Functional Analysis Of Stat3 In The Invasion, Metastasis Of Hepatocelluar Carcinoma And Further Study As A Target For Antisense Therapy

Hepatocellular carcinoma (HCC) mostly warrants a very poor prognosis caused by the recurrence and metastasis after resection. The majority of recurrence after resection occurs due to invasion-related spreading. Thus, it is imperative to investigate the controlling mechanism of HCC metastasis and invasiveness and to explore the effective therapeutic strategies. The recent study in our institute indicates that the expression signature of primary HCC with accompanying metastasis is very similar to that of their corresponding metastases, implying that genes functioning for metastatic progression are initiated in the primary tumors. So, it is important to understand the fundamental molecular mechanisms involved in invasiveness and metastasis of HCC. The signal transducer and activator of transcription 3 (Stat3) regulates gene expression in response to the signaling from cytokine and growth factor receptors. However, Stat3 protein is constitutively activated in a wide variety of human tumors with high frequency. Persistent signaling of Stat3 has been demonstrated to contribute to oncogenesis through their intimate connection to the signaling relating to growth, apoptosis, or angiogenesis. Stat3 as a novel drug target for therapeutic intervention of human cancers has been shown to be valuable. Recent studies have indicated that Stat3 protein or phospho-Stat3 (Tyr705) protein is overexpressed in HCC, however, it is mostly unknown at present how the signaling of Stat3 is implicated in HCC carcinogenesis and development and how the efficacy of treatment targeting Stat3 on HCC. As a novel gene-based therapeutic approach , antisense oligonucleotides have been used as the targeted molecular treatment of various diseases, especially malignant tumors. Antisense approaches to cancer therapy have been explored for a myriad of targets, including gene products involved in tumor invasiveness, the anti-apoptosis cascade, multidrug resistance and telomerase. At present, several antisense oligonucleotides have entered clinical use or clinical trials. On the basis of the first generation phosphorothioate antisense oligonucleotide, the second generation 2'-O-(2-methoxyethyl) (2'-MOE)-modified phosphorothioate gapmer antisense oligonucleotide has been developed which has increased both resistance to nuclease degradation, activation of RNase H, and binding affinity for target mRNA , metabolic stability. The favorable biochemical and pharmacokinetic characteristics of 2'-MOE-modified antisense oligonucleotide make it as a promising therapeutic drug entering clinical use. In the present study, we have investigated the role of the signaling of Stat3 in HCC growth, invasiveness, and metastastasis; and evaluated the efficacy of 2'-O-(2-methoxyethyl)-modified antisense Stat3 oligonucleotides on growth, invasiveness of human high metastatic hepatocellular carcinoma in vitro and in vivo; and studied the molecular mechanisms. Our study provides a basis for further evaluation of 2'-MOE modified antisense Stat3 oligonucleotides as a anticancer drug; and provides a novel pathway to understand the mechanism of HCC invasiveness and metastasis.PartⅠEfficacy of 2'-O-(2-Methoxyethyl)-Modified Antisense Stat3Oligonucleotides on Human Hepatocellular Carcinoma Cells with High Metastatic Potential and Further Study of the Molecular MechanismsThe objective of the present study was to explore how the 2'-O-(2-methoxyethyl) modified antisense Stat3 oligonucleotides specially inhibited the expression of Stat3 in HCCLM3 cells with high metastatic potential; and to investigate the efficacy of the drug on the growth, invasiveness, and angiogenesis of the cells in vitro; and to analyze the molecular changes caused by this drug using cDNA array; so as to provide a basis for further study of this drug and to provide a novel pathway to understand the mechanism of HCC invasiveness and metastasis.1．