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HYD-PEP06 suppresses hepatocellular carcinoma metastasis, epithelial–mesenchymal transition and cancer stem cell-like properties by inhibiting PI3K/AKT and WNT/β-catenin signaling activation

HYD-PEP06, an endostatin-modified polypeptide, has been shown to produce effective anti-colorectal carcinoma effects through inhibiting epithelial–mesenchymal transition (EMT). However, whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma (HCC) remained unknown. In this study, HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro. Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3K/AKT pathway. Moreover, HYD-PEP06 exerted anti-metastasis effects in HepG2 cancer stem-like cells (CSCs) via suppressing the WNT/β-catenin signaling pathway. Finally, in HCCLM3 tumor-bearing BALB/c nu/nu nude mice, HYD-PEP06 substantially suppressed tumor growth, lung metastasis and HCC progress. Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well, and thus has the potential as an agent for HCC treatment.


Introduction
Hepatocellular carcinoma (HCC), as one of the most worldwide malignancies, makes up of 90% primary liver cancer and its incidence continues to rise worldwide 1 . Surgical removal of nidus is the major strategy for HCC, accompanied by high recurrence rate and intrahepatic and extrahepatic metastasis. Sorafenib, a novel multi-targeted oral drug, was authorized as a unique target medicament for advanced HCC by the U.S. Food and Drug Administration in 2007, and it acts by attenuating tumor angiogenesis, delaying tumor progress and inducing apoptosis 2,3 . However, due to the development of resistance of HCC to sorafenib and the serious adverse reactions caused by this drug, the overall therapeutic effect of HCC is far from satisfactory 4,5 . Thus, it is imperative to explore new drugs for better treatment of HCC.
HYD-PEP06 is an RGD-modified endostatin-derived synthetic polypeptide of 30 amino acids. The RGDRGD fragment attached at its N-terminus can specifically bind to different integrins in endothelial cells. HYD-PEP06 is designed and synthesized by our group and it has been demonstrated to exert anti-tumor effects on colorectal carcinoma and oral squamous cell carcinoma 6,7 . However, whether HYD-PEP06 can also suppress HCC progression and metastasis remained untested.
Epithelial mesenchymal transition (EMT) is a critical mechanism for many pathological processes, including tumor invasion and metastasis. During EMT, epithelial cells discard their epithelial properties and acquire the properties of mesenchymal cells in both morphology and biological characteristics 8,9 . Typical EMT malignant tumor cells are characterized by increased migration, invasion, mesenchymal characteristics, along with downregulation of expression of cell adhesion molecules and upregulation of mesenchymal marker genes 10,11 . EMT plays a key role in conferring the metastatic and invasive properties of HCC 12,13 . Several signaling pathways, such as mitogen-activated protein kinase/extracellular regulated protein kinases (MAPK/ ERK), nuclear factor kappa-B (NF-kB) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), are known to regulate EMT 14e16 , of which, the PI3K/AKT pathway is of pivotal importance in controlling tumor invasion and metastasis. Moreover, the PI3K/AKT signaling pathway can induce EMT by dual mechanisms: increasing expression of matrix metalloproteinases and transcription factor Snail 17e21 .
Cancer stem cells (CSCs) belong to a subgroup of tumor cells which possess self-regeneration capacity, extensive proliferation and high carcinogenic potential and are considered to be responsible for cancer occurrence, development, chemo-resistance, metastasis and recurrence 22 . Additionally, CSCs have been identified in several types of human cancers including HCC 23 . Accumulating evidence suggests that tumor cells acquire CSClike characteristics through activating EMT 24,25 . It has been proposed that eliminating CSCs may be a more efficient approach for cancer therapy. Indeed, the self-proliferation and expansion of liver CSCs can activate the WNT signaling pathway to enhance tumor aggressiveness and resistance to chemo-therapeutic agents 26,27 . WNT signaling pathway promotes EMT and metastasis, being considered as a significant mediator of HCC metastasis and a marker of poor prognosis as well 28,29 .
The aims of the present study are to characterize the effects of HYD-PEP06 on EMT and metastasis of HCC with both in vitro and in vivo models and to delineate the underlying signaling mechanisms. Our results indicate that HYD-PEP06 is an anti-HCC agent suppressing metastasis and EMT via inhibiting the PI3K/AKT and WNT/b-catenin signaling pathways. The findings suggest that HYD-PEP06 might be considered as a novel shortpeptide agent for HCC therapy.

