Background: It is essential to understand the mechanisms responsible for hepatocellular carcinoma (HCC) progression and chemoresistance in order to identify prognostic biomarkers as well as potential therapeutic avenues. Recent findings have shown that SLIT3 appears to function as a novel tumor suppressor gene in various types of cancers, yet its clinical correlation and role in HCC has not been understood clearly. Methods: We determined the transcript levels of Slit3 in tumor and adjacent normal tissues within two cohorts (N =40 and 25) of HCC patients, and correlated the gene expression with the clinicopathological data. Subsequently, the functional effects and underlying molecular mechanisms of Slit3 overexpression and/or repression were studied using cell-line and mouse models. Results: Our results demonstrated a repression in Slit3 expression in nearly 50% of the HCC patients, while the overall expression of Slit3 inversely correlated with the size of the tumor in both cohorts of patients. Stable down-regulation of Slit3 in HCC cell-lines induced cell proliferation in vitro and tumor growth in vivo, while stable Slit3 overexpression repressed these effects. Molecular investigations showed that the stable Slit3 repression-induced cell proliferation was associated with a higher expression of β-catenin and a repressed GSK3β activity. Moreover, Slit3-repression induced chemoresistance to sorafenib, oxaliplatin and 5-FU through impairment of β-catenin degradation and induction of cyclin D3 and survivin levels. The effects induced by stable Slit3-repression were diminished by transient repression of β-catenin by siRNA approach. Conclusion: This study suggests that Slit3 acts as a tumor suppressor in HCC by repressing the tumor growth and thus tumor progression. Low Slit3 level indicates a poor response of HCC cells to chemotherapy. Restoration or overexpression of Slit3 is a potential therapeutic approach to repress the tumor growth and enhance the efficacy of chemotherapeutic agents. Keywords: Slit3, β-catenin, GSK3β, Chemoresistanc, Sorafenib, Oxaliplatin, 5-FU * Correspondence: firstname.lastname@example.org; email@example.com Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ng et al. BMC Cancer (2018) 18:621 Page 2 of 13 Background Review Board and the patients were well-informed and Hepatocellular carcinoma (HCC) is the second leading consented prior to inclusion. cause of cancer death worldwide . Most HCC patients die from locally advanced or metastatic disease in a relatively short period of time, and the mechanisms RNA extraction, cDNA synthesis and quantitative real-time responsible for HCC progression and metastasis remain polymerase chain reaction a major challenge to researchers in this field. It is well- To minimize the influence of heterogeneous expression believed that the elucidation of molecular mechanisms of Slit3 in different regions of the tumor, tissue sections involved in HCC progression and metastasis is important from several parts of the tumor and adjacent non-tumor for the identification of prognostic biomarkers as well as liver were homogenized together, and used for RNA therapeutic targets. This study will demonstrate the role extraction. Total RNA was extracted using Trizol of a secretory protein involved in the Slit/Roundabout reagent and Purelink® RNA mini kit (Life Technologies, (Robo) signaling pathway, namely Slit3, in HCC develop- Carlsbad, CA) as previously described . Total RNA ment and progression. (2.0 μg) was reverse-transcribed with SuperScriptII RT- The Slit family of guidance cues interacts with the Robo PCR kit (Invitrogen, Carlsbad, CA) in accordance with family of transmembrane receptors in a wide variety of the instructions of the manufacturer. Real-time PCR was physiological processes requiring cell migration . They performed in a final volume of 15 μl containing 1 μlRT were first identified as an important regulator in axon transcript, 0.2 μM of each primer, 1X ROX reference guidance and cell migration in Drosophila and vertebrates dye and 7.5 μl of FastStart Universal SYBR Green Master . Three Slit proteins (Slit1, 2 and 3) have been identified (ROX) (Roche Diagnostics, Switzerland, Basel). A no RT so far. While the expression of Slit1 is confined to neu- transcript control was included for each gene to ensure rons, Slit2 and Slit 3 are widely expressed in mammalian the signal was truly driven by target gene amplification. tissues  and their deregulations have been identified in The primer sequences used are: Slit3-Forward Primer: malignant tissues. Slit2 is frequently inactivated in human 5′- AGCGCCTTGACCTGGACA -3, Slit3-Reverse cancers including lung cancer , breast cancer [6, 7], Primer: 5′- TCGGCGTGCTCTGGAAAA -3′; Actin- colorectal cancer , ovarian cancer , glioma and Forward Primer: 5′- CGAGCATCCCCCAAAGTT-3′; HCC [11, 12] and its tumor suppressive role that inhibits Actin-Reverse Primer: 5′- GCACGAAGGCTCATCA cancer cell invasion and migration [10, 13–17], angiogen- TT-3′. Real-time PCR was carried out using the ABI esis [18, 19] and growth [8, 20–22], has been well-studied. 