*5.3.1. PI3K/Akt/NF-κB pathway*

**5. Application prospect of targeting CLU gene**

**Group n Average (μg/mL)** *t* **value** *P* **value Positive, n (%)** *χ***<sup>2</sup>**

III & IV 44 122.93 ± 17.31 36 (81.82)

B&C 20 123.63 ± 21.23 16 (80.00)

**Table 4.** Clinicopathologic features of serum sCLU expression in HCC patients (Mean ± SD).

I & II 31 113.93 ± 14.36 2.375 0.02**<sup>d</sup>** 20 (64.52) 2.878 0.09

A 55 117.60 ± 14.56 1.395 0.167 40 (72.73) 0.410 0.522

 **value** *P* **value**

**5.2. Reversal MDR by specific shRNA-targeted sCLU**

ASO is a useful technique to inhibit specific-targeted CLU genes, with a small synthetic natural nucleic acid analogue, that can complementary to CLU mRNA that induce degradation or inhibit translation into protein [52]. It is considered to be a potent inhibitor of sCLU expression in vitro, in vivo, and in human clinical trials, with no apparent effect on the expression of nCLU. Custirsen (OGX-011, 5′-CAGCAGCA GAGTCTTCATCAT-3′, 50 nM) is a novel 2′-methoxy-ethyl-modified phosphorothioate ASO, which is a 21-nucleoside complement to target the translation initiation site of CLU gene exon II mRNA translation initiation site with one CpG motif [53]. Hence, OGX-011 plays the role of chemosensitization by influencing the anti-apoptotic protein sCLU instead of the proapoptotic protein nCLU. Xiu et al. have reviewed the current state of research on clusterin, to predict future research directions and to analyze the potential of the clinical application of custirsen in HCC [54]. However, the median overall survival of HCC cases were ms 23.4 mo in the group of the OGX-011 combining anti-cancer drugs (docetaxel/prednisone) and ms 22.2 mo in the other group of cases with docetaxel/prednisone alone. No significant difference was found between two groups. Some potential key factors might contribute to its results, and still want to do more clinical trials to be ongoing [55].

Resistance of tumor cells to chemotherapy continues to be a major clinical obstacle to extend the survival rate of patients with HCC. Recently, one of the major strategies for liver cancer is surgical resection with adjuvant anti-HCC drug chemotherapy [10]. However, the HCC patients always tend to acquire MDR during tumor progression. MDR-related P-glycoprotein (P-gp), encoded by MDR1 gene is positively linked closely with chronic liver diseases, because of its

**5.1. Antisense oligonucleotide (ASO) therapy**

AFP, α-fetoprotein; TNM, tumor node metastasis.

138 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

With the tumor size ≥5 cm group.

The lymph node metastasis group.

With the multifocal group. <sup>d</sup>With TNM III & IV group.

**TNM stage**

a

b

c

**Child classification**

Previous data revealed that CLU promoted cell survival through the PI3K/Akt pathway and induced MMP-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways [32]. CLU could increase p-Akt and MMP13 expression. A positive correlation between CLU expression and p-Akt level was observed in cohort of HCC tissues. Where CLU knockdown using OGX-011 significantly decreased p-Akt and MMP13 levels and suppressed HCC metastasis in two metastatic models through inhibiting EIF3I/Akt/MMP13 signaling. The related signaling molecule blockade of the PI3K-Akt pathway could significantly inhibited MMP13 expression in human HepG2-CLU or HCCLM3 cells [38, 58]. Decreased level of CLU accompanied with downregulation of MMP13 and p-Akt was observed in tumors derived from HCCLM3 shCLU group, revealed that p-Akt level was significantly correlated with poor prognosis and indicated that CLU may play a crucial role in HCC metastasis [38].

#### *5.3.2. Wnt/β-catenin pathway*

Wnt canonical pathway is often constitutively active in neoplastic cells, although, normally β-catenin is negatively regulated by GSK-3β that phosphorylates β-catenin to drive it for proteosomal degradation [30, 59]. Previous data emphasized sCLU modification after being exposed to Wnt⁄β-catenin inhibitor, and expressions of the crucial β-catenin and GSK-3β genes were detected in cases of sCLU depletion. The data indicated that sCLU suppression might lead to the inhibition of Wnt/β-catenin pathway in reverse. Hence, sCLU might play an important role in chemoresistance of HepG2/ADM cells together with Wnt/β-catenin signaling molecules [31, 56].