Cell lines, culture conditions and reagents
Human HCC cell line HCCLM3 was purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Human HCC cell line HepG2 and human umbilical vein endothelial cells (HUVEC) were purchased from the American Type Culture Collection (ATCC).

Evaluation of cell viability by CCK8 assay
Cell Counting Kit-8 (CCK-8, MCE, USA) assay was used to evaluate cell viability. HCCLM3 and HepG2 cells (5 Â 10 3 /well) were plated into 96-well plates. After the cells had reached 80% confluence, they were starved for 6 h and incubated with different concentrations of HYD-PEP06 for 24 and 48 h. After drug treatment, 10 mL CCK8 was added to each well of the 96-well plate and incubated for 1.5 h. Then the absorbance value was measured at 450 nm by using PowerWave HT microplate spectrophotometer (BioTek, USA). The data of each group was normalized to that of the control group.

In vitro cell migration assay
Wound healing and Transwell assays were conducted to evaluate tumor cell migration in vitro. For wound healing, HCCLM3 and HepG2 cells (5 Â 10 5 /mL) were planted onto a 6-well plate. Then, the cells were incubated with different concentrations of HYD-PEP06 for 24 h. A dense monolayer of cells was scraped from the 6-well plate, and the floating cells were washed with PBS. GS was used as a negative control. Photographs were obtained with a digital Single Lens Reflex Camera. Cell migration area was analyzed by Image J software. Each measurement was conducted at least five times.
Transwell filter chambers (Costar, Corning, NY, USA) were used for migration analysis. HCCLM3, HepG2 and cancer stemlike cells (5 Â 10 4 /well) were planted into upper chambers in basal medium without serum. Then, 600 mL serum was added to the lower chambers. After 24 h culturing, the chambers were covered with methanol for 30 min, followed by staining with 0.1% crystal violet for 20 min. The area of stained cells was photographed under a microscope. For each group, five randomly selected microscopic fields were enumerated and analyzed. Each measurement was conducted in triplicate.

Immunofluorescence (IF) assay
IF staining was conducted to analyze the effects of HYD-PEP06 on EMT and cancer stem-like cells. HCCLM3, HepG2 and cancer stem-like cells were seeded on the sterile coverslips laid on 24-well plate, incubated with HYD-PEP06 at 200 mg/mL for 24 h, and then fixed in 4% PFA for 10 min followed by penetration with 0.5% Triton X-100 (Leagene, China) for 15 min and subsequent blocking with goat serum for 1 h. Next, the cells were incubated with primary antibodies against E-cadherin, vimentin, b-catenin or GSK-3b at 4 C overnight. CD133 (AF5120, Affinity Biosciences, China) and CD44 (DF6392, Affinity Biosciences, China) were used to identify and verify cancer stem-like cells. Then, the cells were incubated with secondary antibody, Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen Thermo Fisher, USA) for 1 h. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) at room temperature for 20 min. Finally, images were obtained under a fluorescence microscope (Zeiss, Jena, Germany). All measurements were performed in triplicate.

Cignal finder pathway reporter array
Cignal Finder Reporter Array (Qiagen, Dusseldorf, Germany) was carried out to investigate the signaling pathways mediating the effects of HYD-PEP06 in HCCLM3 cells and cancer stem-like cells. Briefly, adherent cells (1 Â 10 5 /well) were digested from logarithmic growth phase in 96-well plates for 16 h. The cells were transfected with transcription factor responsive reporter constructs of different signaling pathways. Next, the culture medium was replaced by the fresh medium supplemented with 0.5% FBS and 1% antibiotics. After 24 h, 200 mg/mL HYD-PEP06 was added into 96-well plates.
To study the effect of HYD-PEP06 on HCC, luciferase assay was performed by using the Dual-Luciferase Reporter Assay System (Promega, WI, USA) following the instruction handbook. Firstly, a volume of 100 mL Dual-Glo Reagent was added to each well of the 96-well plate after it was removed from the incubator. Waiting for at least 10 min, cell lysis was allowed to occur. Then the firefly luminescence was measured in a luminometer. After that, a volume of 100 mL Dual-Glo Stop & Glo Reagent was added. Waiting for at least another 10 min, Renilla luminescence was measured and the luminescence ratio of HYD-PEP06 group to the control group was calculated. Finally, relative response ratios were calculated from the normalized ratios.

