**4. Circulating sCLU as diagnostic marker**

The observations were in accordance with the early literature showing that upregulation of sCLU-positive expression might be associated with poor clinical outcome in HCC patients [21, 48]. According to previous clinical studies, average serum sCLU level was significantly higher in the HCC group more than that in any of groups with cirrhosis, chronic hepatitis, or healthy control (**Table 2**). The area under receiver operating characteristic (ROC) curve and diagnostic sensitivity were 0.75 and 74.7% in serum sCLU, and 0.74 and 58.7% in serum AFP, respectively. The incidence of both combining detection rose up to 90.7% for HCC diagnosis (**Table 3**). High-circulating sCLU levels were observed in HCC patients, consistent with a recent study using a three-step serum proteome analysis, which showed that serum sCLU levels in HCC were significantly higher than those in benign liver diseases [42].

**sCLU level > 104.2 μg/mL (%) AFP level > 50 ng/mL (%) Both (%)**

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Sensitivity (%) 74.67 58.67 90.67 Specificity (%) 66.67 75.00 60.00 Accuracy (%) 71.11 65.93 77.03 Positive predictive value (%) 73.68 74.58 73.91 Negative predictive value (%) 67.80 59.21 83.72

AFP, α-fetoprotein; sCLU, secretory clusterin.

**Age (years)**

**AFP (μg/L)**

**Tumor size**

**Portal vein invasion**

**Lymph node metastasis**

**Differentiation**

**Gross classification**

**Table 3.** Assessment diagnostic validity of serum sCLU or AFP level for HCC.

>60 28 121.09 ± 17.16 23 (82.14)

>50 44 118.55 ± 16.50 32 (72.73)

Without 47 119.14 ± 17.97 34 (72.34)

>5 cm 41 123.00 ± 14.52 35 (85.37)

With 31 125.40 ± 16.91 27 (87.10)

Poor 18 124.84 ± 16.90 14 (77.78)

Multifocal 39 124.00 ± 13.20 33 (84.62)

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

≤60 47 118.09 ± 16.45 0.751 0.455 33 (70.21) 1.320 0.251

≤50 31 120.15 ± 17.12 0.405 0.686 24 (77.42) 0.212 0.645

With 28 119.34 ± 14.52 0.050 0.960 22 (78.57) 0.360 0.548

≤5 cm 34 114.63 ± 18.10 2.221 0.029**<sup>a</sup>** 21 (61.76) 5.473 0.019

Without 44 114.84 ± 15.21 2.826 0.006**<sup>b</sup>** 29 (65.91) 4.316 0.038

Well & moderate 57 117.43 ± 16.34 1.662 0.101 42 (73.68) 0.121 0.728

Unifocal 36 114.02 ± 18.57 2.698 0.009**<sup>c</sup>** 23 (63.89) 4.251 0.039

CLU is related to reverse cholesterol transport, platelet degranulation and human immune response pathways [49]. Protein-protein interaction analysis and pathway assessment showed a closed molecular relationship between cirrhosis and HCC [50]. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1., 3 and 6 weeks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Circulating sCLU was significantly up-regulated during cancer progression and metastasis. The expression of sCLU was significantly higher in metastatic HCC cells and samples from HCC patients. Serum sCLU was highly increased with tumor size, tumor number, lymph node infiltration (**Table 4**) [42], and showed that the ROC area under curve value was 0.95 in sCLU more than that (0.85) in AFP. If 128 μg/mL as a cutoff value, the sensitivity or specificity of serum sCLU level for predicting HCC was 90 or 87%, respectively. The data indicated that circulating sCLU is a promising molecular marker of diagnosis or predicting metastasis for HCC [22, 51, 52].


Serum sCLU values >104.0 μg/mL or AFP values >50 ng/mL were considered positive.

sCLU, secretory clusterin; LC, liver cirrhosis; CH, chronic hepatitis; NC, normal control; and AFP, α-fetoprotein.

\* *P* < 0.05, compared with HCC group.

\*\**P* < 0.01, compared with HCC group.

**Table 2.** Serum sCLU or AFP levels among patients with chronic liver diseases and comparative analysis of both diagnostic values for HCC.

