Nestin Overexpression Research Articles

Nestin overexpression reduces the sensitivity of gastric cancer cells to trastuzumab.

Trastuzumab (TRA) shows significant efficacy in patients with human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). While TRA can help treat HER2-positive breast cancer, TRA resistance is a key clinical challenge. Nestin reportedly regulates the cellular redox homeostasis in lung cancer. This study aimed at identifying the functions of Nestin on the TRA sensitivity of HER2-positive GC cells. Real-time polymerase chain reaction (PCR) and Western blotting (WB) were performed to explore the association between the mRNA and protein expression profiles, respectively, of Nestin and the Keap1-Nrf2 pathway. The influence of Nestin overexpression on the in vitro sensitivity of GC cells to TRA was explored by Cell Counting Kit-8 (CCK-8) assay, colony formation assay, reactive oxygen species (ROS) detection, and flow cytometry. TRA treatment caused Nestin downregulation in two HER2-positive GC cell lines (MKN45 and NCI-N87). Nestin overexpression reduced the sensitivity of GC cells to TRA. The expression and activity of Nrf2 and relevant downstream antioxidant genes were increased by Nestin overexpression. Nestin overexpression also significantly suppressed TRA-induced apoptosis and ROS generation. In vivo tumor growth experiment with female BALB/c nude mice indicated that Nestin upregulation restored the tumor growth rate which was inhibited by TRA treatment. Collectively, the inhibitory effect of Nestin on the TRA sensitivity of cells to TRA was confirmed in this study. These results imply that the antioxidant Nestin-Nrf2 axis may play a role in the mechanism underlying the resistance of GC cells to TRA.

Nestin protein affects the maintenance of stem characteristics of colorectal cancer cells based on p53 dependent pathway.

Colorectal cancer (CRC) is a tumor with high incidence and poor prognosis. An increasing number of studies have shown that intermediate filament proteins, such as nestin, participate in the regulation of tumor progression. However, the mechanism related to CRC is complex, and the role and underlying mechanism of nestin have not been elucidated in CRC. We conducted quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blot analyses to examine the mRNA and protein levels in CRC and normal tissues. siRNAs targeting Nestin were transfected into CRC cells and then cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), sphere formation, and transwell analyses were used to assess the role of nestin in the proliferation, stem activity, migration, and invasive ability of CRC cells. Afterwards, nestin was overexpressed in CRC cells and P53 was overexpressed as a rescue group. CCK-8, EdU dyeing, sphere formation, and transwell assay was used to evaluated the role of Nestin/p53 axis in CRC cells. We found high nestin expression and low p53 expression in CRC tissues and cells. Functionally, silencing of nestin suppressed the multiplication, stemness, and metastatic ability of Caco-2 and RKO cells. Encouragingly, rescue experiments suggested that overexpression of p53 partly restored the impacts of nestin overexpression on the viability, proliferation, and metastatic ability of CRC cells. We confirmed that nestin and p53 play a functional role in the progression of CRC, and they may act as potential therapeutic targets for CRC treatment.

Vitamin D Receptor/Vitamin D Response Element Directly Modulate Nestin Transcription to Ameliorate PAN-Induced Podocyte Morphological Changes

Background: The change of podocyte morphology is a pathologic feature of chronic kidney disease. Several studies have suggested that vitamin D plays a role in the protection of podocytes, but the underlying mechanism remains unclear. Methods: The effects of paricalcitol on podocyte injury were tested in a puromycin aminonucleoside (PAN)-induced rat model and cultured mouse podocytes. Proteinuria, podocyte foot process (FP) effacement, and the expression of nestin and vitamin D receptor (VDR) were evaluated. VDR-siRNA or plasmids containing VDR-shRNA were transfected into podocytes to silence VDR expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to verify the connection between VDR and nestin gene expression. Results: Paricalcitol significantly alleviated proteinuria and podocyte FP effacement in PAN-induced nephrosis, which was accompanied by increased VDR expression in the glomeruli. Paricalcitol also inhibited PAN-induced nestin overexpression in the glomeruli. In an in vivo study, PAN significantly inhibited VDR protein expression, stimulated nestin protein expression, and resulted in nestin filament derangement in mouse podocytes, while paricalcitol treatment abolished these effects. In contrast, downregulation of VDR resulted in derangement and overexpression of nestin. ChIP assays demonstrated the presence of a vitamin D response element (VDRE) in the nestin promoter, and paricalcitol enhanced the binding of VDR to VDRE. Furthermore, luciferase reporter assays of the nestin promoter fragment showed that paricalcitol effectively repressed nestin reporter gene expression after PAN treatment, and mutation of VDRE abolished this effect. Conclusions: Paricalcitol directly regulates nestin transcription through the interaction of VDR/VDRE, thereby preventing morphological changes of podocytes in PAN nephropathy.

