USP37 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) was utilized to evaluate potential mechanism of USP37 in breast cancer. The USP37 expression in breast cancer tissues and breast cancer cell lines were detected by immunohistochemistry and western blotting. Sorting of breast cancer stem cells (BCSCs) were by using MACS assay. In vitro and in vivo assays were performed to examine the biological functions of USP37 in breast cancer cells. MG132, CHX chase, immunofluorescence staining and co-immunoprecipitation assays were used to test the interaction between USP37 and Gli-1.
Bioinformatics analysis demonstrated that USP37 gene was elevated in breast cancer tissues and its overexpression was strongly correlated with the increased mortality rate. GSEA analysis showed that USP37 expression was positively associated with cell growth and metastasis while negatively related to cell apoptosis in the TCGA breast cancer samples. USP37 expression was elevated in breast cancer tissues and breast cancer cell lines. Moreover, we also detected that USP37 was overexpressed in BCSCs. USP37 regulated the ability of cell invasion, epithelial-mesenchymal transition (EMT), stemness and cisplatin sensitivity in breast cancer cell lines. Additionally, USP37 knockdown inhibited tumorigenicity and increased anticancer effect of cisplatin in vivo. Knockdown of USP37 significantly decreased hedgehog (Hh) pathway components Smo and Gli-1. Gli-1 was stabilized by USP37 and they interacted with each other. Further studies indicated that USP37 knockdown could inhibit the stemness, cell invasion and EMT in breast cancer via downregulation of Hh pathway.
These findings reveal that USP37 is highly expressed in BCSCs and is correlated with poor prognosis in breast cancer patients. USP37 can regulate the stemness, cell invasion and EMT via Hh pathway, and decreased USP37 confers sensitivity to cisplatin in breast cancer cells. USP37 is required for the regulation of breast cancer progression, as well as a critical target for clinical treatment of breast cancer.
USP37
Stemness
Cisplatin
Breast cancer is the deadliest form of carcinoma affecting women, with nearly a quarter-million cases diagnosed in 2016 [1]. Although there are effective treatments against some types of breast carcinoma, such as those for subtypes with abnormal overexpression of the HER2/Neu oncogene, the majority breast cancers remain incurable. Breast cancer stem cells (BCSCs) possess self-renewal and differentiation capabilities, leading to tumor recurrence, metastasis and therapeutic resistance [2, 3]. CD44+/CD24− or aldehyde dehydrogenase1 (ALDH1) phenotypes are efficient in the identification of BCSCs from breast cancer populations. However, there is a small overlap between CD44+/CD24− and ALDH1 stem phenotypes, as well as less stem markers in differentiation of different breast cancer subtypes [4]. Therefore, it is necessary to identify more discriminatory biomarkers of distinct molecular subtypes for the isolation and identification of the BCSCs subpopulation.
EMT describes the process by which epithelial cells detach from neighboring cells and are transferred to other tissue sites via dissolution of basement membrane and passage through the extra-cellular matrix [5, 6]. EMT could also facilitate the generation of cancer stem cells from more differentiated cancer cells [7]. The EMT process during breast carcinogenesis is considered to be controlled by a series of signaling pathways, including Notch, Wnt/β-catenin and Hedgehog [8, 9]. The Hedgehog (Hh) pathway is responsible for the maintenance of stem cells and EMT, which can contribute to the evolution of breast cancer [10].
Ubiquitination describes a highly conserved and reversible modification process of protein degradation, which is involved in nearly all aspects of cell biology [11]. Deubiquitinating enzymes (DUBs) can prevent ubiquitin-mediated degradation of target proteins [12]. Importantly, dysregulated DUBs expression is frequently associated with the tumorigenesis process, specifically cell self-renewal, apoptosis and EMT [13, 14]. It has been confirmed that DUBs are essential for the regulation of stem cell-related markers and controlling various steps of metastatic progression, including invasion, dissemination and eventual metastasis to distant organs [14, 15]. Ubiquitin specific peptidase 37 (USP37), a novel DUB, is a member of ubiquitin-specific processing proteases family. Human USP37, localized mainly in the cytoplasm, is composed of 979 amino acids harboring three ubiquitin-interacting motifs between the Cys box and His box of the primary sequence [16, 17]. The function of USP37 was initially identified as a potent regulator of the cell cycle where it could accelerate the G1/S transition with exceptional high expression [18, 19]. Previous studies have found that USP37 could regulate the stem cell-related marker SOX2 by binding with its promoter region at the transcriptional level [20]. Pan et al. reported that high levels of USP37 gene expression in lung cancer promoted cell viability as well as the Warburg effect via deubiquitination and stabilization of pluripotent factor c-Myc protein [21]. These advances implicated that USP37 gene may be associated with the stemness of tumor cells facilitating cancer progression. Recent studies indicate that USP37 is a potential factor involved in breast cancer progression [16]. However, the biological function of USP37 in the direct regulation of BCSCs and EMT remains unexplored.
In this study, we found that USP37 expression was upregulated in breast cancer tissues compared with surrounding tissues and its overexpression was significantly correlated with increased rates of mortality. We demonstrated that USP37 was highly expressed in BCSCs. The knockdown of USP37 could inhibit the stemness, cell invasion and EMT via downregulation of Hedgehog pathway. USP37 also interacted with and stabilized glioma-associated oncogene 1 (Gli-1) protein. Additionally, USP37 knockdown enhanced the sensitivity of breast cancer cells to cisplatin in vitro and in vivo. We postulate that USP37 may represent a novel molecular target for breast cancer treatment.
Bioinformatic analysis
Gene data extracted from invasive breast tumor samples was obtained from the TCGA Data Portal. Results of analysis were used to create figures via GraphPad Prism. According to PAM50 gene expression signature, information about four breast cancer subtypes (basal-like, Luminal A, Luminal B, and enriched Her-2) were classified. A scatter plot diagram, where each dot indicated an individual sample, was synthesized using the All Complete Tumors of Breast Invasive Cancer dataset [22, 23]. For survival analysis, clinical data related to invasive breast carcinoma were downloaded from the TCGA database [22]. Kaplan–Meier curves were analyzed by GraphPad Prism.
Gene set enrichment analysis (GSEA) was utilized to evaluate potentially biological mechanism associated with USP37 mRNA expression levels in the TCGA breast cancer samples. GSEA software which was obtained from the Broad Institute draws the result pictures automatically.
Cell culture and animals
Human normal breast epithelial cells (MCF-10A) and breast cancer cell lines (MCF-7, MDA-MB-231, BT549 and T47D) were obtained from the Laboratory of Pathology at Dalian, Medical University. The composite culture of MCF-10A cells used DMEM/F12 media supplemented with insulin (10 μg/mL), cholera toxin (100 ng/mL) (Sigma-Aldrich), EGF (20 ng/mL) (R&D systems, Wiesbaden, Germany), hydrocortisone (500 ng/mL), and L-glutamine (Invitrogen, Gaithersburg, MD, USA). MCF-7 cells were cultured in DMEM/HIGH GLUCOSE (Hyclone, Logan, UT, USA) with 10% FBS. MDA-MB-231 cells were cultured in MEM Alpha Modification medium (Hyclone) supplemented with 10% FBS. In addition, BT549 and T-47D cells were cultured in RPMI-1640 medium (Hyclone) supplemented with 10% FBS. Growth condition for all cells was 37 °C at 5% concentration of CO2.
BALB/c Nude mice (6 to 8 weeks old) were purchased from Vital River Laboratory Animal Technology Company (Beijing, China) and reared according to the Animal Care and Use Committee of Dalian Medical University.
Plasmids, antibodies and reagents
The pEZ-M35-USP37 plasmid was created by FulenGen Co. (Guangzhou, China). Anti-Gli-1 and anti-Smoothened antibodies were obtained from Abcam (Cambridge, MA, USA); all other antibodies were obtained from Proteintech Group, Inc. (Wuhan, China). Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Cycloheximide (CHX), MG132, cisplatin and purmorphamine reagents were obtained from MedChemExpress (Shanghai, China).
siRNA, shRNA, lentivirus
The design and synthesis of siRNAs were completed by RiboBio company (Guangzhou, China), and transfection of siRNAs against USP37 was performed with Lipofectamine 2000. MCF-7 and MDA-MB-231 cells were transfected with USP37 siRNAs or a negative control (NC) siRNA. Lentivirus vectors including short hairpin RNA against USP37 (shUSP37#2) and the negative control (shScramble) were purchased from GenePharma Company (Shanghai, China). The transfected cells were treated with puromycin (Clontech, USA) for selection. To overexpress USP37 in MCF-7 cells, the pEZ-M35-USP37 plasmid (2 μg) were infected into MCF-7 cells via Lipofectamine 2000. Expression was confirmed by RT-qPCR and western blotting. The siRNA or shRNA sequences were listed in Additional file 1: Table S1.
BCSCs isolation
Here, 1 × 107 MCF-7 cells were grown with primary antibody against CD24 following the manufacturer's protocol for the CD24 Microbead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Labeled cells were then incubated with goat anti-mouse IgG microBeads (Miltenyi Biotec) and magnetically separated with MiniMACS columns (Miltenyi Biotec). Acquired CD24− cells were incubated with CD44 microbeads (Miltenyi Biotec) at 4 °C for 15 mins. Cells were again washed and magnetically separated.