Tube network formation
Tube formation assay was conducted in Matrigel for assessing angiogenesis in vitro. The 24-well plate was covered with growthfactor reduced Matrigel (250 mL/well) under cooling condition and incubated for coagulation at 37 C for 30 min. Serum starved HUVEC cells (1.5 Â 10 5 /mL) were resuspended in Endothelial Basal Medium-2 supplemented with 0.5% FBS or in tumor stem cell enrichment medium, and then seeded onto Matrigel-coated well after incubation with 200 mg/mL HYD-PEP06 for 8 h. Next, the morphological changes of the tube formation were imaged in multiple fields per well under a microscope (Olympus, Tokyo, Japan). Mean capillary tube length and the number of branching nodes were quantified by Image J software at three random fields for 5 times.

Culture and isolation of hepatocellular cancer stem-like cells
HepG2 cells were cultured with DMEM medium containing 10% FBS, 1% penicillin and streptomycin in 5% CO 2 at 37 C. At the logarithmic growth phase, the cells were trypsinization collected by centrifugation and resuspended in FBS-free CSC culture medium Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 (DMEM/F12, Hyclone, UT, USA) containing 20 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 4 mg/mL heparin sodium, 20 mL/mL B27 serum substitute supplement, and 1% antibiotic. Then the cells were seeded into polyhydroxy ethyl meth-acrylate pretreatment cell culture flask for two weeks. The adherent cells were discarded. Then the suspending cells were collected and cultured in stem cell culture medium. Cell digestion and passage were conducted every 10 days, the sphere formation was observed under the microscope every 3 days. The cells without the efficient sphere-forming capacity deposited at the bottom of ultra-low attachment surface plates were discard carefully, sphere-like cells were collected for further use when spheroids reached a diameter of 100 mm in about 2 weeks.

Formation and proliferation of 3D spheroids
Cancer stem-like cells (1 Â 10 4 /well) were planted onto Ultra-low adhesion 96-well plates (S-BIO, New Hampshire, USA). Threedimensional (3D) spheroids were photographed every 24 h, and sphere area was measured with Image J software. To evaluate 3D spheroid proliferation in the presence of HYD-PEP06, the photographs were taken under a microscope (Olympus, Tokyo, Japan) and analyzed using Image J software.

Postsurgical residual tumor xenograft models and antitumor effect assay
Adult female 6e8 weeks old athymic BALB/c nu/nu nude mice (15e20 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. [Certificate No. SCXK(Jing) 2016-0006]. The animals were raised in a constant temperature of 23 AE 2 C under a 12 h day and night cycle by feeding with sterilized food and sterile water. A mouse model of postsurgical residual tumor was used to imitate the recurrence of solid tumor, and HCCLM3 cells were digested and collected. First, a traditional tumor xenograft nude mouse model was established by injecting 200 mL 1 Â 10 6 /mL HCCLM3 cell suspension subcutaneously into the right axillary of mice. Once the average volume of tumors had reached 1000e1500 mm 3 , they were aseptically removed from the mice and cut into small pieces. Then, the tumor chunks were subcutaneously inoculated to the tumor xenograft nude mice. After the average volume of the tumors had reached to 300e350 mm 3 , the mice were intravenously injected 1.2% avertin solution for anesthetization. Residual tumor model of average volume 60e100 mm 3 was aseptically constructed by removing parts of tumors. After 24 h, mice were categorized into four groups (n Z 10) based on tumor volume, which included the control groups, a low concentration of 5 mg/kg HYD-PEP06, a medium concentration of 10 mg/kg HYD-PEP06 and a high concentration of 20 mg/kg HYD-PEP06. Each group was daily injected with either GS or HYD-PEP06 intravenously for 14 days. The width and length of tumors were calculated by caliper every other day. Tumor volume was calculated on the basic of the width and length of tumors. On Day 14 after injection of HYD-PEP06, the groups were euthanized. Then the tumors were cut, weighted, and immersed in 4% paraformaldehyde at 37 C for Western blot analysis. The tumor volume inhibitory rate was calculated according to Eq. (1): where V 0 presents the control tumor volume and V t is the HYD-PEP06 groups tumor volume. The tumor weight inhibitory rate was calculated according to Eq. (2): where W t and W 0 presented the tumor weights of the HYD-PEP06 groups and control groups. The protocols for animal experiments were approved by the Institution Animal Care and Use Committee of Harbin Medical University (Harbin, China), and the procedures conformed to the Guide for the Care and Use of Laboratory Animals, and exactly following with the People's Republic of China Legislation Regarding the Use and Care of Laboratory Animals.