Gold nanorods decorated polycaprolactone/cellulose acetate hybrid scaffold for PC12 cells proliferation

Synthetic and natural polymers have recently received considerable attention due to the exclusive potential for supporting the regenerative cellular processes in peripheral nerve injuries (PNIs). Gold nanorods (GNRs) decorated polycaprolactone (PCL)/cellulose acetate (CA) nanocomposite (PCL/CA/GNR) were fabricated via electrospinning to improve PC12 cells attachment and growth or scaffold cues. Transmission electron microscopy (TEM) corroborated the GNR distribution (23 ± 2 nm length and 3/1 Aspect ratio) and suitable average dimension of 800 nm for the fibers; also, scanning electron microscopy (SEM) represented block-free and smooth fibers without perturbation. Because of gold nanorods incorporation, electrical conductivity of PCL/CA/GNR increased ~21%. Water contact angle data emphasized PCL/CA/GNR surface is more wettable that PCL/CA (<90° at 62 s). Real-time PCR technique (RT-PCR) demonstrated overexpression of β-tubulin and microtubule-associated protein 2 (MAP2) on PCL/CA/GNR compared to PCL/CA composite. Additionally, evaluated of the maturation and neurogenic differentiation of PC12 cells emphasized overexpression of nestin and β-tubulin by Immunocytochemistry staining onto PCL/CA/GNR in comparison to PCL/CA composite. Notably, these recently developed hybrid scaffolds could be considered for peripheral nerve injury (PNI) regeneration.

Immunohistochemical Expression of p27Kip1, p57Kip2, Cyclin D1, Nestin, and Ki-67 in Ependymoma.

p27 and p57 are tumor suppressors that are dysregulated in many cancers. We investigated the immunohistochemical expression of p27 and p57 in ependymoma, with a secondary emphasis on cyclin D1, nestin, and Ki-67. Sixty-five patients diagnosed with ependymoma were included. Clinical and tumoral data were retrieved, and the expression of p27, p57, cyclin D1, nestin, and Ki-67 was measured. Pearson’s χ2 test was used to measure associations and the Kaplan–Meier method was used for survival analysis. p27 underexpression was significantly associated with pseudopalisading necrosis in tumors with foci of necrosis (p = 0.004). Cyclin D1 overexpression was associated with intracranial (p = 0.044), recurrent (p = 0.022) and grade 3 tumors (p = 0.016); nestin overexpression was associated with supratentorial (p = 0.025), mitotically active (p < 0.001), and grade 3 tumors (p = 0.004); Ki-67 overexpression was associated with supratentorial (p = 0.044) and grade 3 tumors (p < 0.001) and the 3 main features of anaplasia. None of the markers were intercorrelated or predictive of overall survival. In conclusion, p27 underexpression in tumors with foci of necrosis signals a pseudopalisading pattern. Cyclin D1, nestin, and Ki-67 are useful markers in ependymoma, but evidence-based cutoff values are required to standardize this interpretation.

Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept.

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.

Nestin represents a potential marker of pulmonary vascular remodeling in pulmonary arterial hypertension associated with congenital heart disease