Colony formation assay and cell viability assay
Colony formation assay of breast cancer cells was carried out by plating infected cells at a density of 1000 cells/well in a 6-well plate and then different concentrations of cisplatin were added. After 2 weeks of incubation, cells were washed three times with phosphate buffered saline, fixed with methanol and stained with 1% crystal violet. Visible colonies were stained violet and counted for data analysis. The detection of cell viability was performed in accordance with the Cell Counting Kit 8 assay (Dojindo Laboratories, Kumamoto, Japan).
Cell invasion assay
Cells were similarly cultured in MEM Alpha Modification medium for 24 hours. Briefly, 1 × 105 cells were seeded without serum into 24-well insert Transwell chambers (8 μm pore size, Corning, USA) and pretreated with Matrigel (BD, Bioscience, San Jose, CA, USA). Medium supplemented with 20% serum was added into the lower chamber. After 12–16 hours of seeding, cotton swabs were used to clean the upper cells. The cells on the other side of the membrane were stained with 1% crystal violet. A randomly selected area was counted with an optical microscope.
Wound healing assay
Cells were incubated in 12-well plates. When cellular density reached nearly 100%, the cell monolayer was wounded with a 200 μl micro-pipette tip. The wound areas were washed three times with phosphate buffered saline (PBS). Then the medium was changed to MEM Alpha Modification without FBS. The wound areas were micrographed at 0, 12, 24 and 48 h. All assays were performed in triplicate.
Cold PBS was used to wash the transfected cells. Then cells were harvested and treated with a mixture of lysis and 1 × RIPA buffers (Sigma Chemicals, St. Louis, MO, USA). Protein concentrations were estimated using the Easy IIProtein Quantitative Kit (Transgen, China). Thirty to forty microgram of each protein lysate was separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% non-fat milk dissolved in TBST (TBS and 0.01% Tween-20) for 2 h at room temperature, then incubated with the specific antibodies solution overnight at 4 °C. Finally specific antibodies were diluted as follows: USP37, 1:500; Gli-1, 1:500; Smoothened, 1:500; E-cadherin, 1:1000; Snail1, 1:500; N-cadherin, 1:1000; Vimentin, 1:500 and GAPDH (loading control), 1:1000. Finally, the transfer membranes were incubated with anti-IgG secondary antibodies (1:16000 in TBST) for 1 h at 37 °C. The protein band images were captured with a ODYSSEY infrared imaging system.
RNA extraction and real-time quantitative PCR analysis
Total RNA was extracted from cultured cells with Trizol reagent (Transgen, China). In total, 1 μg of total RNA was reverse-transcribed with the All-in-one First-Strand cDNA Synthesis SuperMiX kit (Transgen, China). The mRNA expression levels of USP37, ALDH1, CD24 and CD44 were quantified by real-time PCR. RT-qPCR was analyzed with the iCycler™ Real Time System and a SYBR Premix EX Tag Master mixture kit (Transgen, China) according to the manufacturer's instructions. The relative expression levels of mRNA were evaluated by using the 2−ΔΔCt method. Primer sequences are listed in Additional file 1: Table S1.
Immunofluorescence analysis
Cells were grown to 3 × 103 in 24-well plates at 37 °C and fixed with 100% methanol. Next, cells were cleaned two to three times using PBST (PBS and 0.5% Triton X-100). After blocked with 3% BSA for 2 h at 4 °C, cells were incubated with the following primary antibodies (San Ying Biotechnology, China) overnight at 4 °C: USP37, 1:100; Gli-1, 1:100; Flag, 1:100; E-cadherin, 1:100 and N-cadherin, 1:100. Subsequently, cells were incubated with fluorescence conjugated secondary antibody (Sigma, 1:200) for 1 h and stained the cell nuclei with DAPI (Beyotime, China). Images were viewed with CKX41 Inverted Microscope (Olympus, Japan).
CHX chase assay and co-immunoprecipitation (co-IP) assay
For the CHX chase assay, cells were treated with CHX (50 μg/ml) and harvested at the indicated timepoints. Treated cells were lysed, and the lysates were analyzed by western blotting with anti-USP37 or anti-GAPDH antibodies. For the Co-IP assay, 8 × 106 MCF-7 cells were harvested and lysed in IP lysis buffer containing protease inhibitor. The experiment based on manufacturer's protocols (IP Kit, Proteintech Group) was performed.
Sphere formation assay
Cells (MCF-7, MDA-MB-231, BT549 or T47D) were inoculated into ultra-low attachment 6-well plates (Corning, New York, USA) at a density of 1000 cells/well, and then grown in DMEM/F12 supplemented with B27 (1:50, Invitrogen), 20 ng/ml human recombinant EGF (Sigma-Aldrich, St. Louis, Missouri, USA), 20 ng/ml bFGF (Sigma-Aldrich, St. Louis, Missouri, USA), 4 μg/ml heparin (Sigma-Aldrich, St. Louis, Missouri, USA), and 5 μg/ml insulin (Sigma-Aldrich, St. Louis, Missouri, USA) for 14 days. Cell colonies larger than 60 μm in diameter were counted under an inverted microscope (Olympus Corporation, Tokyo, Japan). After cultivation for 28 days, spheroid cells were collected for western blotting.
Mouse xenograft mode
Animal experiments and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH). Twenty BALB/C nude mice were randomly divided into four groups. The negative control group (shScramble) and shUSP37#2-transfected MCF-7 cells (5 × 105) were resuspended in 100 μl PBS and injected into mammary fat pads. When the size of tumor reached approximately 100 mm3, animals were randomly treated with cisplatin (2 mg/kg) or 0.9% saline. Mice were then retreated with their assigned treatment once every 2 days. The tumor sizes were measured with a Vernier caliper and recorded every other day. The tumor volume was calculated using the formula: tumor volume [mm]3 = (length [mm]) (width [mm])2 × 0.5. After the inoculation of tumors for 3 weeks, xenografted tumors were excised from sacrificed mice then analyzed by immunohistochemistry and western blotting.
Immunohistochemical analysis
Tumor tissues were obtained from mouse xenograft mode. Paraffin-embedded tissue was cut into 5 μm thick slices that were fixed onto glass slides. All human breast cancer tissue arrays were purchased from Shanghai Outdo Biotech (Shanghai, China), including 60 cases of cancer tissues and surrounding tissues (HBre-Duc060CS-03; HBre-Duc060CS-04). These tissue sections were immunostained with corresponding antibodies, and then deparaffinized in xylene and rehydrated with ethanol. Tissue sections were preincubated with 10% normal goat serum, followed by incubation with primary antibody solution overnight at 4 °C. After washing with PBS, slides were incubated with the secondary antibody at 37 °C for 10 min, then cleaned with cold PBS and treated with peroxidase conjugated-biotin streptavidin complex for 10 min. Finally stains were examined with 3,3′-diaminobenzidine and hematoxylin. As previously described [23], the immunostained tissues were scored by multiplying the intensity (0–3) and extent (0–100) of staining.
This study was conducted with the approval of the Ethical Committee and Institutional Review Board of Dalian Medical University.
Statistical analysis was performed with SPSS software version 11.0. Data are expressed as mean ± SD. Differences between two groups were evaluated by Student's t-test. One-way ANOVA was used when comparing multiple groups. P < 0.05 was considered statistically significant. Clinical data analysis of survival and relevant correlations were performed with GraphPad Prism.
USP37 is commonly overexpressed in breast cancer
USP37 has been previously confirmed to be overexpressed in lung cancers cells and tissues [21]. In order to investigate the role of USP37 in tumorigenesis, we examined the breast cancer database of The Cancer Genome Atlas (TCGA) to evaluate the differential expression of USP37 [22]. Analysis of the TCGA database indicated that cancer with USP37 transcripts (n = 517) had a significantly higher expression level than normal samples (n = 29) (P < 0.0001) (Fig. 1a). Similar results were also found via immunohistochemistry analysis (Fig. 1g). To further confirm the overexpression of USP37 gene in breast cancer, we utilized a series of human breast cancer cells (MCF-7, MDA-MB-231, BT549 and T47D) and human normal breast epithelial cells (MCF-10A) to examine USP37 protein expression by western blotting (Fig. 2a). These results confirmed that USP37 was also overexpressed in breast cancer cells compared to normal breast epithelial cells.
Overexpression of USP37 in human BC was correlated with poor-disease outcome. a Cancer with USP37 transcripts apparently had higher expression (n = 517) than normal breast tissues (n = 29) from the TCGA database [22] (***P < 0.0001). The median and interquartile range are represented by black lines. b USP37 transcripts among different subtypes are divided into five distinct molecular subtypes. The parameters were analyzed with the PAM50 gene expression profiling [22]. The median with interquartile range is represented by black lines. c Breast cancer (n = 738 patients) were evaluated with USP37 mRNA levels and results were correlated with overall survival over 25 years. The red line indicates patients with high USP37 transcript (n = 369) and blue line indicates patients with low USP37 transcript (n = 369). P value was analyzed by Kaplan-Meier analysis using GraphPad Prism. d-f GSEA analysis showed that USP37 expression was positively associated with metastasis (d) and cell growth (e) while negatively related to cell apoptosis (f) in the TCGA breast cancer samples. g The USP37 protein level in breast cancer tissues and surrounding tissues are shown by immunohistochemistry (IHC) (Brown: USP37). Scale bars: 100 μm. h USP37 IHC staining scores in breast cancer tissues (n = 60) and surrounding tissues (n = 60) are shown. **P < 0.01