7900HT Fast Real-Time PCR System (Applied Biosys- In conjunction, hypermethylation and subsequent down- tems, Foster, CA) at 95 °C for 10 min, followed by 40 - regulation of Slit3 has been reported in several cancers, cycles at 95 °C for 15 s and at 56 °C for 1 min. Each including thyroid cancer, colorectal cancer, gastric cancer, assay was done in triplicate. The expression level of nasopharyngeal carcinoma, cervical cancer, ovarian cancer target mRNA was normalized to the expression of actin and pancreatic ductal adenocarcinoma [23–32]. Import- within the same tissue. antly, Slit3 has been shown to suppress tumor growth of breast cancer in a mouse model  and impair cancer cell invasion and migration [24, 28, 34] through modula- Slit3 over-expression plasmid construction tion of the expressions of E-cadherin, Vimentin, MMP2 Slit3 full-length coding sequence was amplified from PLC and MMP9 .These findings demonstrate the tumor cell-line RNA by Platinum® Taq DNA Polymerase High suppressive role of Slit3 in multiple types of tumor, Fidelity (Life Technologies) using Slit3-HindIII-Forward although a comprehensive analysis addressing the clinico- Primer 5’-CCCAAGCTTATGGCCCCCGGGTGGGCA pathological and functional significances of Slit3 is still -3′ and Slit3-XbaI-Reverse Primer 5’-CTAGTCTAGATT lacking. AGGAACACGCGAGGCAG-3′. The PCR cycle was 94 ° Subsequently, this study aims to investigate the clini- C for 2 min, followed by 30 cycles of 94 °C for 30 s, 56 °C copathological association and biological significance of for 30 s and 68 °C for 5 min. The Slit3 PCR product was Slit3 in HCC. purified by Qiagen PCR purification system (Valencia, CA, USA), digested with HindIII and XbaIrestriction enzymes (New England Biolabs), ligated within these Methods restriction sites in the pcDNA3.1 vector by DNA ligase Patients and specimens (New England Biolabs) and transformed into DH5α Fresh tumor specimens were collected from patients who competent cells (Life Technologies) according to the underwent surgical resection of primary HCC at the manufacturer’s instructions. The plasmids were extracted Department of Surgery, Queen Mary Hospital, The Univer- by the Qiaprep® Spin Miniprep Kit (Qiagen) and the sity of Hong Kong. The study was approved by Institutional sequence fidelity was confirmed by Sanger sequencing. Ng et al. BMC Cancer (2018) 18:621 Page 3 of 13 Cell lines, tissue culture, transfections and reagents Immunohistochemical study of mice specimens HCC cell lines LM3, PLC, Hep3B, 97 L, HepG2 and Formalin-fixed and paraffin-embedded specimens were Huh7 were cultured in DMEM medium supplemented cut into 5 μm thick sections by microtome and mounted with 10% heat-inactivated FBS, 5 U/ml penicillin and on pre-coated slides. The sections were deparaffinized in 50 μg/ml streptomycin (Life technologies), at 37 °C in a xylene and rehydrated in serial dilutions of ethanol. fully humidified atmosphere of 5% CO and were Antigen retrieval was done by microwave treatment at low passaged according to the manufacturer’s recommenda- power for 10 min in a preheated citrate buffer. The tions. Plasmids for stable knockdown of Slit3 were endogenous peroxidase and biotin activity were blocked purchased commercially (Origene). The shRNA sequence using a biotin blocking kit (Dako). The sections were then was synthesized as per the Slit3 siRNA sequence pre- blocked with horse serum for 30 min and incubated with scribed by us (sense: CGCGAUUUGGAGAUCCUUAtt; the primary antibodies against Slit3 (Novus Biologicals), anti-sense: UAAGGAUCUCCAAAUCGCGca) and was p27 (Santa Cruz biotechnology, Santa Cruz, CA), CD31 cloned into a pGFP-V-RS vector downstream to the U6 (Abcam, Cambridge, MA), phospho-GSK3β (ser9) (Cell promoter by the manufacturer. Stable transfections of Signaling Technology) and β-catenin (BD Biosciences) Slit3-shRNA and the negative control plasmid (Origene) overnight at 4 °C in a moist chamber. After incubation, into LM3 and PLC cells were performed using Lipofecta- slides were rinsed thrice with TBS-Tween20 and twice mine 2000 reagent (Invitrogen) following puromycin with TBS, and were probed with biotinylated secondary selection. Stable transfections of pcDNA-Slit3 and the antibody (Dako) for an hour. The washing steps were vector control into Hep3B cells were performed using repeated and the slides were then incubated with avidin- Lipofectamine 2000 reagent (Invitrogen) following G418 HRP (Dako) for an hour. Sites of bound antibody were selection. Transient transfections of siRNA against β- visualized using liquid DAB+substrate-chromogen system catenin (Invitrogen) and control siRNA were performed (Dako) and the sections were counterstained with Gill’s using Lipofectamine 3000 reagent (Invitrogen). Hematoxylin and mounted using DPX mountant (BDH Laboratory, UK). Protein extraction and western blot analysis Protein extraction was performed by resuspending the Cell viability assay cells in RIPA buffer (Cell Signaling Technology, Danvers, Ten thousand LM3 and PLC-shSlit3 and shCTL stable MA) containing 1 mmol/L phenoylmethylsulfonyl fluor- cells were seeded in a 96-well plate for 24 h and then ide. Following 1 h incubation on ice and centrifugation subjected to treatment with 10 μM Sorafenib, 10 μM at 14,000 x g for 10 min, the protein in supernatant was oxaliplatin or 100 μM 5-FU. Cell viability was assessed mixed with sodium dodecyl sulfate sample buffer, after 72 h of drug treatment using the MTT reagent denatured, resolved in sodium dodecyl sulfate–polyacryl- (Life technologies). amide gel electrophoresis, and transferred to PVDF membranes (GE Healthcare, Piscataway, NJ). Antibody Statistical analysis against Slit3 was purchased from Novus Biologicals Data analysis was performed using SigmaStat 3.5 (Systat (Littleton, CO). Antibodies against GSK3β and phospho- Software Inc., San Jose, CA, USA). Fisher exact test was GSK3β (ser9) were purchased from Cell Signaling used to compare clinicopathological parameters between Technology. Anti-actin was from Santa Cruz biotechnol- high and low Slit3 patients. The Mann-Whitney test or ogy (Santa Cruz, CA). Anti-β-catenin was purchased student t-test was used to analyze differences between from BD Biosciences (San Diego, CA). Protein experimental groups of clinical specimens and cell-line expression levels were quantified using ImageJ software models. Spearman’s correlation test was applied to deter- (imagej.nih.gov/ij/) and normalized to the expression of mine correlations. A p-value< 0.05 was considered statis- actin. Each experiment was repeated at least 3 times and tically significant. representative western blots are shown in each case. Results Animal work Frequent Slit3 mRNA repression and its association with The protocol was approved by the Committee on the Use tumor size of HCC patients of Live Animals in Teaching and Research (CULATR) of Quantitative PCR was applied to determine the Slit3 TheUniversityofHongKong. Tumors were allowedto gene expression in synchronous primary HCC tumors grow in nude mice by injection of LM3-shCTL/shSlit3 and and the adjacent non-tumorous liver (N = 40). Although, Hep3B-pcDNA/Slit3 cells subcutaneously into flank regions no significant difference was seen in Slit3 gene expres- with 5 × 10 cells per site. Sixth week post-operation, the sion between HCC and adjacent non-tumorous livers, mice were sacrificed, and tumors were excised, measured we observed that 42.5% (N = 17) of the HCC patients and processed for immunohistochemical study. showed Slit3 down-regulation (tumor/non-tumor< 1), Ng et al. BMC Cancer (2018) 18:621 Page 4 of 13 suggesting that Slit3 repression was a frequent event expression in another cohort of HCC patients (N =25). In associated with HCC. Subsequently, we sorted the pa- line with the results obtained in the first cohort, Slit3 tients into high and low Slit3 groups as per the median down-regulation (tumor/non-tumor< 1) was frequently (tumor/non-tumor) Slit3 expression (low≤ Fold change observed 56% (N = 14) of the patients. The median expres- = 1.203 > high) and compared their clinicopathological sion in the second cohort was 0.593. Within the high Slit3 parameters (Table 1 and Additional file 1). Statistical expression (>Fold change = 0.593) group, 50% of the analyses using Fisher exact test showed that Slit3 expres- patients (N = 6) showed a large tumor (size > 5 cm), while sion correlated with the HCC tumor size. Within the the percentage significantly increased to over 90% (N =12) high Slit3 expression group, 55% (N = 11) of the patients in patients with low Slit3 expression (≤Fold change = 0.593; had a large tumor (size > 5 cm), and this number signifi- p = 0.003). Patients with a lower relative Slit3 gene expres- cantly increased to 85% (N = 17) within the low Slit3 ex- sion showed a significantly larger tumor (median: 10 cm) pression group (p = 0.048). Furthermore, patients with a when compared with those with a higher Slit3 gene expres- lower relative Slit3 gene expression showed a signifi- sion (median: 5.25 cm, p = 0.004; Fig. 1c), indicating a cantly larger tumor (median: 9.25 cm) when compared significant inverse correlation between Slit3 expression and with those with a higher Slit3 gene expression (median: size of the HCC tumor (R = − 0.457, p = 0.022; Fig. 1d). 