ObjectiveReportedly, nestin was re-expressed in proliferative synthetic-type pulmonary artery smooth muscle cells (PASMCs) and obligatory for PASMC proliferation in pulmonary arterial hypertension (PAH). Accordingly, nestin is increased in pulmonary vascular lesions of congenital heart disease (CHD)-associated PAH patients. We tested the hypothesis whether nestin was re-expressed in proliferative synthetic-type PASMCs and associated with pulmonary vascular remodeling in CHD-PAH. Materials and methodsNestin expression was tested using lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV) shunt-induced PAH rats, human PASMCs (HPASMCs), and pulmonary artery endothelial cells (PAECs) and PASMCs from MCT-AV-induced PAH rats. The role and possible mechanism of nestin on HPASMC proliferation, apoptosis, cell cycle and migration were investigated by assays of CCK-8, EdU, TUNEL, flow cytometry, transwell chamber and immunoblotting assays. ResultsNestin was solely expressed in proliferative synthetic-type PASMCs, but rarely detected in PAECs. Nestin was barely detected in normal pulmonary arterioles and occlusive pulmonary vascular lesions. Its expression was robustly increased in developing pulmonary vasculature, but returned to normal levels at the late stage of pulmonary vascular remodeling in lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats. Besides, nestin peaks were consistent with the histological features in lung tissues of MCT-AV-induced PAH rats. Moreover, nestin overexpression effectively promoted HPASMC phenotypic transformation, proliferation, apoptosis resistance and migration via enhancing Wnt/β-catenin activation. ConclusionsThese data indicated that nestin was re-expressed in proliferative synthetic-type PASMCs and might represent a potential marker of pulmonary vascular remodeling in CHD-PAH.

Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension.

Uncontrolled proliferation of endothelial cells is essential to the pathogenesis of pulmonary arterial hypertension (PAH). Both proliferation and cytoskeleton reorganization are associated with upregulation of the intermediate filament protein Nestin. Recently, accumulation of Nestin-expressing cells was found in pulmonary vascular lesions of PAH patients. The goal of this study is to determine if Nestin expression contributes to endothelial proliferation in pulmonary arterial hypertension, using both lung tissues and endothelial cells. Here we found that endothelial cells from complex and plexiform lesions of PAH patients expressed Nestin. These Nestin+ cells further stained positive for the angiogenic factors CXC chemokine ligand 12 and Wnt1. Likewise, in the chronic hypoxia/SU5416 animal model of pulmonary hypertension, Nestin+ endothelial cells were found in occlusive pulmonary vascular lesions. In vitro, both growing rat and human lung endothelial cells expressed Nestin protein. When Nestin was overexpressed in endothelial cells (both rat and human), Nestin overexpression promoted proliferation and expression of CXC chemokine ligand 12. Nestin overexpression further increased angiogenic tube formation in vitro. Conclusions: We found increased Nestin expression from endothelial cells of occlusive lung vascular lesions in severe pulmonary hypertension. Elevated Nestin expression likely contributes to unchecked pulmonary vascular proliferation and angiogenesis, possibly via induction of CXC chemokine ligand 12. Additional studies are required to determine whether targeting Nestin would be beneficial to treat PAH.

Abstract 4620: Expression of nestin affect resistance to chemotherapy and clinical outcome in small cell lung caner

Abstract Background: Small cell lung cancer (SCLC) is an aggressive and highly metastatic lung cancer subtype. Nestin is a member of the intermediate filament family and serves as a potential proliferative and multipotency marker in neural progenitor and stem cell. Aberrant expression of nestin is linked to malignant characteristics and poor prognosis in different cancers, including non-small cell lung cancer. However, in SCLC, the association between nestin expression and clinicopathological feature or prognosis has remained unclear. In this study, we examined whether the expression of nestin affect malignant feature and clinical outcome in SCLC. Material and methods: To assess the function of nestin, a short hairpin RNA (shRNA) targeting nestin and a forced expression vector of the nestin gene were transfected into SCLC cell lines (DMS53 and SKLC17). And three lung cancer cell lines (PC6, PC9 and PC14) and their cisplatin-resistant sub-lines were also analyzed. Nestin expression levels were assayed using Western blotting. Cell proliferation assay was performed using both nestin knock-down and overexpressed cell lines. Growth in vivo was assessed in SCID mice using a nestin knock-down cell line. Chemosensitivity was expressed as the drug concentration for IC50 in MTS cell proliferation assay. Then we retrospectively examined 84 SCLC patients. Correlations between immunohistochemical expression of nestin and various clinicopathological features were evaluated. Result: In nestin knock-down and overexpression cell lines, nesin expression was related to cell proliferation in vitro and vivo and resistance to anticancer drugs (cisplatin, etoposide and SN-38). Nestin expression levels of three cisplatin-resistant cells were increased than their parent cell lines. In 84 SCLC patients (16 patients were surgically resected, and 68 patietns were biopsied), 24 patients had nestin positive tumor. Nestin positive ratio tended to be higher in operated patients than biopsied patients. Nestin expression was not associated with age, sex, smoking status, disease stage and serum pro-gastrin releasing peptide (ProGRP) levels. There were no significant differences between nestin positive and negative patients for response rate (RR), progression free survival (PFS) and overall survival (OS) in 1st line chemotherapy. However, positive expression of nestin was associated with shorter PFS in 2nd line chemotherapy (median PFS; nestin positive 81 days vs negative 117 days, P=0.03). Conclusion: Nestin expression lead to resistance to chemotherapy and worse outcome in SCLC patients. Citation Format: Kazuki Sone, Ken Maeno, Eiji Kunii, Osamu Takakuwa, Akira Takeuchi, Satoshi Fukuda, Takamitsu Asano, Yoshihiro Kanemitsu, Hirotsugu Ohkubo, Masaya Takemura, Yutaka Ito, Tetsuya Oguri, Akio Niimi. Expression of nestin affect resistance to chemotherapy and clinical outcome in small cell lung caner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4620.

