Follistatin‐like protein 5 inhibits hepatocellular carcinoma progression by inducing caspase‐dependent apoptosis and regulating Bcl‐2 family proteins

Abstract Hepatocellular carcinoma (HCC) is one of the most common and deadly malignant tumors in the world, especially in China. Follistatin‐like protein 5 (FSTL5) is a member of the FSTL family, which is involved in cell proliferation, migration, differentiation, and embryo development. We aimed to investigate the function and underlying mechanism of FSTL5 in HCC. FSTL5 expression was determined by immunohistochemistry staining in a liver cancer tissue microarray (TMA) and the correlation between FSTL5 and the prognosis of HCC patients was analysed. Further proliferation assay, colony formation assay, flow cytometry, and xenograft tumor model were performed to investigate the bioeffects of FSTL5 in HCC in vitro and in vivo. We found that FSTL5 expression was downregulated in HCC tissues and positively correlated with the prognosis of patients with HCC at tumor node metastasis stage I/II. Overexpression of FSTL5 efficiently impaired HCC growth both in vivo and in vitro with an exogenous manner. Mechanistic investigation demonstrated that FSTL5 promoted HCC cell apoptosis in a caspase‐dependent manner and regulated Bcl‐2 family proteins. These results indicate that FSTL5 may be a potential novel target for HCC treatment, and a biomarker for tumor prognosis.

Therefore, it is of great importance to find novel diagnostic biomarkers and therapeutic targets for HCC.
FSTL5 was first discovered in adult and infant brain tissue in 1993, and it was partially sequenced. 9 Not until 2002 was the complete FSTL5 gene sequence submitted to the Mammalian Gene Collection Program (MGC). 16 FSTL5 is located on human chromosome 4q (mouse chromosome 3), and its 4867-bp cDNA encodes a 93-kD protein with 847 amino acid residues. 16 FSTL5 was suggested to denote poor prognosis in medulloblastoma, which suggests an oncogenic role for this protein. 17 Furthermore, Kim et al revealed that FSTL5 mutations are associated with resistance to XPO1 inhibitors in KRAS-mutant lung cancer. 18 Recently, Zender et al demonstrated that FSTL5 is a potential tumor suppressor gene in HCC by in vivo RNAi screening. 19 Additionally, Zhang et al found that FSTL5 is downregulated and correlates with favourable prognosis in HCC. 20 In this study, to further investigate the potential clinical significance, FSTL5 expression was determined by immunohistochemistry staining in a liver cancer tissue microarray (TMA) and the correlation between FSTL5 and the prognosis of HCC patients was analysed.
Furthermore, we explored the biological effect and underlying mechanism of FSTL5 on HCC growth with proliferation assay, colony formation assay, flow cytometry, western blotting, and xenograft tumor model in vitro and in vivo. Our study would provide a novel therapy target and prognosis indicator for HCC.

| Immunohistochemistry
Tissue microarray was analysed for FSTL5 expression by immunohistochemistry (IHC) according to the manufacturer's recommendations (Vector Lab Inc., Burlingame, CA, USA). The antibody used is anti-FSTL5 (Abnova, Taipei, TW, China). Immunohistochemistry score was evaluated by two pathologists who were blinded to clinical information of the patients independently. The expression of FSTL5 was evaluated by Semi-quantitative analysis. IHC was scored according to both the proportion of positively stained cells and the intensity of staining. The proportion of cells was scored as follows: 0 (<5%), 1 (5%-25%), 2 (25%-50%), 3 (50%-75%), 4 (>75%). The intensity of staining was graded according to the following criteria: 1 (weak), 2 (moderate), 3 (strong). The final score was calculated as the product of the proportion of positive cells × the staining intensity score (range from 0 to 12) and was divided in 4 grades: -(score of 0, 1 and 2), + (score of 3 and 4), ++ (score of 6 and 8) and +++ (score of 9 and 12).

| RNA extraction and reverse transcription PCR
Total RNA from the cell lines and human tissues was extracted using

| Quantitative PCR and Western blotting
Quantitative PCR (qPCR) and western blotting were performed as described previously. 21

| Xenograft tumors in nude mice
Studies were performed in accordance with institutional guidelines concerning animal use and care. Bel7404 and SMMC7721 cells

| Caspase-3 activity assay
Caspase-3 activity was measured by determining the level of chromophore p-nitroaniline (pNA) after cleavage from the labelled substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) using Caspase-3 Activity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's protocols. Collected cells were lysed with the lysis buffer followed by centrifugation (16 000 g for 15 minutes at 4°C). After that, the supernatant was transferred to pre-cooled centrifuge tube.
Caspase-3 activity was assessed following the proteolytic cleavage of Ac-DEVD-pNA. Samples absorbance were measured at 405 nm.

