CLU

Gene Summary

Gene:CLU; clusterin
Aliases: CLI, AAG4, APOJ, CLU1, CLU2, KUB1, SGP2, APO-J, SGP-2, SP-40, TRPM2, TRPM-2, NA1/NA2
Location:8p21-p12
Summary:The protein encoded by this gene is a secreted chaperone that can under some stress conditions also be found in the cell cytosol. It has been suggested to be involved in several basic biological events such as cell death, tumor progression, and neurodegenerative disorders. Alternate splicing results in both coding and non-coding variants.[provided by RefSeq, May 2011]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:clusterin
HPRD
Source:NCBIAccessed: 27 February, 2015

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CLU (cancer-related)

Miyake H, Fujisawa M
[Antisense oligodeoxynucleotide therapy for castration-resistant prostate cancer].
Nihon Rinsho. 2014; 72(12):2121-5 [PubMed] Related Publications
Because of recent advances in the field of nucleic-acid chemistry, antisense oligodeoxynucleotide (AS ODN) technology can offer an attractive strategy to specifically inhibit expression of target genes causing a wide variety of diseases, including cancers. In prostate cancer (PC), several genes, involved in the acquisition of castration-resistant (CR) phenotype, have been identified, some of which, such as clusterin and heat shock protein 27, are intensively investigated as optimal targets for AS ODN therapy. In this review, we attempted to summarize the progress in the novel therapeutic strategy using AS ODN against CRPC, and to discuss the data of the recently completed and currently conducting clinical trials using AS ODNs as well as the future prospects of this therapy for treating CRPC.

Lee JH, Lee JY, Rho SB, et al.
PACAP inhibits tumor growth and interferes with clusterin in cervical carcinomas.
FEBS Lett. 2014; 588(24):4730-9 [PubMed] Related Publications
Secretory clusterin (sCLU), an anti-apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo-resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt-Raf-ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.

Chun YJ
Knockdown of clusterin expression increases the in vitro sensitivity of human prostate cancer cells to paclitaxel.
J Toxicol Environ Health A. 2014; 77(22-24):1443-50 [PubMed] Related Publications
Clusterin/apolipoprotein J is a secreted heterodimeric glycoprotein that is implicated in several pathophysiological processes, including tissue remodeling, reproduction, lipid transport, and apoptosis. Although previous studies demonstrated that clusterin is able to protect against apoptosis, the role of the clusterin in cellular proliferation remains elusive. To determine whether clusterin plays an important role in cellular proliferation, the function of clusterin was examined using a small interfering RNA (siRNA) in PC3 human prostate cancer cells. Transient transfection with clusterin siRNA resulted in significant suppression of clusterin mRNA and protein expression. Clusterin knockdown resulted in a decrease in protein expression of phospho-Akt and an increase in expression of proteins phosphatase type 2AC (PP2AC) and phosphorylation of p38. However, treatment with PP2AC siRNA exerted minimal effects on clusterin expression. Interestingly, clusterin mRNA expression was reduced in paclitaxel-treated cells, and the cytotoxic effect of paclitaxel was more potent when cells were incubated with clusterin siRNA. In addition, co-treatment with paclitaxel and clusterin siRNA significantly enhanced PP2AC levels. Taken together, these results indicate that clusterin plays a crucial role in PC3 cell proliferation and that clusterin depletion may contribute to enhanced sensitivity of PC3 cells to anticancer agents such as paclitaxel.

Qi X, Yu T, Zhu L, et al.
Ochratoxin A induces rat renal carcinogenicity with limited induction of oxidative stress responses.
Toxicol Appl Pharmacol. 2014; 280(3):543-9 [PubMed] Related Publications
Ochratoxin A (OTA) has displayed nephrotoxicity and renal carcinogenicity in mammals, however, no clear mechanisms have been identified detailing the relationship between oxidative stress and these toxicities. This study was performed to clarify the relationship between oxidative stress and the renal carcinogenicity induced by OTA. Rats were treated with 70 or 210 μg/kg b.w. OTA for 4 or 13 weeks. In the rats administrated with OTA for 13 weeks, the kidney was damaged seriously. Cytoplasmic vacuolization was observed in the outer stripe of the outer medulla. Karyomegaly was prominent in the tubular epithelium. Kidney injury molecule-1 (Kim-1) was detected in the outer stripe of the outer medulla in both low- and high-dose groups. OTA increased the mRNA levels of clusterin in rat kidneys. Interestingly, OTA did not significantly alter the oxidative stress level in rat liver and kidney. Yet, some indications related to proliferation and carcinogenicity were observed. A dose-related increase in proliferating cell nuclear antigen (PCNA) was observed at 4 weeks in both liver and kidney, but at 13 weeks, only in the kidney. OTA down-regulated reactive oxygen species (ROS) and up-regulated vimentin and lipocalin 2 in rat kidney at 13 weeks. The p53 gene was decreased in both liver and kidney at 13 weeks. These results suggest that OTA caused apparent kidney damage within 13 weeks but exerted limited effect on oxidative stress parameters. It implies that cell proliferation is the proposed mode of action for OTA-induced renal carcinogenicity.