Melatonin disturbs SUMOylation-mediated crosstalk between c-Myc and nestin via MT1 activation and promotes the sensitivity of paclitaxel in brain cancer stem cells.

Here the underlying antitumor mechanism of melatonin and its potency as a sensitizer of paclitaxel was investigated in X02 cancer stem cells. Melatonin suppressed sphere formation and induced G2/M arrest in X02 cells expressing nestin, CD133, CXCR4, and SOX-2 as biomarkers of stemness. Furthermore, melatonin reduced the expression of CDK2, CDK4, cyclin D1, cyclin E, and c-Myc and upregulated cyclin B1 in X02 cells. Notably, genes of c-Myc related mRNAs were differentially expressed in melatonin-treated X02 cells by microarray analysis. Consistently, melatonin reduced the expression of c-Myc at mRNA and protein levels, which was blocked by MG132. Of note, overexpression of c-Myc increased the expression of nestin, while overexpression of nestin enhanced c-Myc through crosstalk despite different locations, nucleus, and cytoplasm. Interestingly, melatonin attenuated small ubiquitin-related modifier-1 (SUMO-1) more than SUMO-2 or SUMO-3 and disturbed nuclear translocation of nestin for direct binding to c-Myc by SUMOylation of SUMO-1 protein by immunofluorescence and immunoprecipitation. Also, melatonin reduced trimethylated histone H3K4me3 and H3K36me3 more than dimethylation in X02 cells by Western blotting and chromatin immunoprecipitation assay. Notably, melatonin upregulated MT1, not MT2, in X02 cells and melatonin receptor inhibitor luzindole blocked the ability of melatonin to decrease the expression of nestin, p-c-Myc(S62), and c-Myc. Furthermore, melatonin promoted cytotoxicity, sub-G1 accumulation, and apoptotic body formation by Paclitaxcel in X02 cells. Taken together, these findings suggest that melatonin inhibits stemness via suppression of c-Myc, nestin, and histone methylation via MT1 activation and promotes anticancer effect of Paclitaxcel in brain cancer stem cells.

The clinicopathological parameters significance of CD133 and Nestin in epithelial ovarian cancer: a meta-analysis.

This meta-analysis was conducted to evaluate the association of CD133 and Nestin with epithelial ovarian cancer. Databases (PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure, Wanfang) were searched for relevant studies updated in August 2017. CD133 and Nestin expression were estimated by immunohistochemistry. Statistical analysis was performed by RevMan. A total of 18 studies were included in this meta-analysis. High expression of both CD133 and Nestin was associated with late International Federation of Gynecology and Obstetrics stage (p<0.00001), larger size of residual cancer (p <0.05). CD133 overexpression was also associated with higher histological grade (p=0.0006) and lymph node metastases (p <0.00001). Nestin overexpression was associated with a higher rate of treatment resistance (p=0.0007). Positive expression of CD133 and Nestin may be associated with aggressive biological behaviors in epithelial ovarian cancer.