USP37 is highly expressed in breast cancer stem cells. a USP37 expression levels were detected in human normal breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7, MDA-MB-231, BT549 and T47D) via western blotting. *P < 0.05, **P < 0.01. b Protein expression levels of USP37 were tested in spheroid cells and adherent cells by western blotting. *P < 0.05, ***P < 0.001. c mRNA expression levels of USP37 confirmed in MCF-7 cell groups sorted by MACS by CD24 or CD44 marker by quantitative RT-PCR. ***P < 0.001. d Immunofluorescence staining of USP37 in BCSCs and non-BCSCs sorted by MACS with CD24 or CD44 marker in MCF-7 cells (Scale bar: 100 μm)
For investigation of the correlation between USP37 gene and the breast cancer heterogeneity, we also tested USP37 expression in the four cell subtypes using PAM50 gene expression profiling. First, we observed an starkly different tendency within USP37 gene expression among different pathological subtypes of breast cancer cells, including the normal-like subtype having the lowest, and Luminal B type endowed with the highest expression levels of USP37 (p < 0.0001) (Fig. 1b). We next estimated the effect of USP37 as an oncogenic biomarker for overall survival of patients diagnosed with breast cancer. Clinical data from the TCGA database were divided into two groups according to the differential expression of USP37 gene. The results indicated that cancer with higher expression levels of USP37 was significantly correlated with the elevated rates of mortality (P < 0.05) (Fig. 1c). Samples with high USP37 expression also had shorter survival duration than those with low USP37 expression. Moreover, GSEA analysis showed that high USP37 expression was positively associated with metastasis, cell growth and anti-apoptosis in the TCGA breast cancer samples (Fig. 1d–f).In brief, these data confirmed that USP37 gene could act as a cancer biomarker in predicting a worse outcome for breast cancer patients. Collectively, these data suggest that USP37 is abnormally overexpressed in human breast cancer patients and cell lines.
USP37 is highly expressed in breast cancer stem cells
CD24−/CD44+ cells and ALDH1+cells are widely considered to be breast cancer stem cells [24]. To validate the expression levels of the USP37 gene in breast cancer stem cells, we isolated CD24−/CD44+ cell populations from MCF-7 cell lines by magnetic activated cell sorting (MACS). We detected that USP37 was significantly overexpressed in CD24−/CD44+ cells compared to their counterpart CD24+ or CD24−CD44− cells in regards to mRNA levels (Fig. 2c). Immunofluorescence staining further confirmed the upregulation of USP37 expression in BCSCs (Fig. 2d). Additionally, we used breast cancer cell lines to perform sphere-formation experiments and measured the differential expression of the USP37 gene. We confirmed that protein levels of USP37 were evidently elevated in spherical cells compared to their counterpart adherent cells (Fig. 2b). Taken together, these results indicated that elevated expression of USP37 was enriched in breast CSCs and was a novel feature of breast cancer stem cell-like subpopulation.
USP37 knockdown suppresses breast cancer cell migration and invasion by promoting mesenchymal-epithelial transition
Currently, the underlying biological mechanism accounting for the elevated expression of USP37 in breast tumors remains unclear. To investigate the effects of USP37 on breast cancer progression, we used siRNA oligonucleotides to knockdown endogenous USP37 in MCF-7 and MDA-MB-231 cells. USP37 mRNA expression levels significantly decreased with two different siRNAs treated breast cancer cells compared to levels in the control group cells (Fig. 3b). Moreover, USP37 protein was also reduced significantly with siRNA#2 and siRNA#3 treatment of MCF-7 and MDA-MB-231 cells as evidenced by western blotting (Fig. 3a). These results confirmed that USP37 gene expression could be effectively downregulated by siRNA#2 and siRNA#3.
Effect of USP37 expression on breast cancer cell EMT, migration and invasion. a MCF-7 and MDA-MB-231 cells were transfected with USP37 siRNAs or NC siRNA for 48 h. Western blotting analysis of USP37 knockdown efficiency in MCF-7 and MDA-MB-231 cells. Relative expression levels were analyzed by Image-Pro Plus 6.0 software. b Real-time quantitative PCR analysis of USP37 knockdown efficiency in MCF-7 and MDA-MB-231 cells. c, d USP37 knockdown significantly decreased N-cadherin, Snail1 and Vimentin as well as increased E-cadherin expression in MCF-7 and MDA-MB-231 cells. e Immunofluorescence staining of E-cadherin and N-cadherin after transfection with NC siRNA or USP37 siRNA#2 (Scale bar: 50 μm). f, g Representative images of migration assays for MCF-7 and MDA-MB-231 cells after downregulation of USP37 are shown at 12, 24 and 48 h (Scale bar: 200 μm). **P < 0.01, ***P < 0.001. h Representative images of Matrigel-based Transwell assay for MCF-7 and MDA-MB-231 cells after USP37 knockdown are shown at the 48 h time point (Scale bar: 200 μm).*P < 0.05. i MCF-7 cells were transfected with control plasmid (CTL) and a USP37 overexpression plasmid (USP37) for 48 h. USP37 upregulation significantly decreased E-cadherin and increased N-cadherin, Snail1 and Vimentin expressions in both MCF-7 and MDA-MB-231 cells. j Immunofluorescence staining of E-cadherin and N-cadherin after USP37 upregulation (Scale bar: 50 μm). k, l Representative images of migration and Matrigel-based Transwell assay for MCF-7 and MDA-MB-231 cells after USP37 upregulation (Scale bar: 200 μm).*P < 0.05, **P < 0.01, ***P < 0.001
The expression of USP37 gene has been demonstrated to be elevated in patients with a recurrence of cancer, indicating that USP37 levels may be closely related to breast cancer distant metastasis [16]. In order to elucidate the function of USP37 in the migration and invasion of breast cancer cells, we conducted wound healing and Transwell assays to detect the role of USP37 in cell migration and invasion. Knockdown of USP37 evidently inhibited cell migration capacity in both MCF and MDA-MB-231 cells (Fig. 3f, g). The transwell assay revealed that downregulation of USP37 obviously decreased the invading capacity of MCF-7 and MDA-MB-231 cells. In addition, depletion of USP37 in MCF-7 and MDA-MB-231 cells displayed a greater reduction of invasion capacity compared to that of the control cells (Fig. 3h).
Epithelial-mesenchymal transition (EMT) is a dynamic process in which epithelial cells acquire enhanced mobility and invasive properties by losing cell-cell adhesion structures and polarity. To further detect the biological mechanism of USP37 on cell migration and invasion, we focused on the identification whether USP37 affected EMT. We examined typical EMT markers (E-cadherin, N-cadherin, Snail1 and Vimentin) by western blotting. As shown in Fig. 3c and d, knockdown of USP37 significantly decreased Snail1, N-cadherin and Vimentin expression, but increased E-cadherin expression levels. These results indicated that downregulation of USP37 induced the mesenchymal–epithelial transition (MET) process in breast cancer cell lines. Similar results were also found via immunofluorescence analysis (Fig. 3e).
Conversely, USP37 overexpression in MCF-7 cells promoted a mesenchymal phenotype with the downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and Snail1 as evidenced by western blotting (Fig. 3i). Immunofluorescence assay showed that overexpression of USP37 further inhibited E-cadherin expression and upregulated N-cadherin expression (Fig. 3j). Furthermore, upregulation of USP37 markedly promoted invasion and migration of breast cancer cells (Fig. 3k and l). Based on this experimental data, we were able to further demonstrate that USP37 is involved in regulating EMT in breast cancer progression and promotes the migration and invasion capacity of breast cancer cells.
USP37 is essential for the maintenance of cell self-renewal and chemo-resistance
Since USP37 was highly expressed in breast cancer stem cells, we further examined whether USP37 played a role in breast cancer stem cells behavior. We knocked down USP37 in MCF-7 and MDA-MB-231 cells via infection with lentivirus expressing USP37#2 shRNA or control lentivirus (shScramble); we confirmed USP37 gene expression by examining protein levels (Fig. 4a, b). The Hh pathway is considered to control the self-renewal of CSCs in breast cancer [25]; therefore, we tested the expression of representative Hh signaling factors (smoothened and Gli-1) and main stem cell markers (ALDH1 and OCT4) in USP37 knockdown cells and control cells by western blotting analysis. We observed that expression of smoothened, Gli-1, ALDH1 and OCT4 were decreased in USP37 knockdown cells compared with their counterpart cells, indicating that inhibited stemness was accompanied by a decrease in USP37 expression (Fig. 4a and b). In addition, USP37 knockdown significantly inhibited the formation of spheroids, as well as their size and volume, compared to the control cells in MCF-7 and MDA-MB-231 cells (Fig. 4c and d). With regard to chemical sensitivity, the cytotoxic effects of different concentrations of cisplatin on USP37 knockdown cells and their respective control cells were detected after 48 h treatment of cisplatin by CCK-8 assays. The results indicated that USP37 knockdown cells had increased sensitivity to cisplatin-induced growth inhibition and as evidenced by decreased colony formation compared with the control MCF-7 and MDA-MB-231 cells (Fig. 4f–i, k and l). Furthermore, the expression of Smo, Gli-1, ALDH1 and OCT4 were elevated in USP37 overexpression plasmid transfected MCF-7 cells compared with the control cells (Fig. 5a). USP37 overexpression also increased the formation of spheroid and chemo-resistance to cisplatin-induced growth inhibition (Fig. 5b, d).