5.25 cm, p = 0.005; Fig. 1a); consequently suggesting a Taken together, results from both the HCC patient cohorts significant inverse correlation between Slit3 expression showed that the expression of Slit3 in HCC inversely corre- and tumor size (R = − 0.352, p = 0.023; Fig. 1b), indicat- lated with the size of the tumor. ing that Slit3 closely and inversely associated with HCC tumor growth. Although, we found that Slit3 was not af- Slit3 negatively regulates cell proliferation of HCC in vitro fected by any other clinicopathological parameter. The results from the patient samples indicated a potential In order to validate the association between Slit3 expres- tumor suppressive role of Slit3 in HCC. To test this sion and HCC tumor size, we investigated the Slit3 gene hypothesis, we examined the functional effect of Slit3 in HCC cell-lines. The expression of Slit3 in several HCC cell- lines was determined initially (Fig. 2a). While LM3 and Table 1 Clinicopathological correlation of Slit3 expression in HCC patients PLC showed a relatively higher Slit3 expression, HepG2, Huh7, Hep3B and 97 L showed a low Slit3 expression. Slit3 p-value overexpression (T/N) Based on this result, we generated Slit3-downregulated low high stable clones from LM3 and PLC, by stable transfection of Age < 55 10 7 0.523 Slit3-shRNA, in order to examine the effect of Slit3 repres- sion on cell proliferation. The relative cell number was >=55 10 13 determined by MTT in terms of absorbance and growth Gender Male 19 15 0.182 rate was expressed as the percentage of cell absorbance on Female 1 5 day3with referencetothatonday 1. As showninFig. 2b HBV infection No 3 3 1.000 and c, LM3-shSlit3 and PLC-shSlit3 demonstrated a signifi- Yes 15 16 cantly higher growth rate when compared with their corre- Prior liver-directed No 17 18 0.345 sponding vector control LM3-shCTL (434.4% vs 355.9%, treatments p = 0.034) and PLC-shCTL (447.4% vs 337.9%, p = 0.039), Yes 4 1 respectively, suggesting that Slit3 repression significantly Differentiation Well 3 5 0.682 induced HCC cell proliferation. Similarly, the effect of Slit3 Moderate/Poor 11 10 overexpression on HCC cell proliferation was demonstrated Cirrhosis No 14 10 0.313 by stable transfection of Slit3-expression construct in Yes 5 8 Hep3B, which expressed low level of Slit3 (Fig. 2d). Hep3B- Tumor Size <=5 3 9 0.048 Slit3 stable cells showed a significantly lower growth rate when compared with Hep3B-pcDNA control cells (282.9% > 5 17 11 vs 309.2%, p = 0.039), suggesting that Slit3 over-expression Microvascular invasion No 10 13 0.741 significantly reduced HCC cell proliferation. Yes 8 7 Western blot results showed that the induced cell prolif- Stage 1 to 2 9 11 1.000 eration in Slit3-repressed PLC and LM3 cells was accom- 3to 4 9 9 panied with an induction of cyclin D3 and survivin (Fig. 3), Distant metastasis No 9 12 1.000 suggesting that Slit3 down-regulation induced HCC cell growth through activation of G1/S phase transition and Yes 8 9 a inhibition of apoptosis. Subsequently, we hypothesized that In some categories, the total number of patients was less than 40 due to incomplete information Slit3 regulated HCC tumor growth through manipulation Ng et al. BMC Cancer (2018) 18:621 Page 5 of 13 Fig. 1 Slit3 repression correlated with HCC tumor growth. a Tumor size of HCC patients cohort 1 (n = 40) with high or low Slit3 expression. b Relative Slit3 expression inversely correlated with tumor size of HCC patients cohort 1 (n = 40). c Tumor size of HCC patients cohort 2 (n = 25) with high or low Slit3 expression. d Relative Slit3 expression inversely correlated with tumor size of HCC patients cohort 2 (n = 25) of the GSK3β/β-catenin pathway which is commonly asso- Slit3 overexpression was found to significantly repress ciated with the development of HCC and other liver dis- Hep3B tumor growth in mice when compared with 3 3 eases . Our western blot results showed that the stable Hep3B-pcDNA cells (250.5 mm vs 3975.3 mm , p = 0.001). repression of Slit3 induced the expression of GSK3β (ser9) Taken together, these results indicated that Slit3 negatively in LM3 and PLC cells (Fig. 3), indicating that GSK3β which regulated HCC tumor growth in vivo. plays an important role in β-catenin degradation, was inac- IHC staining was applied to determine the expression of tivated through phosphorylation of GSK3β on ser9 residue Slit3, p27 (tumor suppressor), CD31 (vascular marker), upon Slit3 repression. Consequently, we also noted that β-catenin and phospho-GSK3β (ser9) in the tumors β-catenin was induced in PLC-shSlit3 cells when compared formed by LM3-shCTL/shSlit3 and Hep3B-pcDNA/Slit3 to their shCTL cells (Fig. 3). These results showed that Slit3 stable cells. The expression of Slit3 appeared weaker in repression inactivated GSK3β and thus resulting in induc- LM3-shSlit3 tumors in comparison with the LM3-shCTL tion of GSK3β/β-catenin pathway. tumors (Fig. 4c), and stronger in Hep3B-Slit3 tumors in comparison with the corresponding Hep3B-pcDNA Effect of Slit3 alteration on HCC tumor growth in vivo tumors (Fig. 4d), confirming that the Slit3 protein was To investigate the effect of Slit3 repression on tumor indeed silenced or overexpressed in the respective tumors. growth in vivo, 5 × 10 LM3-shCTL and LM3-shSlit3 cells