Morphine delays neural stem cells differentiation by facilitating Nestin overexpression

BackgroundMorphine is used as an analgesic although it causes important secondary effects. These effects are triggered by several mechanisms leading to the dysregulation of gene expression. Here we aimed to study these alterations on neural stem cells (NSC) during CNS development. MethodsAB strain and tg nestin:GFP zebrafish embryos, zebrafish primary neuron culture and mouse embryonic stem cells were used to assess the effect of morphine by qPCR, time lapse microscopy and western blot. ChIP-qPCR and bisulfite conversion assay were performed to determine the changes exerted by morphine in a Nestin candidate enhancer. ResultsMorphine increases GFP in nestin:GFP embryos and overexpresses the NSC marker Nestin. Morphine also exerts a hyperacetylation effect on H3K27 and decreases DNA methylation within a region located 18 Kb upstream nestin transcription starting site. Here, a binding site for the transcription factor complex Sox2/Oct4/Nanog was predicted. These factors are also upregulated by morphine. Besides, morphine increases the histone acetyl transferase p300. The inhibition of p300 activity decreases Nestin. ConclusionsMorphine facilitates Nestin increase by several mechanisms which include hyperacetylation of H3K27, decreased DNA methylation and the overexpression of the transcription factors sox2, oct4 and nanog. It has also been demonstrated that nestin levels depend on p300 activity. The facilitated Nestin expression delays the normal differentiation of neural stem cells. General significanceThe present work provides novel evidence of the effects induced by morphine in the normal differentiation of NSCs, altering Nestin through changes on p300, H3K27ac, DNA methylation and Oct4, Sox2, and Nanog.

Neuronal Differentiation Capability of Nasal Polyps of Chronic Rhinosinusitis.

Chronic rhinosinusitis with nasal polyps is considered a subgroup of chronic rhinosinusitis and a significant health problem, but the pathogenesis remains unclear to date. Therefore, we investigated the stemness to determine the role of stem cells in nasal polyps, with additional analysis of the neuronal differentiation potential of nasal polyp cells. We determined gene and protein expression profiles of stem cells in nasal polyp tissues, using whole genome microarray, quantitative real-time PCR (qPCR), immunohistochemistry, and flow cytometry. To evaluate the neuronal differentiation potential of nasal polyp cells, we used an efficient xenogeneic co-culture model with unsliced adult rat brain biopsies, followed by qPCR, immunohistochemistry, and growth factor antibody arrays. During gene expression analysis and immunohistochemistry, we were able to detect different stem cell markers, like Oct-4, Sox2, Klf4, c-Myc, ABCG2, Nanog, CD133, and Nestin, which confirmed the existence of stem cell like cells within nasal polyps. In addition, co-culture experiments give evidence for a guided differentiation into the neuronal lineage by overexpression of Nestin, Neurofilament, and GM-CSF. Our study demonstrated the expression of stem cell-related markers in nasal polyps. Furthermore, we characterized, for the first time, the stemness and neuronal differentiation potential of nasal polyp cells. These results gave new insights into the pathogenesis of nasal polyps and its therapeutic effectiveness could represent a promising strategy in the future.

Nestin suppression attenuates invasive potential of endometrial cancer cells by downregulating TGF-β signaling pathway.

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

Abstract 4428: Nestin suppression attenuates invasive potential of endometrial cancer cells by down regulating TGF-β signaling pathway

Abstract Nestin, an intermediate filament protein and a stem cell marker is expressed in several cancers. Little is known about the expression level and role of Nestin in endometrial cancer. Compared to immortalized endometrial epithelial cell line EM-E6/E7/TERT, endometrial cancer lines express high to moderate levels of Nestin. Endometrial tumors and tumor cell lines have a cancer stem-like cells population expressing CD133. The AN3CA and KLE cells showed more CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE, and overexpression in Ishikawa cells was associated respectively with attenuation and enhancement of CD133+ cells. Knockdown of Nestin increased cells in G0/G1 phase and decreased in S phase, whereas overexpression decreased cells in G0/G1 phase and increased in S phase of cell cycle. Nestin knockdown cells showed increased expression of p21, p27 and PNCA and decreased expression of cyclin-D1 and D3. Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by down regulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating E-cadherin. Conversely, Nestin overexpression enhanced cell invasiveness by upregulating TGF-β signaling components, MMP-2, MMP-9, mesenchymal markers and downregulaing epithelial marker, E-cadherin. Nestin knockdown inhibited and overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone and a nitric oxide donor inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment. Citation Format: Amber A. Bokhari, Batsukh Dorjbal, Tabari M. Baker, Sana Waheed, Christopher M. Zahn, Chad A. Hamilton, George L. Maxwell, Viqar Syed. Nestin suppression attenuates invasive potential of endometrial cancer cells by down regulating TGF-β signaling pathway. [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 4428.

Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance.

BackgroundNestin expression has been reported to be associated with the prognosis of many solid tumors including human hepatocellular carcinoma (HCC). The present study aimed to identify the role, if any, of Nestin in the chemotherapeutic treatment of HCC.MethodsWe determined Nestin expression in nine HCC cell lines and 220 tissue samples of advanced HCC patients (retrospectively registered) treated with FOLFOX regimens. We examined the correlations between Nestin expression and clinicopatholgical variables and HCC prognosis. Also, we used in vitro and in vivo methods to determine the effects of Nestin expression on HCC cell invasion, migration and chemosensitivity.ResultsNestin expression was significantly increased in HCC tissues and drug-resistant cell lines, and the presence of high levels of Nestin was associated with poor survival. We also showed that drug-resistance occurred in HCC cells with epithelial-mesenchymal transition (EMT), which in turn enhanced invasion ability. Nestin depletion reversed drug-resistance in the Bel-7402/5-FU and Bel-7402/ADM cell lines. Nestin knockdown enhanced chemotherapeutic efficacy in nude mice. Moreover, Nestin up-regulation in Bel-7402 was associated with the activation of Wnt/β-catenin signaling.ConclusionOur findings suggest that Nestin inhibitors may be useful for the chemotherapy of HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0387-y) contains supplementary material, which is available to authorized users.

Cancer stem-like cell behavior in anaplastic thyroid cancer: A challenging dilemma

AimsAnaplastic thyroid carcinoma (ATC) is an undifferentiated tumor of the thyroid which is characterized with poor prognosis, leading to its aggressive behavior and resistance to conventional therapies. Cancer stem cells (CSCs) are tumor cells that have self-renewal and clonal tumor initiation. Like other cancers, many studies have shown that ATC also has tumor cells with properties like stem cells. To evaluate the concept of cancer stem-like cell theory of ATC, we conducted this study to emphasize both on the concept of cancer stemness origin of these cells and target them for further therapeutic purposes. In the current study, we showed that two ATC cell lines, SW1736 and C643, have subpopulations (SP) that are similar to CSCs. Materials and methodsUsing MACS technique, cells positive for CD133 were isolated and subsequently validated with flow cytometry. For further analysis, expression of some stemness markers was evaluated. Key findingsABCG2, CD133, and Sox2 were significantly up-regulated, while Nestin was down-regulated in CD133pos subpopulation compared to CD133neg cells. In contrast to previous reports that over-expression of Nestin was considered as a marker for thyroid CSCs, we noticed that expression of Nestin was declined in stem cell-like tumor cells, derived from ATC cell lines. SignificanceThis study reconfirmed the concept of cancer stem-like cell identity of SW1736 and C643 cells. Indeed, the characterization of CSCs should not be merely based on surface markers. Cell origin and genetic background should be additionally considered on CSCs subpopulation of ATCs for therapeutics.

WITHDRAWN: Nestin over-expression predicts poor prognosis in patients with solid tumors: a meta- analysis