Mouse model of hepatocellular cancer pulmonary metastasis
Adult female 4 weeks old athymic BALB/c nu/nu nude mice (15e20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [Certificate No. SCXK (Jing) 2016-0011 (No.11400700230989)]. The animals were raised in a controlled aseptic environment as described above.
HCCLM3 cells were cultured in DMEM medium (Hyclone, Utah, USA) added with 10% FBS and 1% antibotic at 37 C in 5% CO 2 . HCCLM3 cells were transfected by self-inactivated lentiviral vector pCDH-luc2-GFP, then added 1 mg/mL puromycin (Ther-moFisher Scientific, MA, USA) to the medium to establish a cell line which stably expressed luciferase 2 and green fluorescent protein (HCCLM3-Luc2-GFP). A total of 1.5 Â To test HYD-PEP06 on the efficacy of the HCC tumor pulmonary metastasis model, mice were randomly categorized into four groups after injecting HCCLM3 tumor cells 5 min ago. Based on the bioluminescence imaging (BLI) signals, the mice (n Z 40) were categorized into four groups: control group, 5 mg/kg, 10 mg/kg and 20 mg/kg HYD-PEP06-treated groups. Each group was daily administered with either GS or HYD-PEP06 by intravenous injection for 28 days. The mice were euthanized on the last day and then the main internal organs were collected for BLI imaging in vitro which included brain, stomach, lung, heart, colon, kidney, spleen and small intestine.

Statistical analysis
Data are presented as mean AE standard error of mean (SEM) for three independent experiments. One-way ANOVA followed by the Tukey procedure was used by comparing the mean values in multiple groups. P < 0.05 indicates statistical significance. Statistical significances between two groups were performed with Student t-test. KaplaneMeier technique was performed to plot survival curves. Results were performed for three times. Statistical analysis was conducted by Prism 7.0c GraphPad Software (GraphPad; San Diego, CA, USA).

HYD-PEP06 inhibits hepatocellular carcinoma metastasis in vitro
The mechanism for anti-tumor drugs can be various, including killing dividing cancer cells, suppressing cell proliferation and blocking the spread of cancer cells 30,31 . To investigate the effect HYD-PEP06 suppresses HCC metastasis, EMT and CSC-like properties of HYD-PEP06 on cancer cells, CCK8 assay was used to assess the viability of HCCLM3 and HepG2 cell lines. There were no significant differences of the cell viability after treatment with either glucose or HYD-PEP06 at concentrations from 12.5 to 400 mg/mL. In contrast, the viability of HCCLM3 and HepG2 cells was significantly reduced by 5-fluorouracil (5-FU, Fig. 1AeD).
Next, wound-healing and Transwell migration assays were performed to investigate the effects of HYD-PEP06 on metastasis of HCC. As depicted in Fig. 2A and B, treatment of different concentrations of HYD-PEP06 (50, 100 and 200 mg/mL) for 24 h inhibited the migration of both HCCLM3 and HepG2 cells in a dose-dependent fashion (P < 0.001, Fig. 2C). Similar results were observed with the Transwell migration assay: HYD-PEP06 (200 mg/mL) reduced the migration of HCCLM3 and HepG2 cells by 72.1% and 82.1%, respectively ( Fig. 2D and E). These data suggest that HYD-PEP06 inhibits metastasis without affecting viability in HCC.