| FSTL5 expression is downregulated in HCC tissues and cell lines
To investigate the expression of FSTL5 in liver cancer, IHC staining was performed to detect FSCT5 expression in a liver cancer TMA These findings indicated that FSTL5 is downregulated in HCC.

| FSTL5 expression indicates favourable prognosis of patients with HCC at tumor node metastasis stage I/II
To explore the clinical significance of FSTL5 in HCC, survival analysis of TMA was conducted, which showed that FSTL5 expression indicated favourable prognosis in patients with HCC ( Figure 2A). Moreover, FSTL5 expression was associated with tumor node metastasis (TNM) stage in patients with HCC (Figure 2B). Therefore, we further studied the prognostic value of FSTL5 expression in patients with HCC at different TNM stages.
As shown in Figure 2C, FSTL5 expression was positively correlated with prognosis of HCC at TNM stage I/II (n = 95, P < 0.01) ( Figure 2C), and there was no significant correlation with the prognosis of patients with HCC at TNM stage III/IV (n = 75, P = 0.43) ( Figure 2D). Then, we found that FSTL5 expression level had no significant correlation with gender (n = 180), age (n = 179), liver cirrhosis (n = 180), and tumor number (n = 179) in HCC (Table 1). Nevertheless, FSTL5 expression was significantly associated with tumor size (P = 0.0024), TNM stage (P < 0.0001), and histological grade (P = 0.0037) ( Table 1). The univariate and multivariate analysis of factors associated with overall survival of patients with HCC showed that FSTL5 also can be used as an independent prognostic factor (P = 0.003) ( Table 2). These findings suggested that FSTL5 could be an independent prognostic biomarker in HCC.  Bcl-2 family proteins including pro-apoptotic proteins Bax, Bad, and Puma, and the anti-apoptotic protein Bcl-2 were also investigated.
FSTL5 downregulated the levels of the anti-apoptotic protein P-Bcl2 (T56) and P-Bcl2(S70), and upregulated the expression of the proapoptotic proteins Bax, Bad, and Puma in the mitochondrial pathway ( Figure 5B). The caspase inhibitor Z-VAD-FMK effectively reversed the elevated Caspase-3 activity caused by FSTL5 overexpression in SMMC7721 cells and prevented FSTL5-induced apoptosis ( Figure 5C and D). The above results showed that FSTL5 promoted cell apoptosis in a caspase-dependent manner through regulating Bcl-2 family protein in HCC.
The above results showed that the FSTL5 recombinant protein promoted HCC cell apoptosis through regulating the caspase pathway and Bcl-2 family protein in a dose-dependent manner.

| FSTL5 inhibits HCC tumor growth in vivo
To examine whether FSTL5 expression affected HCC growth in vivo, we employed a gain-of-function approach to study FSTL5 function in

| DISCUSSION
FSTL family members show altered expression in cancer. FSTL1 was found to be downregulated in nasopharyngeal cancer 22 but upregulated in primary glioma. 23 The expression of FSTL3 in endometrial cancer is decreased at early stages, and increased in tumor capillaries. 24 Furthermore, FSTL3 was elevated in the liver cancer, 25  | 6199 responds to a series of apoptotic factors by inhibiting the release of mitochondrial cytochrome c, which can promote cell survival. 38 In the process of apoptosis induced by T lymphocyte glucocorticoids, Bcl-2 mutation at Thr56 or Ser87 can inhibit its anti-apoptotic activity. 39 The phosphorylation of Bcl-2 induced by interleukin 3 and JNK at Ser70 site is necessary to enhance anti-apoptosis activity. 40 Bax and Bad are important component of the process of inducing intrinsic apoptosis. 41,42 Puma can bind to anti-apoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation. [43][44][45][46] We found that FSTL5 lowered levels of the anti-apoptotic proteins P-Bcl2(T56) and P-Bcl2(S70) and increased levels of proapoptotic proteins Bax, Bad, and Puma with a exogenous manner.
Nevertheless, the mechanism underlying FSTL5 regulating Bcl-2 family proteins requires further study.
In summary, we found that FSTL5 was downregulated in HCC cell and HCC tissue and FSTL5 expression positively correlated with good prognosis in patients with HCC at TNM stages I/II rather than TNM stage III/IV. The results in vitro and in vivo show that the FSTL5 inhibit HCC growth by promoting caspase-dependent apoptosis of HCC cells and regulating Bcl-2 family proteins in HCC, rather than influencing cell cycle. Our study suggested that FSTL5 is a potential target and molecular marker for the diagnosis, treatment, and prognosis predicting in HCC. However, a more detailed molecular mechanism remains to be elucidated in the further study.

CONF LICT OF I NTEREST
The authors confirm that there are no conflicts of interest.