// Huihui Cheng 1 , Saisai Li 1 , Lulu Zhou 1 , Hui Luo 1 , Zhaojun Shen 1 and Xueqiong Zhu 1 1 Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Correspondence to: Xueqiong Zhu, email: zjwzzxq@163.com Keywords : nestin; solid tumors; prognosis; meta-analysis Received: July 19, 2017&nbsp;&nbsp;&nbsp;&nbsp; Accepted: November 14, 2017&nbsp;&nbsp;&nbsp;&nbsp; Epub: January 03, 2018 Abstract Background: Nestin, as a marker of cancer stem cells, has recently been reported frequently in the pathogenesis and development of human solid tumors. However, the prognostic role of Nestin in patients with solid tumors remains inconclusive. Here, we performed this meta-analysis of relevant studies published on the topic to quantitatively evaluate the prognostic significance of Nestin in solid tumors. Methods: We performed systematically electronic and manual searches through the databases of Pubmed and Embase for titles and abstracts. The pooled hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of overall survival (OS) or cancer-specific survival (CSS) or disease-free survival (DFS)/progression-free survival (PFS)/recurrent-free survival (RFS)/ event-free survival (EFS) were calculated to evaluate the prognostic value of Nestin expression in patients with solid tumors. Results: A total of 43 studies involving 6,068 participants were included in the study. Nestin over-expression was associated with statistically significant poor OS (pooled HR = 1.74, 95% CI = 1.48-2.05, P &lt;0.001) and shorter CSS (pooled HR = 2.35, 95% CI = 1.87-2.97, P &lt;0.001) and shorter DFS/ PFS/ RFS/EFS (pooled HR = 1.77, 95% CI = 1.41-2.23, P &lt;0.001) in solid tumors. Further subgroup analyses showed that different analysis model (univariate or multivariate model), sample-size (&le; 105 or &gt;105), ethnicity (Asia or Europe or South America), primary tumor location, and method for data collection (direct extraction or indirect extraction) didn&rsquo;t alter the negative prognostic effect of Nestin over-expression on OS. For DFS/ PFS/ RFS/ EFS, subgroup analyses based on statistical analysis model, sample size and study ethnicity also showed the negative prognostic role of Nestin. Conclusion: Nestin over-expression was associated with shorter survival in human solid tumors. Nestin could be a valuable prognosis biomarker of some solid tumors.

Correlation of overexpression of nestin with expression of epithelial-mesenchymal transition-related proteins in gastric adenocarcinoma.

Nestin is associated with neoplastic transformation. However, the mechanisms by which nestin contributes regarding invasion and malignancy of gastric adenocarcinoma (GAC) remain unknown. Recent studies have shown that the epithelial-mesenchymal transition (EMT) is important in invasion and migration of cancer cells. In the present study, we aimed to investigate the expression of nestin and its correlation with EMT-related proteins in GAC. The expression of nestin and EMT-related proteins was examined in GAC specimens and cell lines by immunohistochemistry and Western blotting. Clinicopathological features and survival outcomes were retrospectively analyzed. Positive nestin immunostaining was most obviously detected in the cytoplasm, nucleus or both cytoplasm and nucleus of tumor cells in 19.2% (24/125) of GAC tissues, which was significantly higher than that in normal gastric mucosa tissues (1.7%, 1/60) (p=0.001). Nestin expression was closely related to several clinicopathological factors and EMT-related proteins (E-cadherin, vimentin and Snail) and displayed a poor prognosis. Interestingly, simultaneous cytoplasmic and nuclear nestin expression correlated with EMT-related proteins (E-cadherin, vimentin and Snail) (p<0.05) and lymph node metastasis (p=0.041) and a shorter survival time (p<0.05), but this was not the case with cytoplasmic or nuclear nestin expression. Nestin, particularly expression in both cytoplasm and nucleus, might be involved in regulating EMT and malignant progression in GAC, with potential as an unfavorable indicator in tumor diagnosis and a target for clinical therapy.

Nestin overexpression promotes the embryonic development of heart and brain through the regulation of cell proliferation

Nestin, an intermediate filament protein, is a key regulator of various extracellular proteins that play important roles in cell growth and differentiation. In recent years, nestin has been widely accepted as a molecular marker for neural stem/progenitor cells. However, its function during embryogenesis remains largely unknown since its depletion is lethal after stage embryonic day 8.5 (E8.5). In order to understand the role of this protein in vivo, we compared the heart and brain tissues of control mice with those of mice overexpressing a human nestin cDNA transgene under the control of a ROSA26 promoter. In these tissues we examined the general histology and cell size, the presence of apoptotic cells by TUNEL assay, and the presence of progenitor cell markers like SOX2. Compared to controls, mouse embryos overexpressing the human nestin transgene have a larger size and display characteristic morphological changes including a larger heart and forebrain. In these tissues we found corresponding increases in the size of cardiomyocytes and brain cells, as well as indications of augmented cell proliferation. In contrast, apoptosis was not significantly altered. Co-staining brain sections with SOX2 and Ki67 showed that most of the proliferating cells in the forebrain were neural stem cells. Moreover, nestin overexpression was responsible for a marked activation of the PI3K/Akt signaling pathway. Taken together, the results of this study indicate that nestin plays an important role in the embryonic development of at least two mouse organs (heart and brain) through the regulation of cell proliferation.