Downregulation of USP37 weakens cell self-renewal and chemo-resistance to cisplatin. a, b MCF-7 and MDA-MB-231 cells were transfected with shUSP37#2 and shScramble, and expressions of USP37, Smo, Gli-1, ALDH1 and OCT4 were confirmed by western blotting. *P < 0.05, **P < 0.01. c, d Comparison of mammosphere formation among inoculated cells in MCF-7 and MDA-MB-231 cells (original magnification, 4× or 20×).*P < 0.05, ***P < 0.001. e MCF-7-ShScramble and MCF-7-shUSP37#2 cells were treated with cisplatin (1 μg/ml) for 48 h, then treated cells were harvested to detect the levels of Bcl-2, Bax and cleaved caspase 9. GAPDH was examined as a loading control. j MDA-MB-231 cells transfected with shScramble or shUSP37#2 were exposed to 3 μg/ml cisplatin for 48 h, and the levels of Bcl-2, Bax and cleaved caspase 9 were detected via western blotting. f, g, k, l CCK-8 assay and colony formation assay showed that cell viability was decreased in MCF-7 and MDA-MB-231 cells after USP37 knockdown. Quantitation of colony formation in (h) MCF-7 and (i) MDA-MB-231. GAPDH was examined as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001
Overexpression USP37 elevates cell self-renewal and chemo-resistance to cisplatin. a Expressions of USP37, Smo, Gli-1, ALDH1 and OCT4 were detected in MCF-7-CTL or MCF-7-USP37 cells by western blotting. *P < 0.05, **P < 0.01. b Comparison of mammosphere formation among MCF-7-CTL or MCF-7-USP37 cells. *P < 0.05. c MCF-7-CTL and MCF-7-USP37 cells were treated with cisplatin (1 μg/ml) for 48 h, then treated cells were harvested in order to detect the levels of USP37, Bcl-2 and Bax. GAPDH was examined as a loading control. *P < 0.05. d CCK-8 assay showed that cell viability was enhanced in MCF-7 after USP37 upregulation compared to the control group. *P < 0.05, **P < 0.01, ***P < 0.01
To further explore the mechanism involved in cisplatin-induced apoptosis caused by USP37 knockdown, we detected apoptosis-related markers Bcl-2, Bax and cleaved caspase-9 in treated cells. We observed the upregulation of Bax and cleaved caspase-9 and downregulation of Bcl-2 in MCF-7 cells and MDA-MB-231 cells after transfection with USP37 shRNA and/ or treatment with cisplatin for 48 h (Fig. 4e, j). In the Bcl-2 protein family, the Bcl-2/Bax ratio has been shown to affect apoptosis induction [26, 27]. Our data indicated that USP37#2 shRNA combined with cisplatin induced the cell apoptosis with an underlying decrease in the Bcl-2/Bax ratio. Consistent with our hypothesis, an increase in the Bcl-2/Bax ratio might be involved in anti-apoptosis activity induced by USP37 upregulation treatment with cisplatin in MCF-7 cells (Fig. 5c). These data demonstrated that USP37 knockdown induced drug sensitivity by dislocating intracellular apoptosis-related proteins.
Downregulation of USP37 inhibits stemness, cell invasion and EMT via hedgehog signaling pathway in breast cancer
It has been verified that tightly controlled Hh pathway ensures proper development and averts tumor formation in the mammary gland [10]. In ER-positive breast cancer, Hh pathway contributes to the maintenance and regulation of CSC invasion and EMT [28]. We were able to further determine the effect of USP37 on the stemness, cell invasion and EMT via Hh signaling pathway. Purmorphamine (PM) is considered to be an Hh agonist. Smo, Gli-1 and USP37 expression post-PM treatment was examined by western blotting analysis and immunofluorescence analysis. Interestingly, our data indicated that there was a time-dependent increase in the expression of Smo, Gli-1 and USP37 expression following 24 h, 48 h PM treatment (0.5 μM) (Fig. 6a). Immunofluorescence assays showed similar results (Fig. 6b).
USP37 knockdown inhibits stemness, cell invasion and EMT via Hedgehog signaling pathway in breast cancer. a, b MCF-7 cells were incubated with 0.5 μM purmorphamine for 24 and 48 h. a Hedgehog pathway constituents were examined via western blotting. GAPDH was examined as a loading control. **P < 0.01,***P < 0.001. b Immunofluorescence staining images of MCF-7 cells showed the expression of USP37 and Hedgehog pathway constituents. c Protein levels of USP37, Smo, Gli-1, ALDH1, OCT4, E-cadherin, N-cadherin, Snail1 as detected by western blotting after the NC siRNA group or the USP37 siRNA#2 group was treated with 0.5 μM purmorphamine for 48 h. GAPDH was examined as a loading control. **P < 0.01,***P < 0.001. d Cell invasion capacity of the NC siRNA group or the USP37 siRNA#2 group treated with 0.5 μM purmorphamine (Scale bar: 200 μm). e Spheroid formation capacity of MCF-7-ShScramble or MCF-7-shUSP37#2 cells treated with 0.5 μM purmorphamine (original magnification, 4×). f Immunofluorescence staining of E-cadherin and N-cadherin after the NC siRNA group or the USP37 siRNA#2 group treated with 0.5 μM purmorphamine for 48 h. (Scale bar: 50 μm). *P < 0.05, **P < 0.01
These findings are further supported by the ability of USP37 to participate in Hh signaling pathway in breast cancer. Figure 6c shows that silencing of USP37 expression could reverse the positive effects of PM on the Hh pathway. Moreover, the effect of downregulating USP37 on CSC traits, including the formation of spheroid, BCSCs markers and cell invasion, was impaired after PM treatment (Fig. 6c, d and e and Additional file 2: Figure S1). Silencing of USP37 expression induced MET including upregulated E-cadherin expression and downregulated N-cadherin expression, while PM reversed MET in silenced USP37 cells. Similar results were observed with immunofluorescence assay (Fig. 6f). Collectively, these data indicate that downregulation of USP37 expression was involved in the attenuation of the BCSCs stemness, cell invasion and EMT by suppression of the Hh pathway.
USP37 interacts with and stabilizes Gli-1 protein
Given that USP37 expression was significantly associated with the Hh pathway, we detected whether endogenous USP37 could also regulate Gli-1 stability. Endogenous USP37 was depleted by lentivirus expressing USP37#2 shRNA in MCF-7 and MDA-MB-231 cell lines. The effect of USP37 knockdown on Gli-1 was evidently reversed by MG132, which is a protease inhibitor (Fig. 7a and b). Furthermore, knockdown of USP37 expression significantly decreased the stability of Gli-1 protein both in MCF-7 and MDA-MB-231 cells (Fig. 7d and e). Additionally, the cycloheximide (CHX) chase assay was used to assess MCF-7 breast cancer cell line for the effect of USP37 upregulation on endogenous Gli-1 protein. As shown in Fig. 7f, upregulation of USP37 expression consistently stabilized Gli-1 protein. Immunofluorescence staining assays showed that USP37 could regulate Gli-1 expression (Fig. 7c). Co-IP assay indicated that USP37 interacted with Gli-1(Fig. 7h). We also found overlapping expression of exogenous USP37 and Gli-1 by immunofluorescence staining (Fig. 7g). Together, these data suggest endogenous USP37 may regulate Gli-1 protein stability in breast cancer.
USP37 interacts with and stabilizes Gli-1 protein. a, b MCF-7 and MDA-MB-231 cells were transfected with shScramble and shUSP37#2, respectively, then treated with 10 μM of MG132 4 h before harvesting. c Immunofluorescence staining of Gli-1 after USP37 knockdown or USP37 overexpression (Scale bar: 50 μm). d, e Cells transfected with shScramble and shUSP37#2 were treated with 50 μg/ml of CHX and harvested at the indicated time point. f MCF-7-CTL and MCF-7-USP37 cells were incubated with 50 μg/ml of CHX and harvested at the indicated time point. Gli-1 expression was analyzed by western blotting and quantified with Image-Pro Plus 6.0 software. g Exogenous USP37 and Gli-1 magnified strong, positive overlapping expression in MCF-7 cells by immunofluorescent staining (Scale bar: 50 μm). h CO-IP assay showed the interaction between endogenous USP37 and Gli-1 in MCF-7 cells
USP37 knockdown inhibits tumorigenicity and increases sensitivity to cisplatin in vivo
To further assess the anti-tumor effect of USP37 downregulation on tumor growth and cisplatin sensitivity in vivo, we established a xenograft tumor models by subcutaneously injecting breast cancer MCF-7 cells, which had been transfected with USP37#2 shRNA or scramble shRNA, into the mammary gland of nude mice (Fig. 8a). Tumor volume and weight measurements were analyzed. Compared with the scramble shRNA control group, the volume and weight of USP37 shRNA group increased at slower rate (Fig. 8b, c). The average tumor volume and weight of cisplatin-treated USP37#2 shRNA tumors was evidently smaller than that of cisplatin-treated tumors or USP37#2 shRNA tumors. We found that lower expression level of USP37 obviously impaired the expression of Hh targets (Smo and Gli-1) and cell proliferation marker Ki-67 in the tumor tissues as seen by immunohistochemical staining (Fig. 8e). Consistent with these results, tissues formed from cisplatin-treated USP37 knockdown cells showed the lowest expression levels of Smo, Gli-1, ALDH1, OCT-4 and Bcl-2 than other tissues by western blotting analysis (Fig. 8d). These results indicated that USP37 downregulation attenuates breast cancer progression and enhances sensitivity to cisplatin in vivo.
USP37 knockdown inhibits tumorigenicity and increases sensitivity to cisplatin in vivo. a, b, c MCF-7 cells transfected with shScramble or shUSP37#2 were subcutaneously injected into BALB/c nude mice treated with 0.9% saline or cisplatin. Tumors were excised from nude mice at day 28. a Images of tumor xenografts and b tumor weight for tumor xenografts treated with 0.9% saline or cisplatin are shown above. *P < 0.05, **P < 0.01. c Tumor growth curves were drawn every other day up to day 25. d Western blotting analysis of USP37, Smoothened, Gli-1, ALDH1, OCT4 and Bcl-2 in tumor xenografts. GAPDH was used as a loading control. *P < 0.05, **P < 0.01. e HE staining and immunohistochemical detection of USP37, Smoothened, Gli-1, Ki-67 in tumor xenografts. Scale bar: 100 μm
