**2. Gene structure and functions of sCLU**

Human CLU gene is a single-copy gene on chromosome 8p21-p12 including 9 exons and 8 introns, encoding an mRNA of 2877 bp and translating to a polypeptide with 449 amino acids (a.a.) [25]. The secretory glycoprotein (1st a.a.) is a signal sequence of hydrophobic leader, and targets the ER protein. CLU gene encodes two isoforms with distinct functions as a result of alternative splicing and post-translational modifications: cytoplasm sCLU (75–80 kDa) and nuclear CLU (nCLU, 55 kDa), which is mainly located in the nucleus. The sCLU molecule is a highly conserved heterodimeric disulfide-linked 449 amino acid polypeptide that represents the major product of CLU gene. The ER-Golgi sCLU is considered to influence immune regulation, transformation, tissue remodeling, lipid transport, membrane recycling, complements cascade, DNA repair, cell adhesion, and cell-cell interactions, indicated that sCLU is widely distributed in tissues and body fluids involved in various physiological processes [11, 26, 27].

**1. Introduction**

132 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

great potential for HCC [8, 9].

sion of sCLU as a promising target for HCC gene therapy.

**2. Gene structure and functions of sCLU**

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide [1]. Growing understanding of the multiple pathogenic factors including hepatitis B or C virus (HBV or HCV) infection, toxic, lipid accumulation, aflatoxin B1 intake, and so on with complex molecular mechanisms underlying HCC reveal that hepatocarcinogenesis is a multistep process including lots of activated or suppressed oncogenes or anticancer genes [2–4]. Some techniques for HCC therapy have experienced great progress. However, the prognosis of HCC patients is still very poor due to the high rates of tumor recurrence and metastasis. Effective therapy of HCC is dependent on early specific diagnosis, therefore, to provide optimal treatment for patients, more precise and effective markers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis [5–7]. Numerous studies have shown the clinical utility of novel blood-based markers, such as circulating tumor cells, key signal molecules, long non-coding RNA, and microRNA with

Molecular chaperones are proteins that response to cellular stresses including genotoxic agents, nutrient starvation, and heat shock, with cellular stresses-induced protein misfolding, aggregation, and denaturation [10, 11]. To date, only few specific markers such as hepatoma-specific γ-glutamyl transferase [12, 13], oncofetal antigen glypican-3 (GPC-3) [14, 15], hepatoma-specific alpha-fetoprotein (HS-AFP or AFP-L3) [16], member 3a of Wingless-type MMTV integration site family (Wnt3a) [17, 18], and molecular chaperones like heat shock proteins (Hsp27 or Hsp90) [19] and clusterin have been developed as valuable biomarkers for primary hepatocellular carcinoma (PHC) diagnosis and surveillance. The clusterin (CLU) that was first detected in HCC tissues by Tobe et al., who found that SP40-40 gene in hepatoma cells was located in human chromosome 8, also designated as apolipoprotein J (APOJ), SP-40, sulfated glycoprotein 2 (SGP2), and testosterone-repressed prostate message 2 (TRPM2) [20]. Following the detection of their complete cDNA cloning, sequencing and comparison, secretory CLU (sCLU) is found to be the mature isoform of cytoplasm endoplasmic reticulum (ER)-Golgi CLU, which is over-expressed in a wide variety of tumors with oncogenicity [21, 22]. Recently, the mechanisms of abnormal sCLU expression and its targeted effects for HCC have been explored [23, 24]. This article summarizes some progres-

Human CLU gene is a single-copy gene on chromosome 8p21-p12 including 9 exons and 8 introns, encoding an mRNA of 2877 bp and translating to a polypeptide with 449 amino acids (a.a.) [25]. The secretory glycoprotein (1st a.a.) is a signal sequence of hydrophobic leader, and targets the ER protein. CLU gene encodes two isoforms with distinct functions as a result of alternative splicing and post-translational modifications: cytoplasm sCLU (75–80 kDa) and nuclear CLU (nCLU, 55 kDa), which is mainly located in the nucleus. The sCLU molecule is a Multiple reports have shown that the cytoplasm sCLU is cytoprotective and anti-apoptotic [28, 29], whereas nCLU protein is proapoptotic. Abnormal oncogenic sCLU expression was reported to correlate closely with HCC progression, such as inducing epithelial-mesenchymal transition (EMT) [30], formation of multiple drug resistance (MDR) [31], distal metastasis of tumor cells, and malignant transformation of hepatocytes, interaction with oncogenes or suppressor genes, and related signal pathways (**Figure 1**) [32, 33]. Because of ischemic or hypoxic microenvironment existence in cancerous tissues, the sCLU are often adaptively over-expressed and closely related with increased tumorigenicity, metastatic potential, and MDR to chemotherapy. As a stress-induced chaperone that inhibits protein misfolding and aggregation in a manner similar to small heat shock proteins (HSPs) [34, 35], its promoter

**Figure 1.** Potential mechanisms of hepatic sCLU in hepatocellular carcinoma. Cyto C, cytochrome c; ER, endoplasmic reticulum; Gene Reg., gene regulation; HIF-1α, hypoxia inducible factor-1α; IGF, insulin-like factor; MDR, multiple drug resistance; MMP, matrix metalloproteinase; nCLU, nuclear clusterin; NF-κB, nuclear factor-κB; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; Scr, sarcoma gene; sCLU, secretory clusterin.

region contains an element recognized by heat shock factor 1 (HSF-1) [36]. Cytoplasm sCLUinhibited apoptosis by interacting with activated Bax, and protects HCC cells from ER stressinduced apoptosis through a physical interaction with glucose-regulated prptein78 (GRP78) [29, 37].

Hepatic sCLU has been confirmed that it was physically associated with eukaryotic translation initiation factor 3 subunit I (EIF3I), and might protect EIF3I protein from degradation. A positive correlation was founded between sCLU and EIF3I, and both of their functions might be as a cooperative unit in HCC. The levels of sCLU and EIF3I expression were investigated in HCC using tissue microarray (TMA) and the patients with high EIF3I level exhibited poor prognosis. After silenced EIF3I, Akt phosphorylation was significantly inhibited. The EIF3I-Akt complex could prevent PP2A-mediated dephosphorylation, which in turn led to a constitutive Akt signal activation, suggesting that the CLU-EIF3I complex might prevent EIF3I degradation, and then contribute to Akt upregulation [33, 38].