Teoh JY, Chan NH, Cheung HY, et al.
Inflammatory myofibroblastic tumors of the urinary bladder: a systematic review.
Urology. 2014; 84(3):503-8 [PubMed] Related Publications
We systemically reviewed the literature on inflammatory myofibroblastic tumors (IMTs) of the urinary bladder and compared between anaplastic lymphoma kinase (ALK)-positive and ALK-negative IMTs. An extensive search of the literature was performed in Medline and Web of Science using the following terms: "inflammatory myofibrolastic tumor," "inflammatory pseudotumor," and "bladder." A manual search was also performed using the web-based search engine Google Scholar. Reference lists of the retrieved articles were reviewed for other relevant studies. Patients' and disease characteristics of each individual case were reviewed. Further analyses were performed to compare between ALK-positive and ALK-negative IMTs. Forty-one studies were identified, and 182 patients were included for review and subsequent analyses. Of the IMTs, 65% were ALK-positive. Local tumor recurrence rate was 4%, and no cases of distant metastases have been reported. Compared with ALK-negative IMTs, ALK-positive IMTs had a female predilection with a sex ratio (male:female) of 1:1.67 (P = .048). ALK-positive IMTs also appeared to occur in younger patients (P = .072). No significant differences were noted in terms of their clinical presentations and histologic features. On immunohistochemical staining, ALK-positive IMTs had more positive results for desmin (P = .042) and p53 (P = .05), and more negative results for clusterin (P = .003). In summary, ALK-positive IMTs of the urinary bladder had a female predilection, appeared to occur more frequently in younger patients, and had different immunohistochemical staining patterns when compared with ALK-negative IMTs. Regardless of its ALK status, IMT of the urinary bladder has a good prognosis after surgical resection.

Wang Q, Cao W, Su Q, et al.
Clusterin silencing inhibits proliferation and reduces invasion in human laryngeal squamous carcinoma cells.
World J Surg Oncol. 2014; 12:124 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (sCLU), which plays important roles in cell survival and death. In laryngeal squamous cell carcinomas (LSCC), sCLU is up-regulated and its expression is related to the invasiveness of these tumors. The purpose of this study was to explore the inhibiting role of sCLU gene silence in the invasive ability and growth of Hep-2 human laryngeal squamous carcinoma cells (Hep-2) by transfection of short hairpin RNA expression plasmids against sCLU (sCLU-shRNA) (in vivo) or small interference RNA (sCLU-siRNA) (in vitro).
METHODS: sCLU-siRNA and the control siRNA were transfected into Hep-2 cells using Lipofectamine 2000. RT-PCR and Western blot were used to detect the effect of siRNA transfection on sCLU mRNA and sCLU protein expression. The invasive activity of sCLU-siRNA-transfected Hep-2 cells was measured with the modified Boyden chamber assay and wound healing assay. The effects of sCLU-siRNA on cell proliferation were evaluated by MTT assay. Apoptosis was measured by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining methods. We next evaluated the effects of sCLU silencing by sCLU-shRNA transfection in vivo on tumor growth and metastatic properties to the lung. Terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) staining was used to observe the apoptosis in the xenografts.
RESULTS: It showed that siRNA-mediated down-regulation of sCLU expression in Hep-2 cells significantly inhibited cell proliferation and promoted apoptosis in vitro. Furthermore, siRNA-mediated down-regulation of sCLU expression decreases in vitro cell migration and invasion ability. In vivo, the average volume of tumors in the sCLU-shRNA transfected group was significantly lower than in the control group (P<0.01), and the significant apoptosis detected with TUNEL was indicated in the sCLU-shRNA transfected groups (P<0.05). Significantly, we found that sCLU-shRNA could exert marked inhibition of the lung metastasis of Hep-2 cells in nude mice in vivo.
CONCLUSIONS: sCLU gene silence can inhibit invasion and growth of LSCC. sCLU may provide a potential therapeutic target against human LSCC.