DUBs have been shown to participate in ubiquitin cleaving from ubiquitin conjugated protein substrates [29, 30]. USP37, a novel DUB, contains an insert between its catalytic lobe and its ubiquitin-binding lobe while its function could prevent 14-3-3γ degradation, which might contribute to malignant transformation by MAPK signaling [31, 32]. Subsequently, it was verified that USP37 expression modulated the oncogenic fusion protein PLZF/RARA stability and cell transformation potential in PLZF/RARA-associated acute promyelocytic leukemia [33]. Recently, clinicopathological analysis confirmed that USP37 was a poor prognostic factor in breast cancer [16]. However, there was no direct evidence to identify the carcinogenic mechanism of the USP37 gene in breast cancer. In this study, we have demonstrated overexpression of USP37 in Luminal B subtype breast cancer was a predictor of poor outcomes in breast cancer. Supporting our results, previous clinicopathological analysis has demonstrated that overexpressed USP37 is considered to be a poor prognosis in breast cancer [16]. Additionally, GSEA analysis on the TCGA dataset implicated that USP37 expression was positively associated with metastasis, cell growth and anti-apoptosis. Therefore, USP37 levels could potentially serve as a specific oncogene involved in breast carcinoma progression.
Cancer stem cells (CSCs) are endowed with stem cell properties, which include the ability to self-renew and differentiate by symmetrical or asymmetrical cell division. CSCs self-renewal of the cellular population and generation of progenitor cells have been shown to resist radiation and chemotherapy. Therefore, these cells are commonly deemed to be crucial target for cancer therapy [34, 35]. It was verified that CSCs could be maintained in an undifferentiated status and induce tumor-sphere formation in defined serum-free medium [36]. However, limited markers were utilized for the identification of breast cancer stem cells. While researchers have demonstrated that CD44 and ALDH1 are critical biomarkers to identify BCSCs from breast cancer populations [37, 38], our data suggested that the USP37 gene was significantly associated with CSC properties, such as self-renewal, treatment resistance and EMT phenotype as well. Interestingly, elevated mRNA expression of USP37 was detected in CD24−/CD44+ cells and ALDH1+ cells; the protein expression of USP37 was obviously elevated in spheres compared to adherent cells. Knockdown of USP37 suppressed mammospheres formation and inhibited cancer stem markers, such as ALDH1and OCT4 in breast cancer cells. It has been reported that USP37 participates in regulation of CSCs-related proteins, such as SOX2 and c-myc [20, 21]. Our data further indicated that knockdown of USP37 by constructed lentiviral system weakened the stemness in MCF-7 and MDA-MB-231 cells via inhibiting the expression levels of ALDH1 and OCT4. CSCs are considered to be "bad seeds" due to their drug resistance caused by imbalanced pathway and epigenetics in cancer [39]. As shown above, we detected that knockdown of USP37 in breast cancer cells could promote the sensitivity to cisplatin-induced cell death by the CCK-8 assay and colony formation assays. The Bax/Bcl-2 ratio is a commonly used method to determine whether intracellular apoptosis system is activated [40]. We detected that USP37#2 shRNA combined with cisplatin treatment induced cell apoptosis with an underlying decrease in Bcl-2/Bax ratio. On the contrary, the upregulation of USP37 reversed this phenomenon. USP37 knockdown suppressed stemness and chemo-resistance, which may assist clinical oncologists in designing and testing novel therapeutic strategies. In summary, we suggest that USP37 gene expression confers the stemness and potentially acts as a critical marker of CSCs in breast cancer.
The process of EMT was involved in the acquisition of aggressive cellular traits, including motility, invasiveness and anti-apoptosis, resulting in the dissemination of cells and colonization in distance tissues [6, 41, 42]. Here, we showed that USP37 siRNA treatment stimulated the expression of epithelial marker (E-cadherin) but decreased the expression of three known inducers of EMT (Snail1, N-cadherin and Vimentin) (Fig. 6a). Moreover, we found that knockdown of USP37 suppressed cell migration and invasion in breast cancer cells. On the contrary, upregulation of USP37 promoted EMT, migration and invasion. Accumulation evidence has indicated that tumor cells undergoing EMT process are endowed with the trait of cancer stem-like cells [43], which further speculated USP37 as a CSC marker of breast cancer. In summary, our data showed that USP37 could regulate the migration, invasion, EMT of breast cancer cells.
The Hh pathway was found to be required for the maintenance of breast CSCs traits, tumor formation and the EMT [10]. Normally, the ligand binding of Patched (Ptch1), a 12-pass transmembrane receptor of Sonic Hedgehog (SHH), can activate zinc finger transcription factor (Gli-1). It has been suggested that ectopic expression of Gli-1 upregulates expression of the transcription factor Snail1 accompanied with a decrease in E-cadherin, a characteristic of EMT [24]. In fact, Snail expression promotes EMT via repressing E-cadherin [44]. Hh signaling pathway modulates EMT indirectly via Snail [45]. Therefore, it is a promising research to investigate whether USP37 promoted the activation of the Hh pathway.
Our findings suggested that the post transcriptional levels of Smo and Gli-1 were decreased in vitro and in vivo. Upregulation of USP37 could enhance protein expression levels of Smo and Gli-1. In order to understand the mechanism of USP37-induced EMT and stemness via the Hh pathway, we treated MCF-7 cells with PM. We found that activation of Hh signaling pathway was accompanied by elevated expression of USP37 gene as visualized by western blotting and immunofluorescence assay. Furthermore, the effect USP37 downregulation on CSCs traits including the formation of spheroids, BCSCs markers and cell invasion were impaired after PM treatment. Meanwhile, knockdown of USP37 reversed the effect of PM on the EMT markers. These data confirm that USP37 mediates breast cancer stem-like properties, cell invasion and EMT via the Hh pathway. Gli-1 acts exclusively as a transcriptional activator in the Hh pathway and signal outcomes are determined by the balance of activated and inhibitive Gli proteins [46]. Remarkably, we found that USP37 could regulate and stabilize the protein level of Gli-1 (Additional file 2 : Figure S1). As a deubiquitinase, USP37 is involved in the regulation of multiple proteins by deubiquitination, including P27, Cdt1, PLZF/RARA and 14-3-3γ [31, 33, 47, 48]. Future work should aim to determine whether USP37 could stabilize Gli-1 through deubiquitination.
In conclusion, our research demonstrated that USP37 was highly expressed in breast CSCs and was correlated with poor prognosis in breast cancer patients. Knockdown of USP37 expression hampered cell invasion, stemness, EMT and also resulted in the drug sensitivity to cisplatin. Mechanically, USP37 could orchestrate the stemness, cell invasion and EMT via activation of Hedgehog pathway. Further studies detected that USP37 also interacted with and stabilized Gli-1 protein, which is the main activator of Hedgehog target gene. These results indicate that USP37 is essential in the regulation of breast cancer progression suggesting an experimental basis for its use as a cancer biomarker and expands its potential clinical value.
ALDH1:
Aldehyde dehydrogenase1
BCSCs:
Breast cancer stem cells
DUBs:
Deubiquitinating enzymes
EMT:
Epithelial-mesenchymal transition
Gli-1:
Glioma-associated oncogene 1
GSEA:
Gene set enrichment analysis
MACS:
Magnetic activated cell sorting
Mesenchymal–epithelial transition
Purmorphamine
Smo:
Smoothened
USP37:
Ubiquitin specific peptidase 37
This work was supported by Liaoning Provincial program for Top Discipline of Basic Medical Sciences and National Natural Science Foundation of China (81272430, 81650018).
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.
QT, LM and L-LH conceived and designed the study; QT, LB and F-XY performed the experiments; Y-JF, Z-QQ and L-DD analyzed the data; LL, MJ, LY and Y-XT contributed reagents/materials/analysis tools; QT, F-SJ and ZJ discussed results and advised during the completion of the study; QT, SB and L-LH wrote and revised the paper. All authors read and approved the final manuscript.
This research was reviewed and approved by the Ethical Committee and Institutional Review Board of Dalian Medical University. Animal experiments and procedures were approved by the Animal Care and Use Committee of Dalian Medical University and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH).(No. AEE17046). The tissue samples of patients diagnosed with breast cancer were used in this research provided written informed consent, and the study protocol was approved by the Committee for the Ethical Review of Research, The Second Affiliated Hospital of Dalian Medical University.
Additional file 1: Table S1. Sequences of primer, siRNA and shRNA. (DOCX 14 kb)
Additional file 2: Figure S1. Protein level of OCT4 as detected by western blotting after the NC siRNA group or the USP37 siRNA#2 group was treated with 0.5 µM purmorphamine for 48 h. GAPDH was examined as a loading control. (DOCX 80 kb)
Department of Pathology, Dalian Medical University, Dalian, 116044, People's Republic of China
The Key Laboratory of Tumor Stem Cell Research of Liaoning Province, Dalian Medical University, Dalian, 116044, People's Republic of China
Department of Urology, The First Affiliated Hospital of Dalian Medical University, Dalian, People's Republic of China
Teaching Laboratory of Morphology, Dalian Medical University, Dalian, 116044, People's Republic of China
Department of Pathology, Xiangyang Central Hospital, Xiangyang, 441000, People's Republic of China
Department of Dean, Dalian Medical University, Dalian, 116044, People's Republic of China
Department of Oncology, The Second Affiliated Hospital of Dalian Medical University, Dalian, 116023, Liaoning Province, People's Republic of China
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text:
import * as constants from '../../utils/constants';