### *5.3.3. IGF-1/IGF-1R/Src/Mek/Erk pathway*

Hepatic sCLU is a general genotoxic stress-induced, prosurvival gene product implicated in cancer [36, 37]. The regulatory signal transduction processes that control sCLU expression, the induction of sCLU is delayed, peaking 72 h after low doses of ionizing radiation, and is dependent on up-regulating IGF-1 and phosphorylation-dependent IGF-1R activation [23, 27] that stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately

lower than that in the control or the NC-shRNA group; and the sCLU staining in the control or NC-shRNA group was stronger than that in the shRNA-1 group by immunohistochemistry [46]. Specific shRNA-mediated downregulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis *in vivo* [38]. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling pathway, suggested that oncogenic sCLU might promote HCC metastasis via the induction of

Oncogenic Secretory Clusterin: A Promising Therapeutic Target for Hepatocellular Carcinoma

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EMT process and could be a promising candidate target for HCC therapy [32, 61–63].

In conclusion, the upregulation of sCLU expression at early staging of HCC is considered to promote tumor development, which may be related to the phosphorylation of AKT/GSK-3β. An increasing number of reports have provided evidence that sCLU level could be a novel biomarker for HCC diagnosis and prognosis, and there will be of great significance for the individualized treatment in HCC patients. The sCLU regulating signaling pathways could be critical to unraveling the solution for MDR in HCC. Therefore, silencing sCLU gene transcription and inhibiting sCLU expression by specific Custirsen inhibition have provided a new mechanism insight into molecular-targeted therapy for HCC in injected- or orthotopic model, indicated that sCLU gene would be a potential molecular-targeted for HCC therapy. Further study found that sCLU contributed to HCC migration and EMT *in vitro*, and metastasis *in vivo*. Although additional preclinical and clinical trials are necessary to explore the sCLU role in HCC, targeting the oncogenic sCLU could validate the approach as a systemic therapy to

This work was supported by grants-in-aid from Projects of the National Natural Science Foundation (81673241, 81702419), the Jiangsu Health Plans (2014-YY-028 and BE2016698), the Jiangsu Graduate Innovation Plan (KYCX17\_1934), the Nantong Science Foundation of Health and Family Planning Commission (WQ2016083), and the International Science & Technology

**6. Conclusions**

increase chemotherapy sensitivity.

Cooperation Program (2013DFA32150) of China.

**Acknowledgements**

**Abbreviations**

AFP alpha-fetoprotein

HCC hepatocellular carcinoma IHC immunohistochemistry MDR multiple drug resistance

**Figure 2.** Inhibition of sCLU gene transcription by specific shRNA-1 on effects of forming time, growth curve and size of orthotopic xenograft tumors. The control, neg-shRNA, or sCLU-shRNA groups were injected with the plasmid contained specific shRNA-1 into nude mice. (A) The forming time of the orthotopic xenograft tumors from each group. (B) The growth curves of the xenograft tumors from each group. And the tumor volumes were measured at the indicated time point. (C) The representative photographs of the nude mice and corresponding dissected tumors from each group. 1 and 2, the xenografts in the control group; 3 and 4, the xenografts in the neg-shRNA group; 5 and 6, the xenografts in the sCLU-shRNA group. The data are presented as the mean ± SD. Compared with the control group, \*\**P* < 0.01.

transactivate the early growth response-1 (Egr-1) transcription factor. Thus, the ionizing radiation exposure causes stress-induced IGF-1R-Src-Mek-Erk-Egr-1 activation that regulates the sCLU prosurvival cascade pathway for radiation resistance in HCC therapy [60].

#### **5.4. Suppressed HCC growth in vivo by silencing sCLU**

The inhibition of sCLU gene transcription by specific shRNA-1 on effects of forming time, growth curve, and size of orthotopic xenograft tumors after sacrifice of the mice at the 34th day with injection are shown in **Figure 2**. The mean weight of the xenograft tumors in the shRNA-1 group was significantly less than that of the control or NC-shRNA group, respectively. The curves of xenograft tumor growth indicated that tumor sizes in the shRNA-1 group with lower mRNA level were significantly smaller less than those of the control or NC-shRNA group [31, 46]. Consistently, the sCLU protein expression in the shRNA-1 group was also lower than that in the control or the NC-shRNA group; and the sCLU staining in the control or NC-shRNA group was stronger than that in the shRNA-1 group by immunohistochemistry [46]. Specific shRNA-mediated downregulation of sCLU resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary metastasis *in vivo* [38]. Overexpression of sCLU in HepG2 cell line showed increased cell migratory ability. In addition, sCLU also plays an important role in the regulation of TGF-β1-smad3 signaling pathway, suggested that oncogenic sCLU might promote HCC metastasis via the induction of EMT process and could be a promising candidate target for HCC therapy [32, 61–63].
