Advances and Challenges in Basic and Translational Research on Clusterin

Introduction

Clusterin (CLU), known by several other names, has recently drawn much attention because of its association with cancer promotion and metastasis [1–3]. First discovered as serum apolipoprotein J with chaperoning properties for protein stabilization, CLU also can exist within the cell to display either pro-apoptotic or pro-survival function. This diverse set of functions can be attributed to the existence of two alternatively-spliced forms of the CLU gene that encode secretory CLU (sCLU) or nuclear CLU (nCLU). The sCLU form is cytoprotective and its pro-survival function is the basis of current Phase I/II clinical trials against prostate, lung, and breast cancers, where synergy with various cytotoxic agents has been reported [4]. In contrast, nCLU migrates to the nucleus upon cytotoxic stress to trigger cell death [5, 6]. It interacts with the DNA double strand break repair antigen, Ku70, thus blocking its function [6].

The 5^th International Clusterin Workshop, held in Spetses, Greece, on June 2–5, 2008, brought together researchers active in this field to focus on clarifying the mechanisms of this unique protein for executing the fate of a cell. Based on new findings presented, a full debate on whether CLU has a key role in tumor prevention or tumor promotion led to some consensus that it may depend on the context of its expression and the microenvironment.

CLU as a cell death/survival determinant

The intracellular localization of CLU protein isoforms and the effect on cell survival was an active area of research. Dr. Arkadiusz Orzechowski (Polish Academy of Sciences, Warsaw, Poland) reported decreased nCLU levels but increased sCLU levels in vitro or in vivo in colorectal cancers in comparison to normal cells/tissues. Importantly, the sCLU:nCLU ratio was a key factor in COLO 205 colon tumor cell survival. These data confirmed previous findings in human colon carcinoma [1]. Similarly, Dr. Leonardo Fabbri (University of Modena and Reggio Emilia, Modena, Italy) evaluated CLU isoform expression in human normal bronchial (BEAS-2B) epithelial and A549 lung adenocarcinoma cells after cigarette smoke extract (CSE) exposure. Again, the sCLU:nCLU ratio appears to be an important determinant of cell survival. Dr. Evangelos Kolettas (University of Ioannina, Ioannina, Greece) analyzed CLU expression in mutant p53 human HaCaT keratinocytes after vanadyl sulphate (VOSO₄) exposure and reported that apoptosis correlated with up-regulation of c-fos, decreased sCLU:nCLU ratios, and down-regulation of Bcl-2 levels and caspase activity. Overexpression of either c-fos or v-fos induced the same responses in HaCaT cells. Interestingly, sCLU overexpression failed to protect HaCaT cells from VOSO₄, but nCLU overexpression induced apoptosis. Dr. Saverio Bettuzzi (University of Parma, Palma, Italy) measured the half-life of sCLU to be <2 h and its turnover was tightly regulated by ubiquitin–proteasome-mediated degradation.

CLU as a modifier of tumor-suppression

A number of investigators had evidence of the potential role of CLU as a tumor suppressor in several animal and cell models. Dr. Bettuzzi found that siRNA-mediated sCLU knockdown induced cell cycle progression, with elevated proliferative markers. He also reported that TRAMP mice, carrying a probasin-driven SV40 T antigen, had highly aggressive tumor development when crossed with CLU knockout (CLU^−/−) mice. Onset of cancer was earlier, with higher numbers of primary tumors and elevated, poorly-differentiated metastatic tumors per mouse, in comparison to wildtype TRAMP mice. Thus, loss of CLU drives tumor progression suggesting that CLU might be a tumor suppressor. In support of this view, Dr. Arturo Sala (Institute of Child Health, London, United Kingdom) showed that full-length sCLU mRNA was down-regulated in neuroblastomas associated with MYCN gene amplification and had direct evidence that N-MYC suppressed sCLU expression,. The penetrance of neuroblastomas in N-MYC-transgenic mice was significantly increased after deletion of one or both sCLU alleles, and sCLUsiRNA transduced neuroblastoma cells acquired increased metastases when xenografted into mice. Ablation of CLU was accompanied by NF-κB activation in developing neuroblastomas and during epithelial to mesenchymal transition (EMT). To corroborate that CLU inhibits NF-κB, Dr. Gilles Chiocchia (Cochin Institute, Paris, France) showed that specific sCLU sub-fragments bound and stabilized IκB-α, resulting in NF-κB inhibition. Dr. Andrei Thomas-Tikhonenko (University of Pennsylvania, Philadelphia, PA) reported that Myc-transformed epithelial cells down-regulated sCLU and that sCLU could inhibit cell growth in vitro and prevent carcinogenesis in vivo. He further reported that Myc up-regulated microRNAs, specifically, miR-17-92 cluster, which apparently silences sCLU gene transcription and confirmed that mir17-92 blockade can restore sCLU expression to impede tumor cell growth. Thus, Myc downregulation of sCLU may be critical for cell transformation.