const initialState = {
    isLoading: true,
    createEmployeeError: false,
    createEmployee: false,
    employees: [],
};

export default function employeeGet(state = initialState, {type, payload}) {
    switch (type) {
        case constants.EMPLOYEES_GETTING:
            return {
                ...initialState,
                isLoading: true,
            };

        case constants.EMPLOYEES_GOT:
            return {
                ...initialState,
                isLoading: false,
                employees: payload.users
            };

        case constants.EMPLOYEES_ERROR:
            return {
                ...initialState,
                isLoading: false,
            };

            case constants.EMPLOYEE_ADD:
            return {
                ...initialState,
                isLoading: true,
            };

            case constants.EMPLOYEE_ADDED:
            return {
                ...initialState,
                isLoading: false,
                createEmployee: true,
            };

            case constants.EMPLOYEE_ADD_ERROR:
            return {
                ...initialState,
                isLoading: false,
                createEmployeeError: true
            };
        default:
            return state;
    }
}

redpajama_set_name: RedPajamaGithub


text:
Q: Use Xpath To Filter Records I wonder whether someone can help me please.
I'm using the following script to load images from an XML file into a web page.
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

<?php

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  $galleryPath = 'UploadedFiles/';

  $thumbnailsPath = $galleryPath . 'Thumbnails/';

  $absGalleryPath = realpath($galleryPath) . DIRECTORY_SEPARATOR;

  $descriptions = new DOMDocument('1.0');
  $descriptions->load($absGalleryPath . 'files.xml');
?>

  <title>Gallery</title>
  <meta http-equiv="Content-Type" content="text/html; charset=UTF-8" />
  <link href="Libraries/fancybox/jquery.fancybox-1.3.1.css" rel="stylesheet" type="text/css" />
  <link href="Styles/style.css" rel="stylesheet" type="text/css" />

  View Uploaded Images In Folder Structure  ← View All Uploaded Images

  <form id="gallery" class="page">

          <?php if ('Uploaded files' != $current['title']) :?>
          <?php endif;?>

                  <?php for ($i = 0; $i < $descriptions->documentElement->childNodes->length; $i++) :
                          $xmlFile = $descriptions->documentElement->childNodes->item($i);
                          $name = htmlentities($xmlFile->getAttribute('originalname'), ENT_COMPAT, 'UTF-8');
                          $description = htmlentities($xmlFile->getAttribute('description'), ENT_COMPAT, 'UTF-8');
                          $folder = htmlentities($xmlFile->getAttribute('folder'), ENT_COMPAT, 'UTF-8');
                          $source = $galleryPath . rawurlencode($xmlFile->getAttribute('source'));
                          $thumbnail = $thumbnailsPath . rawurlencode($xmlFile->getAttribute('thumbnail'));
                  ?>

                      <a class="fancybox" target="_blank" rel="original" href="<?php echo $source; ?>"><img class="preview"
                        alt="<?php echo $name; ?>"  src="<?php echo $thumbnail; ?>" />

Image Description: <?php echo htmlentities($xmlFile->getAttribute('description'));?>

                            Image contained in folder: <?php echo htmlentities($xmlFile->getAttribute('folder'));?>

                          <?php endfor; ?>

  </form>

Amended Code With XPath - NB Not A Working Solution
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

<?php

  //This variable specifies relative path to the folder, where the gallery with uploaded files is located.
  //Do not forget about the slash in the end of the folder name.
  $galleryPath = 'UploadedFiles/';

  $thumbnailsPath = $galleryPath . 'Thumbnails/';

  $absGalleryPath = realpath($galleryPath) . DIRECTORY_SEPARATOR;

  $descriptions = new DOMDocument('1.0');
  $descriptions->load($absGalleryPath . 'files.xml');

   $xpath = new DOMXPATH($descriptions);
?>

  <title>Gallery</title>
  <meta http-equiv="Content-Type" content="text/html; charset=UTF-8" />
  <link href="Libraries/fancybox/jquery.fancybox-1.3.1.css" rel="stylesheet" type="text/css" />
  <link href="Styles/style.css" rel="stylesheet" type="text/css" />

  View Uploaded Images In Folder Structure  ← View All Uploaded Images

  <form id="gallery" class="page">

          <?php if ('Uploaded files' != $current['title']) :?>
          <?php endif;?>

                  <?php for ($i = 0; $i < $descriptions->documentElement->childNodes->length; $i++) :
                          $name = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@originalname");
                          $description = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@description");
                          $folder = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@folder");
                          $source = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@source");
                          $thumbnail = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@thumbnail");
                  ?>

                      <a class="fancybox" target="_blank" rel="original" href="<?php echo $source; ?>"><img class="preview"
                        alt="<?php echo $name; ?>"  src="<?php echo $thumbnail; ?>" />

Image Description: <?php echo $description;?>

                            Image contained in folder: <?php echo $folder;?>

            <?php endfor; ?>

  </form>

and this is the XML file which is loaded into the page:
  <?xml version="1.0" encoding="utf-8" ?>
- <files>
  <file name="Test 1/_47061196_greatbritainjpg.jpg" source="_47061196_greatbritainjpg.jpg" size="227505" originalname="_47061196_greatbritainjpg.jpg" thumbnail="_47061196_greatbritainjpg.jpg" description="No description provided" userid="1" locationid="1" folder="Test_1" />
  <file name="Test 1/article-0-07D01B74000005DC-138_468x617.jpg" source="article-0-07D01B74000005DC-138_468x617.jpg" size="143110" originalname="article-0-07D01B74000005DC-138_468x617.jpg" thumbnail="article-0-07D01B74000005DC-138_468x617.jpg" description="No description provided" userid="1" locationid="1" folder="Test_1" />
  <file name="Test 1/stags-snow_1544533c.jpg" source="stags-snow_1544533c.jpg" size="21341" originalname="stags-snow_1544533c.jpg" thumbnail="stags-snow_1544533c.jpg" description="No description provided" userid="1" locationid="1" folder="Test_1" />
  </files>

What I would like to do is to make the images loaded user specific, so for example 'userid' of value '1' at 'locationid' value '1' can only view those images with the matching values in the XML file.
I know from the tutorials and articles I've read that an 'Xpath' statement is able to do this. However there appears to be many different ways to do this, and having very, very, little of XML I'm really not sure where to start.
I just wondered whether someone could perhaps offer me some help please to help me to be able to achieve this.
Kind regards

A: No problem!
For your xpath statement, I'm assuming you would want to return the filename of whichever  xml node that contains the userid and locationid you need.
So you would first start at the root node , then check which file has this criteria:
locationid and userid are both attributes of , so you need to use the @ symbol when referencing them in xpath.
$xpath = new DOMXPATH($descriptions) ;
$result = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@name");

This might look a little confusing since your attribute names are the same as the input names, but the variables in the ". ." are referring to the block:
<input name="userid" type="text" id="userid" value="1" />
<input name="locationid" type="text" id="locationid" value="1" />

So this would give you the name of the file for that user, which you could apply to your $source  tag. Just repeat the given xpath and append /@description or /@thumbnail in place of name to get the other values.
Hope this helps, let me know if I need to clarify anything!
Try this:
(not sure where your input form went in the original code, but if you're still using it:)

                  <?php for ($i = 0; $i < $descriptions->documentElement->childNodes->length; $i++) :
                          $name = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@originalname");
                          $description = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@description");
                          $folder = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@folder");
                          $source = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@source");
                          $thumbnail = $xpath->query("files/file[@userid=" . userid . " and @locationid=" . locationid . "]/@thumbnail");
                  ?>

                      <a class="fancybox" target="_blank" rel="original" href="<?php echo $source; ?>"><img class="preview"
                        alt="<?php echo $name; ?>"  src="<?php echo $thumbnail; ?>" />

Image Description: <?php echo $description;?>

                            Image contained in folder: <?php echo $folder;?>

            <?php endfor; ?>

redpajama_set_name: RedPajamaStackExchange


Contact Neaher Law now by filling out the form below or by calling 239-785-3800.
See below for map and directions to our office. Please feel free to call us at 239-785-3800 to schedule your appointment now.
To speak to an attorney about your legal matter, please contact Neaher Law today.
Attorney at Law offering Estate Planning, Probate, Family Law, HOA/Condo Law and Legal Services in Fort Myers, Cape Coral, Bonita Springs, Lee County, Collier County, Port Charlotte County and all of Southwest Florida.
Copyright © 2019. Neaher Law, PLLC All Rights Reserved - Attorney at Law offering Estate Planning, Probate, Family Law, HOA/Condo Law and Legal Services in Fort Myers, Cape Coral, Bonita Springs, Lee County, Collier County, Port Charlotte County and all of Southwest Florida.


text:
DUI Lawyer in Highland, IN
Request a free legal consult from a DUI Lawyer serving Highland, IN
DUI accidents reported in Highland, IN
Highland, IN | Accident | 10 days ago
Alcohol Suspected In accident On 9500 S WENTWORTH AVE, IL, US
This accident, involving 2 vehicles occurred on Monday, January 10th 2022 at approximately 1:00 pm. There were no injuries, to either drivers or passengers reported. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected In accident On 10105 S RHODES AVE, IL, US
This accident, involving 2 vehicles occurred on Saturday, January 8th 2022 at approximately 8:00 pm. There were no injuries, to either drivers or passengers reported. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected In accident On 10800 S WENTWORTH AVE, IL, US
This accident, involving 2 vehicles occurred on Monday, January 3rd 2022 at approximately 3:23 pm. There were no injuries, to either drivers or passengers reported. Snow was covering the roads. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected 1 Injured In accident On 9002 S JEFFERY BLVD, IL, US
This accident, involving 2 vehicles occurred on Monday, December 27th 2021 at approximately 3:40 pm. Injuries were reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected In accident On 9030 S COTTAGE GROVE AVE, IL, US
This accident, involving 2 vehicles occurred on Sunday, December 26th 2021 at approximately 11:30 pm. There were no injuries, to either drivers or passengers reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Highland, IN | Accident | 3 months ago
Alcohol Suspected In accident On 11100 S PERRY AVE, IL, US
This accident, involving 2 vehicles occurred on Wednesday, October 20th 2021 at approximately 2:30 pm. There were no injuries, to either drivers or passengers reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected In accident On 9833 S VAN VLISSINGEN RD, IL, US
This accident, involving 2 vehicles occurred on Thursday, October 14th 2021 at approximately 5:40 pm. There were no injuries, to either drivers or passengers reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected In accident On 10536 S EWING AVE, IL, US
This accident, involving 2 vehicles occurred on Thursday, October 14th 2021 at approximately 11:20 am. There were no injuries, to either drivers or passengers reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected 1 Injured In accident On 9500 S EWING AVE, IL, US
This accident, involving 2 vehicles occurred on Monday, October 4th 2021 at approximately 1:35 pm. Injuries were reported. It was reported that it was raining at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.
Alcohol Suspected 3 Injured In 3 Vehicle Collision On 10612 S TORRENCE AVE, IL, US
This accident, involving 3 vehicles occurred on Friday, September 17th 2021 at approximately 10:33 pm. Injuries were reported. Weather conditions were reported as clear at the time of the accident. An officer from Chicago Police Department was present at the scene and recorded the information contained in this report.