The expression of CD31 was stronger in tumors devel- were subcutaneously injected into the flank region of 5 oped from LM3-shSlit3 cells when compared with that nude mice. After 6 weeks, the tumors were excised from from LM3-shCTL cells, and weaker in tumors developed themiceand thetumor sizesweremeasured(Fig. 4a and from Hep3B-Slit3 when compared with that from Hep3B- b). In line with the in vitro effects, Slit3 repression signifi- pcDNA, suggesting that Slit3 impaired the process of cantly induced tumor growth in mice (1054.9 mm vs 48. angiogenesis during the tumor growth process. On the 2mm , p = 0.002). Similarly, the in vivo effect of Slit3 other hand, the expression of p27 which is a negative overexpression was examined by subcutaneous injection of regulator of cell cycle progression  was lower in LM3- Hep3B-pcDNA and Hep3B-Slit3 cells into 5 nude mice. shSlit3 versus shCTL tumor, and stronger in Hep3B-Slit3 Ng et al. BMC Cancer (2018) 18:621 Page 6 of 13 Fig. 2 Slit3 negatively regulated HCC cell growth. a Expression of Slit3 in HCC cell-lines. b and c Stable Slit3 repressed clones (shSlit3) of LM3 and PLC cells displayed induced cell proliferation when compared with the control cells (shCTL) after 72 h. d Stable Slit3 overexpressed clones (Slit3) of Hep3B cells displayed impaired cell proliferation when compared with the control cells (pcDNA) after 72 h. The protein expression level was quantified by ImageJ software and normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown versus pcDNA tumor, showing that a higher expression of and cell viability was expressed as the percentage of sur- Slit3 suppressed cell cycle progression of HCC cells. Fur- viving cells following treatment of sorafenib/oxaliplatin/5- thermore, β-catenin and phospho-GSK3β (ser9) were in- FU as compared to negative control. As shown in Fig. 5a duced in LM3-shSlit3 tumors versus shCTL tumors and to c (left panel), LM3-shSlit3 cells showed a significantly reduced in Hep3B-Slit3 tumors versus pcDNA tumors, higher percentage of viable cell population than LM3- suggesting that Slit3 repressed β-catenin expression by shCTL cells following exposure to sorafenib (75.35% vs 52. reducing the activity of GSK3β. 81%, p < 0.001), oxaliplatin (68.94% vs 47.44%, p =0.013) and 5-FU (50.42% vs 43.96%, p =0.021). Similarly, Fig. 5a to Slit3 repression induces chemoresistance c (right panel) showed that PLC-shSlit3 cells also exhibited We investigated the effect of Slit3 repression on chemore- a significantly higher cell viability than PLC-shCTL cells sistance of HCC cells by evaluating the growth and following treatment with sorafenib (80.70% vs 54.75%, survival of LM3 and PLC shSlit3 cells, upon treatment of p < 0.001), oxaliplatin (54.69% vs 41.02%, p =0.018) and 5- chemotherapeutic drugs including sorafenib, oxaliplatin FU (48.54% vs 40.88%, p = 0.002). These results indicated and 5-FU for 72 h. The relative number of surviving cells that Slit3 suppression induced chemoresistance in HCC were determined in terms of absorbance by MTT assay, cells towards sorafenib, oxaliplatin and 5-FU. Ng et al. BMC Cancer (2018) 18:621 Page 7 of 13 Down-regulation of β-catenin diminishes the effects of Slit3-repression In order to further study the regulatory effects of Slit3 in HCC, we repressed β-catenin expression in PLC-shSlit3 cells and investigated the downstream effects. As shown in Fig. 7a, PLC-shSlit3 cells transiently transfected with a scrambled siRNA control (PLC-shSlit3 siCTL) showed a significantly higher proliferation rate than siCTL trans- fected PLC-shCTL cells (308.2% vs 259.8%, p = 0.012), however, such an induction was significantly reduced by transient transfection of siRNA targeting β-catenin (243. 3%, p = 0.008). Similarly, the induced chemoresistance in PLC-shSlit3 siCTL cells when compared with PLC- shCTL siCTL cells upon treatment of sorafenib (79.4% vs 43.1%, p < 0.001; Fig. 7b), oxaliplatin (55.4% vs 43.5%, p < 0.001; Fig. 7c) and 5-FU (56.0% vs 42.8%, p < 0.001; Fig. 7d) were also impaired by transient transfection of siRNA targeting β-catenin (59.5% for sorafenib, p <0.001; 45.0% for oxaliplatin, p < 0.001; 48.1% for 5-FU, p =0.003, Fig. 7b - d). Furthermore, following transient downregula- tion of β-catenin, the inductions in protein expression of phospho-GSK3β, cyclin D3 and survivin in PLC-shCTL stable cells were all reduced (Fig. 7e). These results sug- Fig. 3 Molecular mechanism associated with stable Slit3 repression. Protein expression of certain proteins associated with cell cycle gested that the GSK3β/β-catenin pathway served as a progression (cyclin D3), apoptosis (survivin) and GSK3β/β-catenin key regulatory network for the Slit3-regulated effects in pathway (β-catenin and phosopho-GSK3β (ser9) in LM3 and PLC HCC. Slit3 shRNA transfectants (shSlit3) and shRNA control (shCTL). The protein expression level was quantified by ImageJ software and Discussion normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide . Poor prognosis can be attributed to tumor progression as well as a lack of Furthermore, the expression of cyclin D3 was re- promising chemotherapy for treating advanced HCC. pressed by treatment of sorafenib, oxaliplatin and 5-FU There is hence a dire requirement of in-depth research in LM3-shCTL and PLC-shCTL cells, indicating an to further elucidate the underlying molecular pathogen- impairment of the G1/S phase transition within the drug esis of the disease and develop therapeutic targets. treated cells. Such repression was weaker in LM3-shSlit3 Hypermethylation and down-regulation of Slit3 has and PLC-shSlit3 cells (Fig. 6). On the other hand, the been reported in several types of cancers such as thyroid expression of survivin, following treatment with the cancer, colorectal cancer, gastric cancer, nasopharyngeal chemotherapeutic agents, was stronger in LM3-shSlit3 carcinoma, cervical cancer, ovarian cancer and pancre- and PLC-shSlit3 cells as compared to their respective atic ductal adenocarcinoma [23–32]. Additionally, Slit3 shCTL cells; indicating that LM3-shSlit3 and PLC- has been shown to suppress tumor growth in mouse shSlit3 cells were protected from the apoptotic effect models  and impairs cancer cell invasion and induced by the chemotherapeutic agents. We previously migration [24, 28, 34], suggesting that Slit3 functions as reported that repression of β-catenin was observed in a tumor suppressor in a variety of cancers. In HCC, Avci oxaliplatin-treated HCC cells [38, 39]. In this study we et al. compared the expression of Slit3 between 8 tumor- showed that β-catenin degradation following treatment adjacent normal and 35 tumor tissues . Though they with chemotherapeutic drugs, was impaired in Slit3- demonstrated that Slit3 was not differentially expressed, repressed LM3 and PLC cell lines. In conjuncture with 4 of the 8 tumor-adjacent normal tissues showed Slit3 the previous studies that demonstrated that the activa- repression in the HCC tissue, suggesting that Slit3 tion of β-catenin pathway enhanced the chemotherapeu- repression was present in half of the HCC patients. In tic resistance of HCC cells [40, 41], results from the support of this interpretation, this study demonstrated current study suggested that Slit3 repression contributed that Slit3 down-regulation was present in nearly 50% of to the chemoresistance in HCC cells, through its inhibi- both cohorts of our HCC samples, indicating that Slit3 tory effect on β-catenin degradation. repression was indeed a frequent event observed in Ng et al. BMC Cancer (2018) 18:621 Page 8 of 13 Fig. 4 Slit3 negatively regulated HCC cell growth in vivo. a Stable Slit3 repressed clones (LM3-shSlit3) displayed induced tumor growth when compared with the control cells (LM-shCTL) 6 weeks post-injection of cells subcutaneously into flank region of nude mice (n = 5). b Stable Slit3 overexpressed clones (Hep3B-Slit3) displayed impaired tumor growth when compared with the control cells (Hep3B-pcDNA) 6 weeks post-injection of cells subcutaneously into flank region of nude mice (n =5). c Immunohistochemical staining of Slit3, CD31, p27, β-catenin and phosopho-GSK3β (ser9) in tumor formed by LM3-shCTL and LM3-shSlit3 stable cells. d Immunohistochemical staining of Slit3, CD31, p27, β-catenin and phosopho-GSK3β (ser9) in tumor formed by Hep3B-pcDNA and Hep3B-Slit3 stable cells. Representative results from two mice of each group were shown HCC. Moreover, we found that Slit3 repression inversely a stronger IHC staining for β-catenin when compared correlated with tumor size, which was subsequently with LM3-shCTL tumors (Fig. 4). The β-catenin level is reaffirmed in the in vitro and in vivo experiments which regulated via phosphorylation by GSK3β in a multimeric showed that Slit3 negatively regulated HCC cell growth. protein complex comprising of β-catenin, GSK-3β,APC These clinical findings in combination with the functional and AXIN proteins . Repression of Slit3 in HCC cells studies suggested that Slit3 plays an important role in inhibited the activity of GSK3β by the induced phos- inhibiting tumor growth and progression in HCC. phorylation on the Ser 9 residue. Moreover, the effect of Our next step was to investigate the molecular mecha- Slit3 alteration on tumor growth in vivo was more nisms associated with Slit3 repression in HCC. Several obvious than that on in vitro cell proliferation, which studies implicate Slit and Robo in regulating E-cadherin- was possibly due to the effect of Slit3 on tumor angio- dependent adhesion via the Wnt downstream signaling genesis as demonstrated by the CD31 immunohisto- axis, including β-catenin and GSK3β , hence we chemical staining. We believed that Slit3 impaired investigated whether Slit3 regulated its functional effects angiogenesis through its regulatory effect on GSK3β/β- in HCC through the GSK3β/β-catenin