HYD-PEP06 suppresses HCC metastasis by inhibiting epithelial mesenchymal (EMT) transition in HCC cells
It is known that EMT enables non-metastatic tumor cells to infiltrate into surrounding tissues and eventually metastasizes to distant sites 9 . In this process, cells change from their epithelial "cobblestone" phenotype to the spindle-shaped morphological characteristics of mesenchymal cells, causing enhanced motility and invasion 32,33 . Western blot results show that the expression of epithelial markers E-cadherin and a-catenin was significantly increased, along with concomitant downregulation of mesenchymal markers N-cadherin and vimentin after HYD-PEP06 treatment in HCCLM3 (Fig. 3AeE). Similar results were found in HepG2 cells (Fig. 3FeJ). In addition, immunofluorescent staining demonstrated that vimentin expression was significantly decreased by HYD-PEP06 (200 mg/mL) treatment for 24 h, whereas E-cadherin expression was increased (Fig. 3K and L).

HYD-PEP06 inhibits tumor metastasis and EMT in human HCC by blocking the PI3K/AKT pathway
To decipher the mechanisms by which HYD-PEP06 suppresses HCC metastasis and EMT, we conducted the Cignal Finder Reporter Arrays for the comprehensive analysis of 10 signaling pathways to pinpoint the pathways perturbed by 200 mg/mL HYD-PEP06 in HCCLM3 cells after 24 h treatment. Using a cut-off fold change >1.5 and P-value 0.05 criteria, we identified the PI3K/AKT reporter gene to be a negatively regulated signaling pathway (reduced by approximately 4-fold, Fig. 4A). Moreover, Western blot results reveal that HYD-PEP06 significantly decreased the protein level of active or phosphorylated AKT (p-AKT) in HCCLM3 cells without altering the total AKT protein level ( Fig. 4B and C). Furthermore, HYD-PEP06 substantially inhibited the phosphorylation of AKT after adding to AKT inhibitor MK2206 (10 mmol/L). Meanwhile, MK2206 exacerbated the effects of HYD-PEP06 in greater downregulation of vimentin and N-cadherin protein expression, along with greater up-regulation of a-cadherin and E-cadherin protein expression in HCCLM3 cells (Fig. 4DeI). Conversely, SC79 (an AKT activator) reversed the downregulation of the mesenchymal markers and upregulation of the epithelial markers induced by HYD-PEP06 (Fig. 4JeO).

HYD-PEP06 attenuates the metastatic potential of cancer stem-like cells isolated from HepG2 cells
Cancer stem cells (CSCs) contribute to HCC metastasis, progression and recurrence after therapy, by inducing EMT 34e37 . We therefore continued to investigate whether HYD-PEP06 has any effects on CSCs. Our study reveals that HepG2 cells expressed higher levels of CSC markers CD133 and CD44 than HCCLM3 cells (Fig. 5AeC), suggesting that the former possesses high-stemness ability than the latter. Thus, we isolated stem-like cells from HepG2 cells for the subsequent experiments. Cancer 3D spheroid culture is a widely used model for studying CSCs 38 . In the medium with serum, HepG2 cells were polygonal morphology. The tumor spheres were obtained from HepG2 cells, which suspended in FBS-free DMEM/F12. Cells cultured for 10 days demonstrated round shape with few cells exhibiting epithelial-like morphology. We observed the size of the sphere under the microscope on Days 0, 3, 5, 7 and 10. HepG2 cells with stem-like properties have efficient sphere-forming capacity under serum-free conditions, and three-dimensional (3D) spheres growth over time (Fig. 5D). Cells without the efficient sphere-forming capacity deposited at the bottom of ultra-low attachment surface plates were discard carefully. When the suspension of spheres reached a diameter of 100 mm, the sphere-forming efficiency was calculated. And spheres were collected for further experiments. CSC population in solid tumors is identified by typical markers such as CD133/CD44 and characterized by high tumorigenicity and invasiveness 38 . The isolated stem-like cells expressed higher protein levels of both CD133 and CD44 than HepG2 cells, as determined by Western blot analysis (Fig. 5EeG) and immunofluorescence staining (Fig. 5H). Moreover, while HepG2 cells formed smaller tumors in mouse xenograft model, the isolated tumor spheres xenotransplants generated impressively larger tumors (Fig. 5I). The xenograft tumors derived from HepG2 cells and the isolated CSCs were harvested two weeks after subcutaneous inoculation in nude mice. The Western blot results demonstrate that the xenograft tumors derived from CSCs expressing higher levels of CD133 and CD44 than those derived from HepG2 cells (Fig. 5JeL).
Moreover, 3D spheroids formation assay demonstrated treatment of HYD-PEP06 for 24 and 48 h inhibited the formation of 3D tumor spheroids compared with control or glucose solution (GS) group, indicating inhibitory effect of the polypeptide 30 on cancer stemness ( Fig. 6A and B). In addition, Transwell assay exhibited remarkable reduction of CSCs migration in the presence of HYD-PEP06 ( Fig. 6C and D). Furthermore, the effects of HYD-PEP06 on tube formation were evaluated in HUVEC cells. The culture medium collected from CSCs was more favorable to form tube formation relative to that of regular HepG2 cells, as indicated by more dense meshwork of branching capillary-like tubules formed by connection between neighboring cells (Fig. 6EeG). Strikingly, the intercellular tube formation was considerably abrogated by HYD-PEP06 as HUVEC cells being failed to form tubes and essentially staying as separate spherical bodies. These findings indicate that HYD-