Xiu P, Xu Z, Liu F, et al.
Downregulating sCLU enhances the sensitivity of hepatocellular carcinoma cells to gemcitabine by activating the intrinsic apoptosis pathway.
Dig Dis Sci. 2014; 59(8):1798-809 [PubMed] Related Publications
PURPOSE: The purpose of this study was to investigate whether the therapeutic activity of gemcitabine (GCB) in hepatocellular carcinoma (HCC) could be increased by the down-regulation of secretory clusterin (sCLU), a glycoprotein that is considered to play a cytoprotective role in the resistance to chemotherapy.
METHODS: The expression of sCLU was detected in HCC tumor tissues and cell lines. A cell viability and apoptosis assay were performed in parental HCC cells or the same cells transfected with sCLU shRNA and treated with or without GCB. The potential downstream pathways were investigated using the Human Apoptosis RT(2) Profiler™ PCR Array.
RESULTS: The expression levels of sCLU in HCC tissues were significantly higher than in adjacent non-tumor liver tissues and were associated with the histological grade and transarterial chemoembolization. sCLU overexpression was also found in three HCC cell lines and hepatocytes. The depletion of sCLU synergistically increased GCB sensitivity in Bel7402 and SMMC7721 cells and induced cell apoptosis. Based on the PCR array analysis, sCLU depletion also resulted in the up-regulation of BNIP1, GADD45A, TNFRSF10A, and TRADD and down-regulation of AKT1 in Bel7402 and SMMC7721 cells compared with the parental controls. These results were further supported by a Western blot analysis, which showed increased GADD45a protein expression and the decreased expression of phosphorylated AKT. GADD45a overexpression also increased the sensitivity to GCB in the Bel7402 and SMMC7721 cells.
CONCLUSION: Targeting sCLU may be a useful method to enhance the cytotoxic effect of GCB in hepatocellular carcinoma.

Conteduca V, Kopf B, Burgio SL, et al.
The emerging role of anti-angiogenic therapy in ovarian cancer (review).
Int J Oncol. 2014; 44(5):1417-24 [PubMed] Related Publications
The introduction of new therapeutic agents into clinical practice of ovarian cancer, in addition to the role of surgery and chemotherapy, has been the subject of numerous studies because this tumor remains worldwide the most lethal gynecological cancer. It is now known that angiogenesis plays a vital role for ovarian physiology, but also in ovarian carcinogenesis and so it has become the main target of ovarian cancer treatment. In this review, the most common molecular pathways of angiogenesis have been investigated leading to the identification of novel targets, including monoclonal antibodies and tyrosine kinase inhibitors. The fundamental targets of anti-angiogenic drugs are vascular endothelial growth factor receptor and its ligand, but also platelet-derived growth factor, fibroblast growth factor and angiopoietin. Moreover, improved knowledge of angiogenic process allowed the discovery of other molecules, such as semaphorins, neuropilins, clusterin, some transcriptional factors, and the identification of features, including stemness, epithelial-mesenchymal transition, downregulation of certain microRNAs, the alteration of immune system, that contribute to angiogenesis and possibly to resistance mechanisms. The following patent and literature review aim to highlight recent findings of approved and novel anti-angiogenic drugs that make the treatment of patients with ovarian cancer a rapidly growing field of oncology.

Park J, Park SY, Shin E, et al.
Hypoxia inducible factor-1α directly regulates nuclear clusterin transcription by interacting with hypoxia response elements in the clusterin promoter.
Mol Cells. 2014; 37(2):178-86 [PubMed] Free Access to Full Article Related Publications
Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl₂, incubation under 1% O₂ conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl₂ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.

Takeuchi A, Shiota M, Beraldi E, et al.
Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth.
Mol Cell Endocrinol. 2014; 384(1-2):117-25 [PubMed] Related Publications
Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant prostate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsiveness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3-Twist1 signaling pathway. In response to IGF-I, STAT3 was phosphorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcription. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I-induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, suggesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and suggests that this signaling cascade plays a critical role in prostate cancer pathogenesis.

Mydlarz W, Uemura M, Ahn S, et al.
Clusterin is a gene-specific target of microRNA-21 in head and neck squamous cell carcinoma.
Clin Cancer Res. 2014; 20(4):868-77 [PubMed] Free Access to Full Article Related Publications
PURPOSE: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21-specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.
EXPERIMENTAL DESIGN: miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.
RESULTS: miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3'-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression.
CONCLUSIONS: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21.

Liu B, Han MT, Zhang J, et al.
Downregulation of clusterin expression in human testicular seminoma.
Cell Physiol Biochem. 2013; 32(4):1117-23 [PubMed] Related Publications
BACKGROUND: Clusterin, a heterodimeric glycoprotein of approximately 80 kDa, exists extensively in human body fluids. The abnormal expression of clusterin is closely related to the occurrence, progression, and prognosis of tumors. Up to now, few studies have focused on clusterin in human testicular cancer. This study describes an extensive exploration of the presence and expression of clusterin in testicular seminoma.
METHODS: Tumor tissues and normal testis tissues were collected from 13 patients with testicular seminoma and 16 patients undergoing surgical castration for prostate cancer. Real-time polymerase chain reaction (PCR) was performed to detect the expression difference of clusterin mRNA between testicular seminoma and normal testis. Western blot and immunohistochemical analysis were performed to detect the presence and expression difference of clusterin protein between two groups.
RESULTS: Real-time PCR showed the expression of clusterin mRNA in testicular seminoma to be significantly lower than in normal testis (only 13% relative quantification). Western blot analysis indicated marked reductions in the expression of clusterin protein in testicular seminoma. Similar results were observed upon immunohistochemical analysis.
CONCLUSION: In testicular seminoma and normal testis, clusterin exists in its heterodimeric secretory isoform. Clusterin expression is significantly lower in testicular seminoma than in normal testis. This is the first comprehensive study of the presence and expression of clusterin in human testicular cancer.