pathway. This catenin axis which promotes angiogenesis through the study showed for the first time that Slit3 negatively activation of vascular endothelial growth factor signaling regulated β-catenin expression in HCC cells, and such in endothelial cells . an induction was associated with an altered GSK3β For patients with advanced HCC who are not activity (Fig. 3). Though the induction of β-catenin was candidates for surgical resection, liver transplantation, or not obvious in LM3-shSlit3 cells in vitro, we observed localized tumor ablation, systemic chemotherapy re- that tumors formed in vivo by LM3-shSlit3 cells showed mains the mainstay of therapy. Unfortunately, HCC is a Ng et al. BMC Cancer (2018) 18:621 Page 9 of 13 Fig. 5 Stable repression of Slit3 expression enhanced chemoresistance in HCC cells. (Left panel) LM3-shSlit3 and shCTL cells and (right panel) PLC-shSlit3 and shCTL cells were treated with (a)10 μM sorafenib, (b)10 μM oxaliplatin and (c) 100 μM 5-FU for 72 h. The number of cells was determined by MTT assay and expressed as percentage of that under vehicle control treatment. When compared with shCTL cells, shSlit3 cells showed significantly stronger resistance to the treatment of all chemotherapeutic drugs. Each experiment was performed in triplicate and data was obtained from three independent experiments relatively chemotherapy-resistant tumor; therefore, out- identification of biomarkers for predicting the response comes using this mode of treatment are generally unsat- to chemotherapy and development of molecular targets isfactory. A primary area of research in HCC is the to combat chemoresistant HCC. We tested the effect of Ng et al. BMC Cancer (2018) 18:621 Page 10 of 13 Fig. 6 Molecular mechanism associated with Slit3 repression-induced chemoresistance. Protein expression of β-catenin, cyclin D3 and survivin in LM3 and PLC Slit3 shRNA transfectants (shSlit3) and shRNA control (shCTL) following 72-h vehicle control (C), 10 μM sorafenib (SOR), 10 μMoxaliplatin (OXA) and 100 μM 5-FU (5FU) treatment. The protein expression level was quantified by ImageJ software and normalized to the expression of actin. The experiment was repeated for three times and one representative blot was shown Slit3 repression on the response of HCC cell lines to protein and this might reduce the occurrence of unex- sorafenib, an oral multikinase inhibitor that executes its pected side-effects. Indeed, the application of Slit3 as antitumor activities by blocking tumor angiogenesis, therapeutic agent has been demonstrated. A study by targeting the Raf/Mek/Erk pathway and inducing cell Denk et al. showed that the treatment with recombinant apoptosis . Our results demonstrated that HCC cells Slit3 caused a strong inhibition of migration of melan- harboring repressed Slit3 level were more resistant to oma cells in vitro, and down-regulation of AP-1 activity sorafenib treatment. In addition, we examined the effect [34, 50]. A different research group demonstrated that of Slit3 repression upon treatment with oxaliplatin and the in vitro ability of Slit3 to reduce the migratory activ- 5-FU, and our results showed that down-regulation of ity of synovial cells from patients with rheumatoid arth- Slit3 induced oxaliplatin- and 5-FU chemoresistance. In ritis and melanoma cells can be mimicked by small this study, though the Slit3 shCTL cells showed baseline protein fragments derived from Slit3 containing only chemoresistance to all the chemotherapeutic agents, we leucine rich repeat domain 2 [34, 50]. While the thera- showed that Slit3 repression, which was observed in peutic application of a full length Slit3 may not be around 50% of HCC patients of this study, was one of appropriate due to its large size that make them difficult the contributing factors towards the development of to be expressed recombinantly, reducing their stability resistance to a broad range of chemotherapeutic agents. as well as ease of usage in in vivo studies; the recombin- We believe that by having a comprehensive understand- ant Slit3 fragments offer a greater benefit for usage in ing of the molecular mechanisms leading to the cancer therapy. Within HCC, recombinant Slit3 treat- chemoresistant nature of HCC, novel therapeutic ment may benefit at two levels: Firstly, Slit3 can repress avenues to enhance the efficacy of chemotherapy on the growth of HCC tumor or perhaps even cause a HCC patients can be identified and the prognosis of shrinkage of the established tumor; Secondly, Slit3 could HCC patients can be improved. be applied as an adjuvant therapy which enhances the Our study reinforces the importance of Slit3 as a effectiveness of other chemotherapeutic agents such as therapeutic approach for HCC patients through its in- sorafenib, oxaliplatin and 5-FU, as shown in our stable hibitory effect on β-catenin pathway. The deregulation cell-line models. Though the in vitro tumor suppressive