CLU as a modifier of tumor promotion

On the other hand, sCLU also seems to possess the property of an oncogene, by its ability to enhance cell proliferation and inhibit apoptosis. Dr. Sabina Pucci (University of Rome, Rome, Italy) evaluated sCLU levels in sera and stools of healthy donors versus colorectal cancer patients. A relationship between IL-6 and sCLU levels was found, correlating with progressive disease, and screening of both molecules was proposed as in situ and circulating prognostic markers for non-invasive colorectal cancer. Additionally, during colon carcinoma progression, IL-6 and VEGF-A production influenced tumour cell proliferation, favoring apoptotic escape and cell migration by interfering with Ku70-CLU-Bax interactions. It was demonstrated in vitro that these interactions are modulated by IL-6. Moreover, IL-6 together with VEGF inhibited Bax-dependent cell death, promoted cell survival and tumor invasion and increased the production of sCLU, shifting thus death to survival. Dr. Bon-Hong Min (Korea University, Seoul, Korea) reported that sCLU stimulated astrocyte proliferation through EGF receptor (EGFR)-stimulated Erk activation. Knockdown of sCLU via siRNA or Erk inhibition suppressed growth, whereas sCLU overexpression or exogenous treatment with purified sCLU promoted EGFR phosphorylation at Y1173 and enhanced astrocyte proliferation. Dr. Amina Zoubeidi working with Dr. Martin Gleave (University of British Columbia, Vancouver, Canada) showed that elevated sCLU levels promoted prostate tumor cell survival by increasing NF-κB nuclear translocation and transactivation. Significantly, sCLU acted as an ubiquitin-binding protein that enhanced COMMD1 and I-κB proteasomal degradation via interaction with E3 ligase family members. For confirmation, sCLU knockdown was shown to stabilize COMMD1 and I-κB, blocking NF-κB release while sCLU levels positively correlated with expression of known NF-κB-regulated gene signatures. Dr. Ioannis Trougakos (Institute of Biological Research and Biotechnology, Athens, Greece) described changes in signal transduction after the singular loss or overexpression of sCLU in non-stressed human cells, as well as changes in sCLU levels on ageing. Depletion of sCLU signaled stress-inducing, p53-dependent growth retardation, altered pro- to anti-apoptotic Bcl-2 protein ratios and disrupted Ku70-Bax complexes. Bax-activated mitochondrial dysfunction and elevated apoptosis ensued because sCLU functions as a cytosol retention factor for Bax. Moreover, he reported that gene expression signatures induced in response to altered sCLU expression levels included cell cycle, focal adhesion, p53, insulin, mTOR and actin cytoskeleton signaling pathways. Intriguing data were presented by Dr. Claudia Koch-Brandt (University of Mainz, Mainz, Germany) linking Toll-like receptor 3 (TLR3) with transcriptional activation of sCLU leading to cytoprotective and anti-inflammatory responses. In addition to poly I:C that is a ligand for TLR3, CLU gene activation was also stimulated by purified cellular RNA and RNA released from necrotic cells.