Radziwon-Balicka A, Santos-Martinez MJ, Corbalan JJ, et al.
Mechanisms of platelet-stimulated colon cancer invasion: role of clusterin and thrombospondin 1 in regulation of the P38MAPK-MMP-9 pathway.
Carcinogenesis. 2014; 35(2):324-32 [PubMed] Related Publications
Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.

Song HB, Jun HO, Kim JH, et al.
Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells.
Oncol Rep. 2013; 30(6):2713-8 [PubMed] Related Publications
Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma.

Wang X, Luo L, Dong D, et al.
Clusterin plays an important role in clear renal cell cancer metastasis.
Urol Int. 2014; 92(1):95-103 [PubMed] Related Publications
OBJECTIVE: Clusterin (CLU) is implicated in regulating clear renal cell carcinoma (CRCC) progression and metastasis, yet the mechanisms are not elucidated. In the present study, we explored the potential role of CLU in CRCC metastasis.
METHODS: Levels of CLU mRNA and CLU protein were measured by RT-PCR and immunohistochemistry analysis in 22 CRCC with metastasis and 22 without metastasis and 22 samples of normal kidney tissue. After CLU silencing and re-expression, the migration and invasion in vitro and in vivo of Caki-2 cells were determined by wound healing assay, transwell migration assay and pulmonary nodule assay, respectively. The expression of pERK1/2 and MMP-9 were detected by RT-PCR and Western blot assay.
RESULTS: We found a significant increase of CLU and CLU mRNA expression in CRCC, and the expression of CLU is strongly correlated in patients with metastatic disease. We discovered that CLU-rich Caki-2 cells displayed higher invasive ability which prompted us to investigate if CLU silencing could reduce the migration and invasion in Caki-2 cells. Compared with the vector-transfected cells, CLU knocked-down (CLUi) cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of CLU in CLUi cells restored the invasive phenotypes. We found that MMP-9 was downregulated in CLUi cells. We also discovered that levels of activated ERK1/2 correlated with the rich expression of CLU and MMP-9.
CONCLUSION: Our data suggest that CLU may regulate aggressive behavior of human CRCC cells through modulating ERK1/2 signaling and MMP-9 expression.

Tang M, Li J, Liu B, et al.
Clusterin expression and human testicular seminoma.
Med Hypotheses. 2013; 81(4):635-7 [PubMed] Related Publications
Clusterin expression has a positive correlation with the occurrence and progression of various types of tumors from different genetic backgrounds. Clusterin overexpression may protect tumor cells from apoptosis and damage caused by autoimmunity or anti-tumor therapy. Using immunohistochemisty, one previous study showed that clusterin protein expression is downregulated in human testicular seminoma, which is highly sensitive to radiotherapy and chemotherapy. We thus postulate that clusterin expression in human testicular seminoma differs from clusterin expression in other tumors. It may be the cause of the treatable characteristics of testicular seminoma. In the present preliminary study, we detected the abundance of clusterin mRNA in human testicular seminoma and normal testis. The results showed decreased clusterin expression in seminoma at the gene transcription level. Our primary data and summarized previous literature suggest that the downregulation of clusterin expression may be the cause of the high sensitivity of testicular seminoma to radiotherapy and chemotherapy. It may be that the scarcity of clusterin leaves tumor cells with insufficient protection from treatment. This is the first study to focus on the relationship between clusterin expression and human testicular cancer.