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**Table 3.** Assessment diagnostic validity of serum sCLU or AFP level for HCC.

patients [21, 48]. According to previous clinical studies, average serum sCLU level was significantly higher in the HCC group more than that in any of groups with cirrhosis, chronic hepatitis, or healthy control (**Table 2**). The area under receiver operating characteristic (ROC) curve and diagnostic sensitivity were 0.75 and 74.7% in serum sCLU, and 0.74 and 58.7% in serum AFP, respectively. The incidence of both combining detection rose up to 90.7% for HCC diagnosis (**Table 3**). High-circulating sCLU levels were observed in HCC patients, consistent with a recent study using a three-step serum proteome analysis, which showed that serum sCLU levels in HCC were significantly higher than those in benign

CLU is related to reverse cholesterol transport, platelet degranulation and human immune response pathways [49]. Protein-protein interaction analysis and pathway assessment showed a closed molecular relationship between cirrhosis and HCC [50]. Serum samples collected from HCCLM3-R metastatic HCC tumor model at specific stages of metastasis (1., 3 and 6 weeks) were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Circulating sCLU was significantly up-regulated during cancer progression and metastasis. The expression of sCLU was significantly higher in metastatic HCC cells and samples from HCC patients. Serum sCLU was highly increased with tumor size, tumor number, lymph node infiltration (**Table 4**) [42], and showed that the ROC area under curve value was 0.95 in sCLU more than that (0.85) in AFP. If 128 μg/mL as a cutoff value, the sensitivity or specificity of serum sCLU level for predicting HCC was 90 or 87%, respectively. The data indicated that circulating sCLU is a promising molecular marker of diagnosis or

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

liver diseases [42].

predicting metastasis for HCC [22, 51, 52].

136 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

HCC 75 119.21 ± 16.67 56 (74.67)

HCC 75 2177.32 ± 3757.99 44 (58.67)

**sCLU μg/mL**

**AFP ng/mL**

*P* < 0.05, compared with HCC group. \*\**P* < 0.01, compared with HCC group.

diagnostic values for HCC.

\*

**Group n Mean ± SD Positive n (%) χ<sup>2</sup>**

Serum sCLU values >104.0 μg/mL or AFP values >50 ng/mL were considered positive.

LC 30 97.78 ± 19.06 8 (26.67)\*\* 20.744 <0.001 CH 30 106.30 ± 19.22 12 (40.00)\*\* 11.285 <0.001 NC 36 89.96 ± 7.27 0 (0.00)\*\* 54.249 <0.001

LC 30 126.84 ± 244.76 10 (33.33)\* 5.505 0.019 CH 30 30.27 ± 50.09 5 (16.67)\*\* 15.188 <0.001 NC 36 7.1 ± 3.50 1 (2.78)\*\* 31.520 <0.001

sCLU, secretory clusterin; LC, liver cirrhosis; CH, chronic hepatitis; NC, normal control; and AFP, α-fetoprotein.

**Table 2.** Serum sCLU or AFP levels among patients with chronic liver diseases and comparative analysis of both



drug efflux via ATP-binding cassette (ABC) family transporters, which can decrease intracellular concentration of anti-HCC drugs. Targeting sCLU to sensitize cancer cells to chemotherapy has become an attractive new strategy for cancer treatment. Previous studies found sCLU expressed in line with P-gp via immunohistochemical staining. The sCLU expression was analyzed in human hepatoma cells and chemoresistant counter-part HepG2/ADM cells. After transfection of shRNA-1 (5′-GTAAGTACGTC AATAAGGA-3′) into HepG2/ADM cells, the inhibition of CLU expression was 73.68% at mRNA level with obvious enhancement in chemosensitivity, and increasing apoptosis induced by doxorubicin [56]. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-gp. Moreover, silencing CLU led to downregulation of β-catenin, suggesting that downregulation

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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

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].

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

of CLU might be a key point to reverse MDR of HepG2/ADM cells [57].

indicated that CLU may play a crucial role in HCC metastasis [38].

**5.3. Blockade-related pathway**

*5.3.1. PI3K/Akt/NF-κB pathway*

*5.3.2. Wnt/β-catenin pathway*

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

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