Regulation of sCLU expression

In support of the pro-survival and tumor promoting role of sCLU, Dr. David Boothman (University of Texas Southwestern Medical Center, Dallas, TX) reported an insulin-like growth factor-1 (IGF-1)-mediated sCLU induction pathway. The ATM (mutated in Ataxia telangiectasia) kinase, induced by low doses of irradiation or DNA double strand break-inducing agents, activated IGF-1 production that in turn induced sCLU gene transcription. Importantly, all genetically unstable cells analyzed to date (i.e., H2AX^−/−, MDC1^−/−, NBS1^−/−, MMR^−/−, and mTR^−/−) showed elevated basal ATM-IGF-1/IGF-1R-sCLU expression. This pathway was exquisitely regulated by p53 in the cell, whereby IGF-1 promoter transactivation was repressed by p53-NF-Y interactions. Dr. Boothman also elaborated on the role of transforming growth factor (TGF)-β1 in sCLU induction. In contrast to irradiation, where p53 could repress IGF-1-mediated sCLU expression, TGF-β1-treated wild-type p53 cells induced expression of this axis. Molecularly, TGF-β1-induced Hdm2 gene transactivation, in conjunction with AKT over-expression/activation and p-Hdm2-dependent p53 degradation stimulates IGF-1R/SRC/MAPK/ERK signaling, causing Egr-1-mediated sCLU expression. Using sCLU knockdown, Dr. Boothman showed that a positive feedback loop for sCLU expression played a role in survival and EMT transition in human colon and non-small cell lung cancers. He proposed that IGF-1R/Src/MAPK/Erk/Egr-1 signaling was a universal regulatory pathway for this gene’s expression.

Dr. Efstathios Gonos (Institute of Biological Research and Biotechnology, Athens, Greece) reviewed the involvement of sCLU in ageing. Although sCLU mRNA and protein levels were up-regulated in vitro during either replicative- or stress-induced-premature senescence of primary human cells as well as in vivo during normal ageing, serum levels were lower in healthy centenarians versus elderly donors. He proposed that sCLU is central to cellular homeostasis and that during aging sCLU upregulation occurred in response to chronic stress. Dr. Julie Djeu (H. Lee Moffitt Cancer Center, Tampa, FL) analyzed docetaxel-resistance (DR) in androgen-independent PC3 and DU145 prostate tumor cells and found elevated Stat1 and sCLU levels versus parental tumor cells. Stat1 knockdown via siRNA prevented sCLU gene and protein expression, while restoring docetaxol sensitivity. Thus, Stat1 controlled sCLU production, leading to docetaxel resistance. Moreover, TRAIL resistance was also traced to clusterin gene expression. Expression of sCLU was positively regulated by Src/Jak/Stat signaling and inhibition of Src/Jak1 kinases by resveratrol abolished sCLU expression and resensitized cells to TRAIL. Thus, resveratrol may be added to current chemotherapeutic regimens to treat prostate cancer, circumventing docetaxel or TRAIL resistance.

Chaperone activity – Neurological diseases

Another major function of CLU is its chaperoning capacity for protein stabilization and facilitation of clearance of damaged proteins, Dr. Mark Wilson (University of Wollongong, Wollongong, Australia) identified putative endogenous extracellular ligands for the chaperone action of sCLU in human plasma. Clusterin-misfolded proteins complexes were of high molecular weight (>4×10⁷ Da) and could be traced in vivo to be rapidly cleared in the liver and spleen. Putative receptors for these complexes were identified in rat hepatocytes. Thus, CLU may exert a key role in clearing damaged proteins from extracellular spaces and protect the body from potentially dangerous accumulation of aggregated proteins and deposition, such as in Alzheimer’s disease. Dr. Jeong Hun Kim (Seoul National University, Seoul, Korea) showed that sCLU played a critical function in retinal vessel development as a secreted molecular chaperone. This isoform was cytoprotective in ischemia-induced retinal endothelial cell apoptosis and in diabetic retinas in vitro. Analysis of mechanism indicated that sCLU had anti-permeable activity against high-glucose or glycation end-product-induced tight junction breakdown. Since prior data from this and other groups indicated similar functions in cancer cells, sCLU may play a role in oxidative high glucose utilization (i.e., Warburg Effect) and regulate tumor cell metabolism.