Wu K, Xie D, Zou Y, et al.
The mechanism of DAB2IP in chemoresistance of prostate cancer cells.
Clin Cancer Res. 2013; 19(17):4740-9 [PubMed] Related Publications
PURPOSE: The docetaxel-based chemotherapy is the standard of care for castration-resistant prostate cancer (CRPC), inevitably, patients develop resistance and decease. Until now, the mechanism and predictive marker for chemoresistance are poorly understood.
EXPERIMENTAL DESIGN: Immortalized normal prostate and cancer cell lines stably manipulated with different DAB2IP expression levels were used and treated with chemotherapeutic drugs commonly used in prostate cancer therapy. Cell proliferation was measured using MTT assay; Western blot, quantitative PCR, and luciferase reporter assays were used to analyze Clusterin gene regulation by DAB2IP. Immunohistochemical analysis was conducted for evaluating DAB2IP, Clusterin and Egr-1 expression in human prostate cancer tissue.
RESULTS: DAB2IP Knockdown (KD) cells exhibited resistance to several chemotherapeutic drugs, whereas increased DAB2IP in C4-2 cells restored the drug sensitivity. Parallel, DAB2IP KD cells exhibited higher expression of Clusterin, an antiapoptotic factor, whereas elevated DAB2IP in C4-2 cells decreased Clusterin expression. Functionally, knocking down Clusterin by short-hairpin RNA or antisense oligonucleotide OGX-011 decreased drug resistance, whereas overexpressing Clusterin in C4-2 D2 enhanced drug resistance. Mechanistically, DAB2IP blocked the cross-talk between Wnt/β-catenin and IGF-I signaling, leading to the suppression of Egr-1 that is responsible for Clusterin expression. A similar result was observed in the prostate of DAB2IP knockout animals. In addition, we observed a significantly inverse correlation between DAB2IP and Egr-1 or Clusterin expression from clinical tissue microarray.
CONCLUSIONS: This study unveils a new regulation of the Egr-1/Clusterin signaling network by DAB2IP. Loss of DAB2IP expression in CRPC cells signifies their chemoresistance. Clusterin is a key target for developing more effective CRPC therapy.

Matsumoto H, Yamamoto Y, Shiota M, et al.
Cotargeting Androgen Receptor and Clusterin Delays Castrate-Resistant Prostate Cancer Progression by Inhibiting Adaptive Stress Response and AR Stability.
Cancer Res. 2013; 73(16):5206-17 [PubMed] Related Publications
Although androgen receptor (AR) pathway inhibitors prolong survival in castrate-resistant prostate cancer (CRPC), resistance rapidly develops and is often associated with increased stress-activated molecular chaperones like clusterin (CLU) and continued AR signaling. Because adaptive pathways activated by treatment facilitate development of acquired resistance, cotargeting the stress response, activated by AR inhibition and mediated through CLU, may create conditional lethality and improve outcomes. Here, we report that CLU is induced by AR antagonism and silencing using MDV3100 and antisense, respectively, to become highly expressed in castrate- and MDV3100-resistant tumors and cell lines. CLU, as well as AKT and mitogen-activated protein kinase (MAPK) signalosomes, increase in response to MDV3100-induced stress. Mechanistically, this stress response is coordinated by a feed-forward loop involving p90rsk (RPS6KA)-mediated phosphoactivation of YB-1 with subsequent induction of CLU. CLU inhibition repressed MDV3100-induced activation of AKT and MAPK pathways. In addition, when combined with MDV3100, CLU knockdown accelerated AR degradation and repressed AR transcriptional activity through mechanisms involving decreased YB-1-regulated expression of the AR cochaperone, FKBP52. Cotargeting the AR (with MDV3100) and CLU (with OGX-011) synergistically enhanced apoptotic rates over that seen with MDV3100 or OGX-011 monotherapy and delayed CRPC LNCaP tumor and prostate-specific antigen (PSA) progression in vivo. These data indicate that cotargeting adaptive stress pathways activated by AR pathway inhibitors, and mediated through CLU, creates conditional lethality and provides mechanistic and preclinical proof-of-principle to guide biologically rational combinatorial clinical trial design.

Wu J, Xie X, Nie S, et al.
Altered expression of sialylated glycoproteins in ovarian cancer sera using lectin-based ELISA assay and quantitative glycoproteomics analysis.
J Proteome Res. 2013; 12(7):3342-52 [PubMed] Related Publications
Herein, we identify and confirm differentially expressed sialoglycoproteins in the serum of patients with ovarian cancer. On the basis of Sambucus nigra (SNA) lectin enrichment and on an isobaric chemical labeling quantitative strategy, clusterin (CLUS), leucine-rich alpha-2-glycoprotein (LRG1), hemopexin (HEMO), vitamin D-binding protein (VDB), and complement factor H (CFH) were found to be differentially expressed in the serum of patients with ovarian cancer compared to benign diseases. The abnormal sialylation levels of CLUS, CFH, and HEMO in serum of ovarian cancer patients were verified by a lectin-based ELISA assay. ELISA assays were further applied to measure total protein level changes of these glycoproteins. Protein levels of CLUS were found to be down-regulated in the serum of ovarian cancer patients, while protein levels of LRG1 were increased. The combination of CLUS and LRG1 (AUC = 0.837) showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone (AUC = 0.811). In differentiating early stage ovarian cancer from benign diseases or healthy controls, LRG1 showed comparable performance to CA125. An independent sample set was further used to confirm the ability of these candidate markers to detect patients with ovarian cancer. Our study provides a comprehensive strategy for the identification of candidate biomarkers that show the potential for diagnosis of ovarian cancer. Further studies using a large number of samples are necessary to validate the utility of this panel of proteins.