HYD-PEP06 inhibits the metastatic potential of CSC-like cells by suppressing the canonical WNT/b-catenin pathway
To investigate the mechanisms by which HYD-PEP06 suppresses the metastatic potential of CSCs, we conducted the Cignal Finder Reporter Arrays. As illustrated in Fig. 7A, WNT was substantially down-regulated in CSC-like cells after treatment with 200 mg/mL HYD-PEP06 for 24 h. We therefore hypothesized that HYD-PEP06 might attenuate CSC metastasis via the WNT/b-catenin pathway. To test this notion, we measured the protein levels of b-catenin, axin2, p-GSK-3b and total GSK-3b, the mediators of the WNT/b-catenin signaling pathway. As illustrated in Fig. 7BeE, HYD-PEP06 significantly downregulated the expression of b-catenin, axin2, and p-GSK-3b in CSC-like cells. Considering that accumulated b-catenin can be translocated to the nucleus, we conducted immunofluorescent staining to analyze the expression and subcellular distribution of b-catenin in the presence of HYD-PEP06. As expected, CSC-like cells in control group exhibited even distributions of b-catenin in cytoplasm and nucleus, whereas in the presence of HYD-PEP06, b-catenin appeared more restricted to the localization close to cytoplasmic membrane while nuclear b-catenin protein substantially decreased (Fig. 7F). The level of total GSK-3b protein didn't change after HYD-PEP06 HYD-PEP06 suppresses HCC metastasis, EMT and CSC-like properties treatment (Fig. 7G). These results suggest that the anti-metastasis effect of HYD-PEP06 in CSC-like cells might be attributable to its ability to regulate the canonical WNT/b-catenin pathway.

HYD-PEP06 suppresses HCC tumor growth in a postsurgical residual tumor xenograft model
Recurrence caused by metastatic spreads from the primary tumor usually peaks in the first two years after tumor resection and carries a poor prognosis 39 . Additionally, surgical resection may promote the growth of residual tumor. Therefore, a mouse model of postoperative recurrent tumor was constructed to assess the effect of HYD-PEP06 on tumor suppression. Clinical postoperative recurrence model was mimicked by surgically excising a part of the primary HCC tumors with a section of 60 mm 3 tumor tissue. The average tumor width, length and body weights were measured every 2e3 days. Administration of different dosages of HYD-PEP06 (5, 10, and 20 mg/kg) through tail vein injection significantly suppressed HCC tumor growth in xenograft mice, as reported by substantial decrease of residue tumor growth compared with the control group (Fig. 8AeD). Specifically, HYD-PEP06 at 5, 10 and 20 mg/kg resulted in 18.0%, 24.1% and 31.4% decreases in tumor weight, respectively. Consistently, HYD-PEP06 caused dose-dependent reduction of HCC tumor volume (20.59%, 31.17%, and 39.18%, respectively; Table 1). Moreover, the anti-tumor effect of HYD-PEP06 was also supported by a slower increase of the tumor volume. The body weight of the mice was unaffected by the peptide (Fig. 8E). Consistent with the above results, Western blot results indicate that the epithelial protein markers were upregulated while the mesenchymal protein markers were downregulated in the tumor specimens from mice treated with HYD-PEP06 relative to the control group (Fig. 8FeJ).