redpajama_set_name: RedPajamaCommonCrawl


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The controversy on Fort Myers Beach continues over a proposed Grand Resorts development.
A group of business owners and community leaders released a statement Tuesday saying the developer is violating components of the town's comprehensive plan.
Three resorts, a seawall, a boardwalk, a parking garage and a roundabout are all part of Tom Torgerson's grand design, and a spokesperson said the project fits "very nicely" with the town's plan.
But Connie Ramos Williams, spokesperson for the group known as the Voice of Fort Myers Beach, disagrees.
"They are not following the comp plan to a T," she said. "The rooms and units that they are putting in their proposed plan are more than double the number allowed by the town's comp plan."
Indeed, the town's plan says a hotel is allowed 201 units. Grand Resorts is requesting 509, and suggests the project requires compromise from both sides.
"We are nine feet below what we would be allowed on the parking garage, for example," said Tina Matte, a spokesperson for the development project. "So I think it's really looking in context of what the downtown area seeks to do. We could take the same number hotel rooms and flatten it out but then it would take up much more of the beachfront and you wouldn't have that same experience."
Residents, though, say the comprehensive plan was created 17 years ago for a reason -- and it shouldn't be ignored.
"I think we are all willing to compromise," said Doris Grant, "but let's do it according to our comp plan."
The mayor says no formal application has been submitted, so it's too soon to say if the comprehensive plan is being followed or not. The developers say a completed application is expected within three or four weeks.


Conquer Crohn's & Colitis at the Lincoln Marathon & Half Marathon!
Join Team Challenge as we fight IBD at the 41st Annual Lincoln Marathon & Half Marathon on May 6, 2018! We are excited to offer this opportunity to raise funds and awareness in the Nebraska/Iowa Chapter. Join our team. Run or walk. Help cure digestive disease.
Fundraising Goal Deadline: In order to receive your Team Challenge race day jersey you must raise $1,000 by April 16, 2018.
STEPS to REGISTER: Visit www.lincolnmarathon.org and register for the race (Marathon or Half Marathon) on January 6th. CAUTION: Race sold out in 10 hours last year, so we recommend registering by 8am on Saturday, January 6th!
Complete this very brief Team Registration survey below.
You're all set - let's do this!
Want to Learn More? Contact Aaron Castens at acastens@crohnscolitisfoundation.org.


text:
AMC Sets Dates for Premieres of 'Turn' and Return of 'Mad Men,' Slates 'Better Call Saul' for Fall
AMC Sets Dates for Premieres of 'Turn' and Return of 'Mad Men,' Slates 'Better Call Saul' for Fall
@alisonwillmore
AMC has announced dates for its 2014 original programming schedule today, including the premieres of new series "Turn" and the first half of the final season of "Mad Men." But what may be most interesting for people is that "Breaking Bad" prequel "Better Call Saul" is set to debut in the fall.
"Turn," the first of two new dramas the network is debuting this year, will kick off on Sunday, April 6th at 9pm. The series, which stars Jamie Bell, is about George Washington's spy ring during the Revolutionary War. The first episode, which was directed by Rupert Wyatt ("The Escapist"), will run 90 minutes.
The first half of the final season of "Mad Men" will launch one week later, on Sunday, April 13th at 10pm. The 14-episode seventh season is being split into two parts, with the second half airing in 2015.
AMC's second new series, the 1980s tech drama "Halt & Catch Fire" starring Lee Pace, Scoot McNairy and Kerry Bishé, will premiere in June, while western "Hell on Wheels" will kick off its fourth season later in the summer.
"The Walking Dead" will have its season five premiere in October, and after show "The Talking Dead" has been renewed for a fifth season. And yes, "Better Call Saul" is really happening and is on the schedule for fall, with a November premiere date.
This Article is related to: Television and tagged AMC, Better Call Saul, Halt and Catch Fire, Mad Men, Television, The Walking Dead, Turn, TV News


As we all know that the valentine's day is nearly coming to celebrate. It is the event that is considered to be as the lovers day. Boys and girls who are in love must celebrate this event. Many of us, arrange parties and late night gatherings with friends. So, if you are also invited for valentines day party, then you are very busy in preparation of it. There are so many ways to enhance the beauty of woman ion valentine's day. The red color is the theme of valentine's day and from head to toe, girls love to wear red color outfit. Apart of clothes, different accessories are considerably used to enhance the beauty of woman, in which nail art is very well known. Yes, here in this post i am sharing beautiful nail art designs 2015 for valentines day below.
Nail art is one of the most popular way to decorate hands. It is known to be as the fun of art that is widely popular in women. Mostly young girls are always seem to be playing with different nail art designs. There are so many nail art designs that are wearing according to occasion as well as mood. If we talk about the nail art designs 2015 for valentines day, then it must be very artistic and stunning. Nail art designs 2015 for valentines day should be in red color. You can find so many beautiful designs on websites. Here in this post i am also sharing some beautiful nail art designs that are perfect for valentines day. With these nail art designs, you can easily express your love for other.
There are so many nail art patterns such as floral, heart shape, love nail art designs, and so many more. You can also use 3D stickers to make nail art designs awesome and lovely. Let's check out these beautiful nail art designs below.


There were a higher than normal number of changes in the 'Top 40' Chart last month.
This may be because fewer people frond the site through Google searches in May 2014.
Why was that? The EdinPhoto web site still has a page ranking of 5/10 on Google - a ranking that it has had for several years - but since 23 April far fewer viewers have been referred to the site from Google.
The EdinPhoto web site is entirely non-commercial, so I'm not looking for more visitors to do more business, and I'm already receiving more emails than I'm able to cope with, so a reduction in the number of visitors from Google in not likely to cause a problem for me - but it would be interesting to discover the reason for the change.
I believe that Google are continually changing their Search Engine algorithm - 500 times a year, I've read - but the change in April this year seems to have had quite a dramatic effect, at least for the EdinPhoto site!
(a) 187,716 sessions in May (2% more than in April).
(b) 745,083 pages viewed in May (7% fewer than in April)).
(c) The average session was 4.0 pages in May (0.4 fewer than in April).


text:
Chinese Martial Arts and Media Culture
Edited by Tim Trausch
Signs and images of Chinese martial arts increasingly circulate through global media cultures. As tropes of martial arts are not restricted to what is considered one medium, one region, or one (sub)genre, the essays in this collection are looking across and beyond these alleged borders. From 1920s wuxia cinema to the computer game cultures of the information age, they trace the continuities and transformations of martial arts and media culture across time, space, and multiple media platforms.
Rowman & Littlefield Publishers / Rowman & Littlefield International
Pages: 238 • Trim: 6⅜ x 9
978-1-78660-902-1 • Hardback • November 2018 • $135.00 • (£90.00)
978-1-78660-903-8 • eBook • November 2018 • $43.99 • (£29.95)
Series: Martial Arts Studies
Subjects: Social Science / Popular Culture, Social Science / Regional Studies, Social Science / World / Asia
Tim Trausch is a research associate in the Department of East Asian Studies at the University of Cologne. His research focuses on Chinese media culture and aesthetics. He has published essays on Chinese-language film and television, and a book on the aesthetics of martial arts cinema. His current work focuses on photography and modernity in late imperial and Republican-era China.
Introduction: Martial Arts and Media Culture in the Information Era: Glocalization, Heterotopia, Hyperculture (Tim Trausch)
The Demise of the Wuxia Film? - The Mutation of a Genre from Manifestation of Crisis to Postmodern Pastiche and Reaffirmation of Centralized Power (Clemens von Haselberg)
Transposing Jianghu in Chinese Martial Arts Cinema From the 20th Century to the 21st Century (Helena Wu)
A Touch of Sin, Translation, and Transmedial Imagination (Carlos Rojas)
The Effortless Lightness of Action: Hong Kong Martial Arts Films in the Age of Immediacy (Man-Fung Yip)
Imagining Transcultural Mediascapes: Martial Arts, African Appropriation, and the Deterritorializing Flows of Globalization (Ivo Ritzer)
From the Boxers to Kung Fu Panda: The Chinese Martial Arts in Global Entertainment (John Christopher (Chris) Hamm)
Bruce Lee, Bruceploitation, and Beyond: Renegotiating Discourses of Original and Copy (Tim Trausch)
David Henry Hwang's Kung Fu in Cross-Cultural Perspectives (Kin-Yan Szeto)
In Search of the 36th Virtual Chamber - Martial Arts in Video Games From Screen Fighting to Wuxia Worldbuilding (Andreas Rauscher)
The MUD Era: The Origins of Chinese Martial Arts Online Games (Zheng Baochun, Wang Mingwei)
Martial Arts and Media Supplements (Paul Bowman)
Scholars have traditionally separated the study of the 'Asian martial arts' as bodily practice from the examination of the ideas and images that uphold them. This collection expertly problematizes that notion while showcasing innovative insights from media and cultural studies. Fresh and always fascinating, it will be critical reading for anyone curious about the place of the martial arts in the modern world.
— Benjamin N. Judkins, Visiting Scholar, Cornell University East Asia Program and co-author of The Creation of Wing Chun: A Social History of the Southern Chinese Martial Arts
This lively, varied and innovative collection breaks the confines of film and area studies to chart representations of the martial arts as they circulate in our global media culture. Doing so, it offers valuable new insights into both the transnational media landscape and the meaning of the martial arts within this, not only today but also historically.
— Luke White, Senior Lecturer in Visual Culture and Fine Art, Middlesex University, UK
Whether examining the 'traces' of Bruce Lee, the 'effortless' action of Hong Kong martial arts cinema or online wuxia worldbuilding, this book is an essential read for anyone interested in the re-shaping of Chinese martial arts in the mediascapes of the 20th and 21st centuries. Covering film, TV, theatre, literature and gaming, it's a veritable 36th Chamber of interdisciplinary perspectives.
— Leon Hunt, Senior Lecturer in Film and TV Studies, Brunel University
Beginning with the first movies produced in Shanghai during the 1920s, this book mainly addresses the amazing success and manifold metamorphoses martial arts narratives have undergone since global capitalism entered and transmogrified Asian modernities. The virtual universe of today's digital martial arts games is furthermore traced all the way back into antiquity, when the fictional wuxia communities' enchanted heterotopia of the "Rivers and Lakes" (jianghu) first emerged. Besides exploring a range of methodologies for the cultural analysis of the most widely known contemporary representations, readers are also introduced to less popular topics and forms that critically interrogate the contemporary globalized hype. For instance, it is demonstrated how the Orientalist fashioning of the martial arts hero serves ambiguous trajectories in global cultural contexts, as it can be employed to either overcome or redraw ethnic and cultural boundaries. In sum, the book offers timely, sound, multi-scalar scholarship that sheds new light on the major generic, aesthetic, and ideological configurations of martial arts since the early 20th century. Its intellectual rigor and rich material corpus moreover facilitate significant conceptual advances in this and related fields of inquiry.
— Andrea Riemenschnitter, Professor of Modern Chinese Language and Literature, University of Zurich