Trougakos IP
The molecular chaperone apolipoprotein J/clusterin as a sensor of oxidative stress: implications in therapeutic approaches - a mini-review.
Gerontology. 2013; 59(6):514-23 [PubMed] Related Publications
BACKGROUND: Organisms are constantly exposed to physiological and environmental stresses and therefore require an efficient surveillance of genome and proteome quality in order to prevent disruption of homeostasis. Central to the intra- and extracellular proteome surveillance system are the molecular chaperones that contribute to both proteome maintenance and clearance. The conventional protein product of the apolipoprotein J/clusterin (CLU) gene is a heterodimeric secreted glycoprotein (also termed as sCLU) with a ubiquitous expression in human tissues. CLU exerts a small heat shock protein-like stress-induced chaperone activity and has been functionally implicated in numerous physiological processes as well as in ageing and most age-related diseases including tumorigenesis, neurodegeneration, and cardiovascular and metabolic syndromes.
OBJECTIVE: The CLU gene is differentially regulated by a wide variety of stimuli due to the combined presence of many distinct regulatory elements in its promoter that make it an extremely sensitive cellular biosensor of environmental and/or oxidative stress. Downstream to CLU gene induction, the CLU protein seems to actively intervene in pathological states of increased oxidative injury due to its chaperone-related property to inhibit protein aggregation and precipitation (a main feature of oxidant injury), as well as due to its reported distribution in both extra- and, most likely, intracellular compartments.
CONCLUSION: On the basis of these findings, CLU has emerged as a unique regulator of cellular proteostasis. Nevertheless, it seemingly exerts a dual function in pathology. For instance, in normal cells and during early phases of carcinogenesis, CLU may inhibit tumor progression as it contributes to suppression of proteotoxic stress. In advanced neoplasia, however, it may offer a significant survival advantage in the tumor by suppressing many therapeutic stressors and enhancing metastasis. This review will critically present a synopsis of recent novel findings that relate to the function of this amazing molecule and support the notion that CLU is a biosensor of oxidative injury; a common link between ageing and all pathologies where CLU has been implicated. Potential future perspectives, implications and opportunities for translational research and the development of new therapies will be discussed.

Shi H, Deng JH, Wang Z, et al.
Knockdown of clusterin inhibits the growth and migration of renal carcinoma cells and leads to differential gene expression.
Mol Med Rep. 2013; 8(1):35-40 [PubMed] Related Publications
Clusterin (CLU) is a glycoprotein involved in tumor progression, whose expression level correlates with the metastasis of renal cell carcinoma (RCC). However, the mechanism by which CLU plays an oncogenic role in RCC remains unclear. In this study, we used the human renal cancer cell 786-O as an experimental model. We knocked down CLU expression in the 786-O cells using lentiviral vector-mediated delivery of RNAi, and then compared the gene expression profiles between the knocked down CLU 786-O cells and control cells. We observed that CLU knockdown induced apoptosis and inhibited the proliferation and migration of 786-O cells. Microassay analysis revealed changes in the expression of 588 genes between the 786-O cells infected by a si-CLU lentivirus and the control cells, where 356 genes were upregulated and 232 were downregulated. Pathway analysis classified the differentially expressed genes into 17 upregulated and 12 downregulated pathways, including the PI3K/Akt, MAPK and VEGF pathways. In this study, we demonstrated that CLU acts as an oncogene in RCC by promoting cell proliferation and migration and inhibiting apoptosis. Microassay analysis may provide a platform for further characterization of the individual genes implicated in the development of RCC, providing new insights into the oncogenic role of CLU.

Varisli L
Identification of new genes downregulated in prostate cancer and investigation of their effects on prognosis.
Genet Test Mol Biomarkers. 2013; 17(7):562-6 [PubMed] Related Publications
Prostate cancer is the most common noncutaneous malignant neoplasm in men in the Western countries. It is well established that genetic and epigenetic alterations are common events in prostate cancer, which may lead to aberrant expression of critical genes. Most of the studies are focused on the overexpressed or duplicated genes in prostate cancer. However, it is known that some of the differentially expressed genes in prostate cancer are downregulated. Since the inventory of downregulated genes is incomplete, we performed in silico approaches to reveal the novel prostate cancer downregulated genes. Moreover, we also investigated for a possible link between the expression of the downregulated genes and tumor grade, recurrence, metastasis, or survival status in prostate cancer. Our results showed that the expression of GSTP1 and AOX1 are downregulated in prostate cancer, in concordance with previous reports. Moreover, we showed that TPM2, CLU, and COL4A6 mRNA levels are downregulated in prostate cancer. Further, we found a significant negative correlation between the expression of the above-mentioned genes and the prognosis of prostate cancer.