HYD-PEP06 inhibited pulmonary metastasis of HCC in a mice model
To evaluate the efficacy of HYD-PEP06 in inhibiting the metastasis potential of HCC in vivo, the metastatic tumor growth in a mouse model of HCC was investigated by bioluminescence imaging. The accumulation of GFP-Luc2-transfected HCCLM3 cells   in mouse lungs was verified by BLI images after intravenous injection for 5 min. The mice were evenly assigned into four groups (n Z 10 per group) on the basic of equal mean BLI signal intensities. Most tumor cells were cleared by the immune system of the animals on the first seven days with a loss of BLI signals in all groups. However, the control group showed certain conspicuous bioluminescent signals in the lung on Day 14. Concomitantly, only few mice treated with HYD-PEP06 developed obvious signals. From Days 21e28, HYD-PEP06 dramatically decreased the accumulation of BLI signals in the lung ( Fig. 9A and B, and Supporting Information Fig. S1). On the last day (Day 28), the mean BLI signal intensity of the control group was 7.9-fold higher than that on Day 1, while that of the HYD-PEP06 (5, 10 and 20 mg/kg) groups was all markedly lower than on Day 1 (0.31-,  The significance of differences (vs. control) was determined by one-way ANOVA and Student's t-test. Data are presented as mean AE SEM, n Z 10; **P < 0.01, ***P < 0.001. a QD Â 14 days, iv. QD, every day; IR, inhibitory rate; iv represents vein injection daily. 0.24-, and 0.17-fold, respectively; Table 2). The ex vivo organ images also supported that HYD-PEP06 inhibited the formation of lung metastasis with an inhibition rate of 87.8%, 92.7% and 95.3% at the dosage of 5, 10 and 20 mg/kg, respectively (Fig. 9C). In addition, HYD-PEP06 did not show any appreciable adverse effects with a stable increase in body weight of mice during the whole course of experiment (Fig. 9D). Of note, two mice of the control group died on Day 22 and three died on Day 26, while the HYD-PEP06 groups all survived to the end. That is, HYD-PEP06 prolonged the survival time of mice bearing tumors (Fig. 9E).