text:
Update: 11 Year Old Seeks to Advance Protection of Wild Horses and Burros
By R.T. Fitch on August 8, 2012 • ( 11 Comments )
By Maggie Lillis as published in the Las Vegas Review-Journal
"We don't want them to reduce their numbers or to have the horses' watering holes polluted or herds run around by motorists"
Photo © Cat Kindsfather, all rights reserved
One Centennial Hills 11-year-old has turned a nickname, Wild Mustang Robin, into a passion in motion.
Sixth-grader Robin Warren's mission to protect indigenous horses and burros has garnered national and international attention, visits with legislators and a youth advocate title for a preservation group.
For three years, Robin has worked to raise awareness of the Free-Roaming Horses and Burros Act of 1971 and the Bureau of Land Management's exemption to it.
She started a petition and gathered about 2,500 signatures to request that the BLM adhere to the same rules and regulations as the general public in regards to the humane treatment of wild horses and burros.
"We don't want them to reduce their numbers or to have the horses' watering holes polluted or herds run around by motorists," Robin said. "I hope many people see my petition and understand the problem and issues and help us put a stop to this."
Robin was introduced to the cause by the book "Mustang, Wild Spirit of the West," written by Marguerite Henry and illustrated by Robert Lougheed. Her humanitarian spirit was also influenced by "The Puppy Place Special Edition: Puppy and Chica" by Ellen Miles due to its message about puppy mills, said Robin's mother, Denise DeLucia.
"In the end of the book, it says how you can help, and it was to start a petition," she said.
Robin obtained signatures from family, friends and patrons outside area libraries at first. She then made a Facebook page and a series of YouTube videos, and her pleas were viewed globally.
In one video, Robin addressed her peers when she sang, "Come on, you kids out there, sign my petition. You could be a world changer."
DeLucia said Robin's fervor hasn't waned.
"I was taken aback by her zeal and determination," she said. "I wasn't sure how lasting it would be . You know how a child can be."
DeLucia battled health troubles last summer, but Robin continued her fight.
"She encouraged me," she said.
Robin's videos were viewed by people in every state and 33 countries, DeLucia said.
A link to Robin's horse petition is included in each video.
The videos caught the attention of Anne Novak, executive director of Protect Mustangs, a San Francisco Bay Area preservation group. The group's mission is to educate the public about the American wild horse, protect and research wild horses on the range and help those that have lost their freedom, Novak said.
"I noticed her about two years ago and I saw how dedicated this young lady is," Novak said. "She's amazing and brilliant and a great wild horse advocate."
Novak appointed Robin as Protect Mustang's youth campaign director . She is to help with outreach, public appearances and political events.
Robin spoke about the issue with a Nevada senator and addressed crowds at a rally and press conference in front of the Sacramento Federal Courthouse last month.
"She had people in tears," Novak said. "And she had a standing ovation. She's a very strong voice for Protect Mustangs."
Robin said her work with Protect Mustangs is fun and exciting.
"I'm very proud of her," DeLucia said.
There have been massive removals of herds since 2009, Novak said, and a debate about the wild horse and burro population merits.
"We're starting to get to a situation that is dire," she said.
There is varied census information for indigenous horses and burros in the United States.
There were an estimated 51,000 horses before the BLM's Jackson Mountains Herd Management Area roundup in June, a controversial helicopter operation that gathered 424 wild horses.
There are an estimated 20,000 wild horses in 10 most Western states. Eighty percent of them live in Nevada, Novak said.
Wild horses and burros help sustain biodiversity in ecosystems and are a source of ecotourism, Novak said.
Protect Wildlife hopes to highlight the issues for a new generation via Robin.
"Robin is a wonderful leader and inspiration for kids," Novak said. "It's important that even though they aren't voters, they will vote, and we want to have something left for them to see on a ballot."
To view Robin's online content, visit youtube.com/user/wildmustangrobin.
Click (HERE) to visit the Review-Journal and to Comment
Wild Horse and Burro Bait-Trapping: Going … Going … Gone (rtfitchauthor.com)
Mother Nature Gives West Douglas Herd a 30 Day Reprieve (rtfitchauthor.com)
Wild Horse Advocates Level Another Legal Blow Against BLM's Bogus Emergency Gather (rtfitchauthor.com)
Total Removal of Historic Colorado Mustang Herd Denied (rtfitchauthor.com)
Categories: Horse News, Wild Burros, Wild Horses/Mustangs
Tagged as: Bureau of Land Management, Horse, Protect Mustangs, wild burro, Wild Horse
Independent Horse Slaughter White Paper
Raw Video Update: The Takhi, Wild Horses of Mongolia
Jane Heltebrake says:
Good for this "upstanding, caring, young lady." Continue your fight… Expose the BLM, Ken Salazar, for the "mean spirited," individuals, who they are!!!
heavenlyholly says:
our Mustang's hope for the future.
What an awesome girl, videos are wonderful …………..Doesnt it say everything that a little girl has such a brilliant insight to these Beautiful Wild Mustangs, she is filled with the beauty she sees with her eyes and feels in her soul….. Children have such wonderment i am so proud of all children when they display this beauty that they have inside them, this is not taught ,this is innocently inside !!!!! Then to act upon it is divine !!!!!! Onward little lady !!!!! the book, Mustang Wild Spirit of the west should be required reading in every School………………….
Barbara W. says:
And a child shall lead them. I know that pertains to Jesus but I don't think He'll mind me using it. Robin is a gift from God. Bless her and our wild horses.
Valerie Wehmueller says:
Wow! Robin, you are amazing, & should be an inspiration to all, young or old!! This country & this world could use more wise, caring, compassionate people like yourself! Never give up your passion, never let anyone or thing get in your way, stay determined! You go girl!! 🙂
Donna Hilliard says:
It is wonderfu to see such a fine young lady doing such great work for these fine animals.
Keep up the good work, Robin – seeing a young girl who cares enough about the horses & burros to work hard to try to save them should shame the "powers that be" . And they should be ashamed for
sitting back & letting this happen to our wild horses & burros.
jenniebarron says:
Reblogged this on jenniebarron and commented:
Robin Warren, also Wild Horse Robin, has been fighting for the wild horses and burros for three years, at least. That means this young lady has been fighting for wild horses and burros since she was 8 years old. I don't know any politician that would take for granted Robin and Declan's testimony to them; and their testimony is their desire for the horses and burros of this land not to be slaughtered. To take care of them and to be responsible and accountable for our actions as adults means that we leave a better world for them to inherit. This is wisdom and knowledge from Robin, who by any rights as an American citizen who is accountable and responsible as her actions have proven, and honesty, courage and respect she carries with her. I hope the politicians of this day realize that there are young people who are preparing themselves to make decisions as adults by educating themselves accordingly. We should expect nothing less from the adults we call politicians. We support you, Robin, and we are sure your voice will be heard, we are listening!
Jo-Claire Corcoran says:
Robin will be speaking at the Equine Welfare Alliance, International Equine Conference Sept 22-23, in Las Vegas! Robin, Declan, Amber and all the children who have worked very hard on this issue, should be an inspiration to the adults, that clicking "like" on a post, is not being an advocate. Being an advocate means, standing up, being vocal, contacting legislators, lobbying, writing letters, making the videos.. acting on Calls To Actions. Our children are doing this… the adults need to follow suit.
Louie Cocroft says:
Robin, you are showing your power to lead and stay the course at a very young age. It is so very encouraging to see our young people, such as you, coming up and getting ready to take the reins. As young as you are, you already know your purpose in life. Hold on to that vision and keep your focus.
Robin Warren (Wild Mustang Robin) says:
Thank you all for your kind words and encouragement! I am so happy to help make a difference for the horses. If we all work together we can save them!