Fuzio P, Valletti A, Napoli A, et al.
Regulation of the expression of CLU isoforms in endometrial proliferative diseases.
Int J Oncol. 2013; 42(6):1929-44 [PubMed] Related Publications
Clusterin (CLU) is a nearly ubiquitous multifunctional protein synthesized in different functionally divergent isoforms, sCLU and nCLU, playing a crucial role by keeping a balance between cell proliferation and death. Studying in vivo CLU expression we found a higher mRNA expression both in neoplastic and hyperplastic tissues in comparison to normal endometria; in particular, by RT-qPCR we demonstrated an increase of the specific sCLU isoform in the neoplastic and hyperplastic endometrial diseases. On the contrary, no CLU increase was detected at the protein level. The CLU gene transcriptional activity was upregulated in the hyperplastic and neoplastic tissues, indicating the existence of a fine post-trans-criptional regulation of CLU expression possibly responsible for the protein decrease in the malignant disease. A specific CLU immunoreactivity was present in all the endometrial glandular cells in comparison to the other cellular compartments where CLU immunoreactivity was lower or absent. In conclusion, our results suggest the existence of a complex regulatory mechanism of CLU gene expression during the progression from normal to malignant cells, possibly contributing to endometrial carcinogenesis. Moreover, the specific alteration of the sCLU:nCLU ratio associated with the pathological stage, suggests a possible usage of CLU as molecular biomarker for the diagnosis/prognosis of endometrial proliferative diseases.

Li Y, Hu J, Guan F, et al.
Copper induces cellular senescence in human glioblastoma multiforme cells through downregulation of Bmi-1.
Oncol Rep. 2013; 29(5):1805-10 [PubMed] Related Publications
Most human tumor cells, including glioblastoma multiforme (GBM) cells, have aberrant control of cell aging and apoptosis. Subcytotoxic concentrations of oxidative or stress‑causing agents, such as hydrogen peroxide, may induce human cell senescence. Thus, induction of tumor cells into premature senescence may provide a useful in vitro model for developing novel therapeutic strategy to combat tumors. In the present study, we assessed the molecular mechanism(s) underlying senescence in GBM cells induced by copper sulfate. Following pretreatment with subcytotoxic concentrations of copper sulfate, U87-MG tumor cells showed typical aging characteristics, including reduced cell proliferation, cell enlargement, increased level of senescence-associated β-galactosidase (SA β-gal) activity, and overexpression of several senescence-associated genes, p16, p21, transforming growth factor β-1 (TGF-β1), insulin growth factor binding protein 3 (IGFBP3) and apolipoprotein J (ApoJ). We further demonstrated that the Bmi-1 pathway was downregulated in GBM cells in parallel with the induced senescence. The present study for the first time demonstrates the ability of copper to induce GBM cell senescence by downregulating Bmi-1.

Wang C, Jiang K, Gao D, et al.
Clusterin protects hepatocellular carcinoma cells from endoplasmic reticulum stress induced apoptosis through GRP78.
PLoS One. 2013; 8(2):e55981 [PubMed] Free Access to Full Article Related Publications
Clusterin (CLU) is a stress-activated chaperone, which plays an important role in cancer development and progression through promoting cell survival. However, the exact mechanism of how CLU exerts its cell protective role under ER stress condition is still unclear. Therefore, in order to explore the molecular mechanisms by which CLU inhibited ER stress-induced apoptosis, HCC cell lines were treated with tunicamycin (TN), an ER stress inducer. We found that the expressions of both CLU and GRP78 were increased after TN treatment. Knockdown of CLU expression in SMMC7721 and HCCLM3 cells inhibited GRP78 expression after TN treatment and enhanced ER stress-induced apoptosis, whereas over-expression of CLU in HepG2 cells increased GRP78 expression after TN induction and abolished the effect of TN on cell apoptosis. Furthermore, knockdown of GRP78 expression in CLU-HepG2 cells abrogated the protective role of CLU under ER stress condition. Co-immunoprecipitation (co-IP) and confocal microscopy experiments confirmed the direct interaction between CLU and GRP78 under ER stress condition. The effect of CLU knockdown on GRP78 expression and cell apoptosis in HCC tumors were further determined in orthotopic xenograft tumor model. Knockdown of CLU expression in HCCLM3 cells inhibited GRP78 expression in tumor tissues, accompanied with increased number of apoptotic cancer cells. Moreover, the correlation between CLU and GRP78 expression was further determined in clinical HCC specimens. Taken together, these findings reveal that CLU protects HCC cells from ER stress induced apoptosis at least partially through interacting with GRP78.