of β-catenin pathway is a hallmark of several cancers in- effect of Slit3 overexpression in HCC cells was not very cluding HCC . A high therapeutic efficacy of inhibit- strong, but as we observed that the negative regulatory ing β-catenin has already been demonstrated both in effect of Slit3 on tumor growth was much more obvious vitro and in vivo in HCC [46–49], yet there are no clin- in the in vivo model, possibly due to its involvement in ically approved anti–β-catenin agents available. Through tumor angiogenesis as demonstrated by the CD31 the current study, we offer a likely advantage of applying immunohistochemical staining. Based on the results Slit3 to inhibit β-catenin pathway as a novel treatment from this study, we strongly believe that administration strategy in HCC; since Slit3 is a naturally existing of recombinant Slit3 is a novel potential therapeutic Ng et al. BMC Cancer (2018) 18:621 Page 11 of 13 Fig. 7 Down-regulation of β-catenin diminished the effects of Slit3-repression. Stable PLC Slit3-repressed cells (PLC-shSlit3) and control clone (PLC-shCTL) were transiently transfected with siRNA control (siCTL) or β-catenin siRNA (si-β-catenin) to investigate the effect of β-catenin down- regulation on Slit3-regulated effects. a PLC-shSlit3 siCTL cells showed significantly higher proliferation rate than PLC-shCTL siCTL cells whereas such induction was significantly impaired in PLC-shSlit3 si-β-catenin cells. b to d The induced chemoresistance in PLC-shSlit3 siCTL cells when compared with PLC-shCTL siCTL cells upon treatment of sorafenib, oxaliplatin and 5-FU were impaired in PLC-shSlit3 si-β-catenin cells. e The inductions in protein expression of phospho-GSK3β, cyclin D3 and survivin in PLC-shSlit3 siCTL cells when compared with PLC-shCTL siCTL were all reduced in PLC-shSlit3 si-β-catenin cells approach for the treatment of HCC, however further in- Conclusion vestigations are necessary in order to elucidate its This study showed that Slit3 was a potential tumor potency and efficacy in patients with HCC. suppressor in HCC. Slit3 was frequently down-regulated Ng et al. BMC Cancer (2018) 18:621 Page 12 of 13 in HCC tumor tissue and its expression inversely corre- 2. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman CS, Tear G. Roundabout controls axon crossing of the CNS midline and lated with tumor size. Stable Slit3 repression induced the defines a novel subfamily of evolutionarily conserved guidance growth of HCC cells in vitro and in vivo, and induced receptors. Cell. 1998;92(2):205–15. chemoresistance to oxaliplatin, 5-FU or sorafenib, through 3. Brose K, Tessier-Lavigne M. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr Opin Neurobiol. 2000;10(1):95–102. the negative regulatory effect on β-catenin expression. 4. Dickinson RE, Dallol A, Bieche I, Krex D, Morton D, Maher ER, Latif F. Slit3 down-regulation in HCC might indicate a poor Epigenetic inactivation of SLIT3 and SLIT1 genes in human cancers. 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Slit3 expression and patient characteristics from breast cancer patients. Virchows Archiv : Int. J. Pathol. 2011;459(4):383–90. in cohort 1. Table S2. Slit3 expression and patient characteristics in cohort 2. (PDF 78 kb) 8. Dallol A, Morton D, Maher ER, Latif F. SLIT2 axon guidance molecule is frequently inactivated in colorectal cancer and suppresses growth of colorectal carcinoma cells. Cancer Res. 2003;63(5):1054–8. Abbreviations 9. Dong R, Yu J, Pu H, Zhang Z, Xu X. Frequent SLIT2 promoter methylation in 5-FU: 5 fluorouracil; GSK3β: Glycogen synthase kinase 3 beta; the serum of patients with ovarian cancer. J. Int. Med. Res. 2012;40(2):681–6. HCC: Hepatocellular carcinoma 10. Yiin JJ, Hu B, Jarzynka MJ, Feng H, Liu KW, Wu JY, Ma HI, Cheng SY. Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity. Acknowledgements Neuro-Oncology. 2009;11(6):779–89. We would like to thank Miss Tracy Lau and the Centre for Cancer Research 11. Avci ME, Konu O, Yagci T. Quantification of SLIT-ROBO transcripts in of the University of Hong Kong for providing technical support and hepatocellular carcinoma reveals two groups of genes with coordinate equipment for this study. expression. BMC Cancer. 2008;8:392. 12. Jin J, You H, Yu B, Deng Y, Tang N, Yao G, Shu H, Yang S, Qin W. Epigenetic Funding inactivation of SLIT2 in human hepatocellular carcinomas. Biochem Biophys This study is supported by the Small Project Funding from the University of Res Commun. 2009;379(1):86–91. Hong Kong (201209176190) and Central Allocation Group Research Grant 13. Gu JJ, Gao GZ. Zhang SM: miR-218 inhibits the migration and invasion of glioma “Molecular Pathology of Liver Cancer – A Multidisciplinary Study” from the U87 cells through the Slit2-Robo1 pathway. Oncol Lett. 2015;9(4):1561–6. Research Grant Council of Hong Kong (HKU-7-CRF09). 14. 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Published: Jun 1, 2018