Insight into the dual role of Clusterin as a tumor suppressor and a tumor promoter

At the workshop, the idea began to emerge that the dual nature of CLU as a tumor suppressor and a promoter may not be contradictory. The outcome may depend largely on the isoform available, on the time and context that CLU is expressed in the life of a cell, and the microenvironment. In terms of sCLU, including its pre-secretory form, it can be speculated that a normal cell, upon exposure to oxidants or DNA damaging agents, will require sCLU to bind misfolded proteins until they can be properly refolded or recycled through the ubiquination pathway for degradation. Thus, without sCLU, as in the case of CLU^−/− mice, the cell can accumulate damaged proteins, some of which may be critical for cell homeostasis, and result in transformation. Under this scenario, sCLU acts as a tumor suppressor. However, when tumor transformation has already occurred and the tumor cells are being treated with toxic chemotherapeutic agents, cell survival is paramount for tumor maintenance. In this case, tumor cells must have sCLU to protect against DNA-damaging agents and sCLU thus becomes a critical component for cell survival. In addition, growth factors necessary for tumor cell proliferation, such as IL-6, VEGF, IGF-1 and EGF, as well as tumor-derived factors such as TGF-β1, can stimulate sCLU production, which can then mediate tumor or senescent cell survival. In this latter case, sCLU within a transformed cell acts as an oncogene. These concepts are relatively unexplored but are easily testable. In terms of nCLU, it is invariantly pro-apoptotic and it is possible that a balance of sCLU/nClU in a cell dictates its fate. Interestingly, nCLU is often absent in advanced tumors or tumor cell lines.

Suggested terminology for the CLU gene locus protein isoforms

Previously known by various names, including testosterone repressed prostate message-2, sulphated glycoprotein-2, apolipoprotein J, and x-ray-inducible transcript #8, CLU was designated the universally accepted name for this gene at the 1st International CLU workshop. At this workshop, a unifying terminology for the various CLU protein isoforms was adopted based on the two characterized CLU gene transcripts. The main gene transcript identified from the CLU gene locus (Supplemental Fig. 1A), sCLU isoform 2 (NCBI: NM_203339.1 / NP_976084.1, predicted MW: 50.1 kDa) is detected as an ~60 kDa glycosylated pre-secretory form of sCLU and will be referred to as psCLU (Supplemental Fig. 1B). Once it is cleaved and heavily glycosylated to form the mature, secreted heterodimeric ~75–80 kDa protein (Supplemental Fig. 1C), it will be referred to as sCLU. The sCLU protein form is thus composed of sCLUα and sCLUβ chains that are of ~40 kDa (Supplemental Figs. 1B,C). The second gene transcript lacks the endoplasmic reticulum-targeting sequence at exon 2 (Supplemental Fig. 1A, nCLU) and its product is detected as an ~49 kDa non-glycosylated precursor nuclear CLU protein (pnCLU, predicted MW: 48.8 kDa) in the cytosol, and an ~55 kDa glycosylated protein (referred to as nCLU) in the nucleus.

Future directions

CLU expression changes are a sensitive indicator of oxidative stress in mammalian cells [7] and may play a role in genomic instability [8–10] seen during tumor progression (1–3) and in pathological conditions associated with deposition of protein aggregates (e.g., Alzheimer’s disease, inflammation, stroke and heart attacks) [7]. Its function as a chaperone in human serum and its ability to exist in two forms, sCLU to mediate cell survival, and nCLU to induce apoptosis, endows it with unique properties that can decide the fate of a cell. Results suggest that IGF-1-Src-Erk signaling, even in response to TGF-β1 exposure, may be key regulators of sCLU expression, accounting for tumor progression and other chronic diseases. Interestingly, Dr. Ofer Levy (Compugen LTD, Tel Aviv, Israel) presented data for a peptide that specifically bound sCLU and caused growth arrest as well as synergistic cytotoxicity with Taxol against breast, lung, prostate and melanoma cancer cells indicating that sCLU is a “drugable target” in tumor therapy. What is sorely needed is research illuminating roles for sCLU versus non-inducible nCLU in normal and pathological tissues. Understanding the molecular regulation and structure of CLU isoforms is essential, especially given that the ratio of sCLU:nCLU is critical for cancer survival and progression. It is essential to address cell- and tissue-specific CLU functions during tumor initiation and progression. Further research into CLU functions and downstream signaling pathways affected by CLU expression are also clearly needed. Collaborations between groups are needed to enhance clinical opportunities to exploit these findings.