Essabbani A, Garcia L, Zonetti MJ, et al.
Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death.
PLoS One. 2013; 8(2):e54920 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties.
METHODOLOGY: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress.
RESULTS AND CONCLUSIONS: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

Corvetta D, Chayka O, Gherardi S, et al.
Physical interaction between MYCN oncogene and polycomb repressive complex 2 (PRC2) in neuroblastoma: functional and therapeutic implications.
J Biol Chem. 2013; 288(12):8332-41 [PubMed] Free Access to Full Article Related Publications
CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5'-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.

Lubin J, Markowska A, Knapp P
Factors affecting response of chemotherapy in women with ovarian cancer.
Eur J Gynaecol Oncol. 2012; 33(6):644-7 [PubMed] Related Publications
Chemotherapy plays an important role in the treatment of ovarian cancer. Patients' response to chemotherapy is determined by a variety of acknowledged factors, but one might expect that many of them are yet to be described. The aim of this paper was to present the most essential yet still to be generally assessed in clinical practice, factors, which include: E-cadhedrin, hypoxia inducible factor alpha, survivin, COX-2, clusterin, BRCA1 protein, TP53 protein, YY1 protein, multidrug resistance protein, and interleukin-8.

Gottschling S, Granzow M, Kuner R, et al.
Mesenchymal stem cells in non-small cell lung cancer--different from others? Insights from comparative molecular and functional analyses.
Lung Cancer. 2013; 80(1):19-29 [PubMed] Related Publications
BACKGROUND: Cancer-associated fibroblasts (CAF) play a vital role in lung cancer initiation and progression. Although mesenchymal stem cells (MSC) are considered progenitor cells of fibroblasts and show cancer modulating abilities themselves, analyses on their presence and properties in lung cancer are lacking so far.
METHODS: We performed a comparative molecular and functional analysis of MSC derived from non-small cell lung cancer (NSCLC) and corresponding normal lung tissue (NLT) of a total of 15 patients. MSC were identified and selected according to their mesenchymal multilineage differentiation capability and surface marker profile.
RESULTS: Compared to NLT-MSC, NSCLC-MSC showed accelerated growth kinetics and reduced sensitivity to cisplatin. Karyotyping, comparative genomic hybridization and multiplex fluorescence in situ hybridization revealed no chromosomal aberrations. However, gene expression profiling of NSCLC- and NLT-MSC indicated variable expression of 62 genes involved in proliferation, DNA repair, apoptosis, extracellular matrix synthesis, tissue remodeling and angiogenesis. Differential expression of the selected candidate genes butyrylcholinesterase, clusterin and quiescin Q6 sulfhydryl oxidase 1 was validated by quantitative real-time PCR and, on protein level, by immunohistochemical analyses of original tumor tissue. Upon exposure to tumor cell-conditioned medium or transforming growth factor-β, both, NSCLC-MSC and NLT-MSC acquired expression of α-smooth muscle actin (α-SMA), a major characteristics of CAF.
CONCLUSIONS: This study indicates that NSCLC tissue contains MSC with specific molecular and functional properties. These cells might represent a progenitor reservoir for CAF and thus crucially contribute to lung cancer progression.

Xiu P, Dong X, Dong X, et al.
Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma.
Cancer Sci. 2013; 104(3):375-82 [PubMed] Related Publications
Secretory clusterin (sCLU) is expressed in numerous cancers and is associated with the resistance to chemotherapy. However, the role of sCLU in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin (OXA), a recently used third-generation platinum agent, remains unclear. The stable transfectants that are depleted of or overexpress sCLU and OXA-resistant cells were generated using human HCC cells. Overexpression of sCLU abrogated OXA-induced inhibition of cell growth and cell apoptosis, but depletion of sCLU synergized with OXA to inhibit cell growth and enhance cell apoptosis, by regulating proteins involved in mitochondrial apoptosis pathways, such as Bcl-2, Bax, Bcl-xL and caspase-9, and affecting phosphorylation of Akt and GSK-3β. Overexpression of sCLU in either OXA-resistant cells or stable transfectants that overexpress sCLU significantly increased phosphorylated Akt. However, specific inhibition of Akt enhanced sensitivity of sCLU-overexpressing cells to OXA, but had no effect on sCLU expression, suggesting that the regulatory effects between sCLU and pAkt may be in a one-way manner in HCC cells. The expression levels of sCLU affected the therapeutic efficacy of OXA to treat HCC tumors established in immunodeficiency mice. The results have demonstrated that sCLU contributes to OXA resistance by activating Akt pathway, indicating that sCLU may be a novel molecular target for overcoming OXA resistance in HCC.

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