Discussion
In the present study, we provided strong evidence for the anti-HCC and lung metastasis-suppressing effects of HYD-PEP06 both in vivo with mouse models of tumor metastasis and postsurgical residual tumor xenograft and in vitro cellular models. Mechanically, HYD-PEP06 inhibited HCCLM3 cell metastasis and EMT likely by attenuating the PI3K/AKT pathway and hepatocellular CSC-like cells metastasis via suppressing the WNT/b-catenin signaling pathway. These findings suggest the potential of HYD-PEP06 as a novel therapeutic agent for HCC (Fig. 10). HYD-PEP06 is an endostatin-derived synthetic polypeptide consisting of 30 amino acids engineered to attach RGD sequences to the N-terminus. Previous studies have demonstrated that HYD-PEP06 exhibits potent anti-tumor activity in diverse malignancies and is currently undergoing phase I clinical trial for evaluation of its safety and efficacy. Tuguzbaeva et al. 7 reported that HYD-PEP06 could inhibit the av integrin/FAK/SRC signaling pathway and eliminate the cluster-driven metastasis of oral squamous cell carcinoma. Our previous study revealed that HYD-PEP06 significantly decreases the expression of miR-146b-5p through binding to integrin avb3 with its RGD domain and inactivates the miR-146b-5p-Smad4 cascade to suppress colorectal carcinoma (CRC) pulmonary metastasis and residual tumor growth and increases the survival rate of tumor-bearing mice 6,40 .  The significance of differences (vs. control) was determined by two-way ANOVA followed by the Bonferroni procedure. Data are presented as mean AE SEM, n Z 10; *P < 0.05, ***P < 0.001. IR, inhibitory rate.
Endostatin, the prototype of HYD-PEP06, has been demonstrated to decrease the phosphorylation of AKT and inhibit growth and cell invasion in non-small cell lung cancer cells, breast cancer cells and esophagus cancer cells 41e43 . It is known that integrins can recognize RGD motif to affect cell adhesion through regulating the connection between cells and their microenvironment, which is one of the mechanisms by which HYD-PEP06 suppresses tumor metastasis 44 . Mounting evidence supports that HCC invasion and metastasis are positively regulated by multiple subtypes of integrins (avb3, a3b1, and a6b1) through multiple mechanisms, including cell adhesion, cell migration and cell invasion 45,46 . Moreover, the b subunit of integrin regulates the activity of AKT. For instance, inhibition of integrin avb3 significantly prevents the cathepsin B-induced activation of PI3K/AKT thereby the progression of HCC 47 . Ma et al. 48 reported that integrin avb3 suppresses HCC progression via targeting PI3K/AKT signaling. It is therefore possible that integrins underlies the inhibitory effects of HYD-PEP06 on HCC metastasis and EMT via inhibiting the PI3K/AKT pathway. Epithelial to mesenchymal transition is a process tightly linked to CSC biology, including stemness, immune escape and resistance to radio-and chemotherapy 49 . When EMT is aberrantly activated, cancer cells can gain the stem cell-like characteristics attaining self-renewal capabilities and the potential to differentiate into literally all different cell types. The resulting cancer stem cells attain phenotypes of cancer including replicative immortality, resistance to cell death, and invasiveness. Tumor cells in the non-CSC subpopulation can spontaneously undergo EMT-like changes expressing CSC-like cell-surface marker genes and promoting tumor spread. EMT and mesenchymal to epithelial transition (MET) are mutually reversible in cancer cells, enabling the conversions between the CSCs and non-CSCs 50 . In our present study, we suggest that a subset of HepG2 cells displayed CSC-like biochemical and functional characteristics, leading to recurrence, metastasis and chemo-resistance of HCC. Besides, hyperactivity of the WNT/b-catenin signaling pathway is established as a cause of tumor development in some human malignancies. WNT/b-catenin also regulates the self-renewal of cancer stem-like cells and is involved in tumor progression and chemotherapy resistance 51 . This study also revealed that HYD-PEP06 inhibited liver CSCs by suppressing WNT/b-catenin signaling pathway.
CSCs, constituting <1% of the cellular population in the most solid tumors, are a rare subpopulation of highly self-renewal cells with intense tumorigenic potency and resistance to radio-and chemotherapy. Various oncogenic signaling pathways have been implicated in maintaining and regulating the function of CSCs in malignancies. Particularly, the dysregulated signaling pathways such as WNT/b-catenin, Notch, transformation growth factor b (TGF-b), hedgehog, Janus kinase/signal transducers and activators of transcription (JAK/STAT), PI3K/AKT, and NF-kB, have been well elucidated. Interestingly, miRNAs can modulate the expression and functioning of genes and their products associated with the abovementioned signaling pathways for the maintenance, growth, and function of CSCs. Upregulation of miR-19, miR-501-5p, and miR-744 stimulates the activation of b-catenin 52e54 . Conversely, upregulation of miR-708-5p and miR-142-3p inhibits b-catenin activation and prevents its accumulation in the cytoplasm 55,56 . Furthermore, upregulation of miR-21 and miR-23a stimulates the activation of the AKT/PI3K pathway, whereas upregulation of miR-128 inhibits its activation 57e59 . In addition, upregulation of miR-106b is found to act on TGF-b through targeting SMAD 60 . Downregulation of miR-200c stimulates the activation of Notch pathways and, hence, enhances CSC features 61 . Further, downregulation of miR-136 stimulates CSCs as a result of activating various signals including NF-kB, survivin, cyclin D1, and BCL2 62 . Taken together, stem cell features can be stimulated or suppressed by miRNA-regulated expression of signaling proteins. Our previous study has demonstrated that HYD-PEP06 significantly inhibited miR-146b-5p-SMAD4 cascade to suppressed CRC pulmonary metastasis. And this study showed that HYD-PEP06 suppressed HCC by inhibiting the PI3K/AKT pathway. Moreover, HYD-PEP06 exerted anti-metastasis effects in hepatocellular CSCs via suppressing WNT/b-catenin signaling pathway. However, whether miR-146b-5p also exerted therapeutic effect on HCC cells or regulate the stem cell features through these pathways needs further investigation.

Conclusions
This study uncovered that HYD-PEP06 possessed potent anti-HCC and anti-CSC metastasis effects by inhibiting EMT through downregulating the PI3K/AKT and WNT/b-catenin signal pathways. Our findings indicate that HYD-PEP06 might be considered as a potential drug for HCC treatment.