2'-MOE Stat3 ASO significantly inhibited the expression of Stat3 in HCCLM3 cellsOligonucleotides were delivered to HCCLM3 cells in the form of complexes with Lipofectamine^TM2000 (the ratio of oligo:lipo. was 1: 2(w/v)). HCCLM3 cells were treated with 2'-MOE Stat3 ASO (ISIS-113176-3) , base-randomized control oligonucleotides (BRC oligon. ISIS-29848-11) , or Lipofectamine alone as a control. The Stat3 mRNA and Stat3 protein and phospho-Stat3 (Tyr705) protein were assayed by RT-PCR or immunocytochemistry, respectively. The results indicated that the expression of Stat3 mRNA in HCCLM3 was inhibited by 2'-MOE Stat3 ASO in a concentration-dependent and sequence-specific fashion with with an IC50 of about 250 nM (51% inhibiton rate). The expression of Stat3 protein or phospho-Stat3 (Tyr705) protein was also significantly downregulated by Stat3 ASO.2．The effect of 2'-MOE Stat3 ASO on cell morphology and ultrastructure of HCCLM3 cellsThe cell morphology and ultrastructure of HCCLM3 cells was observed using reverse-microscope and electronic microscope respectively. The complex of liposome and oligonucleotides was entered cells in a form of monolayer liposome complex 300nm in diameter. The number mitochondrial critae in HCCLM3 cells transfected with Stat3 ASO was reduced compared with cells treated with BRC oligon.3．2'-MOE Stat3 ASO impeded cell growth and tumorigenicity, induced cell apoptosis and cell cycle S /G2-M phase ArrestThe in vitro growth-inhibitory effect of the 2'-MOE Stat3 ASO on HCCLM3 cells was evaluated using MTT assay. The ability of umorigenicity was detected by in vivo tumorigenecity assay. Apoptosis and cell cycle analysis were performed by flow cytometer. The results showed that Stat3 ASO reduced HCCLM3 cell growth in a sequence-specific and dose dependent manner with a peak of the inhibition at 48h. The tumors induced by HCCLM3 cells pretreated with AS-Stat3 odn growed slower than those induced by cells pretreated with BRC oligon. or lipofectamine only. AS-Stat3 odn resulted in enhanced blockade of cell cycle progression at the S/G2-M checkpoint and induced significant cell apoptosis. CDNA array analysis indicated that key genes associated with cell cycle or DNA damage respair such as BRCA1,BRCA2,cyclinD1,p16ink4,CDC25a,cdk4, p21Waf1 etc. and genes on apoptosis such as Apaf-1,bax,bcl-2,bcl-x,survivin etc. were significantly down-regulated after inhibition of Stat3 by Stat3 ASO. The Fas (Apo-1) gene was upregulated. The changes of multiple genes described above resulted in the efficacy we observed.4．2'-MOE Stat3 ASO inhibited cell invasion of HCCLM3 cellsThe invasive ability of cells was evaluated in 24-well matrigel precoated filters Transwell chambers. Furthermore, we analyzed the secretion of matrix metalloproteinase-2 and-9 (MMP2 and MMP9) using zymogram assay. The results showed that the treatment of 250nM Stat3 ASO (24h) resulted in decreased invasive ability of HCCLM3 cells. The Stat3 ASO significantly reduced the secretion of MMP2 and MMP9, especially MMP2．CDNA array analysis indicated that genes on invasion such as MMP-2, MMP-9, KISS1, Nm23-E4, etc. and genes on adhesion such as Integrinα5, Integrinα3, CD44, MUC-18, Integrinβ3, etc.) were significantly down-regulated by Stat3 ASO. The TIMP1 gene was upregulated by Stat3 ASO. The changes of these genes might be involved in the mechanisms of inhibiting invasiveness.5．2'-MOE Stat3 ASO inhibited angiogenesis induced by HCCLM3 cellsThe intradermal angiogenesis assay in nude mice indicated that angiogenesis induced by cells treated with Stat3 ASO was significantly reduced ,compared with BRC oligon.. The results from RT-PCR and ELISA showed that three isoforms of VEGF (VEGF121, VEGF165, VEGF189) were downregulated by Stat3 ASO treatment. CDNA array analysis demonstrated that genes related to angiogenesis such as ANGPT2, EGFR, FGF2, IFNA1, IGF-1, PDGFα, thrombospondin2, VEGF, etc. were downregulated by inhibiton of Stat3． In addition, some key genes related to signal transduction pathway such as akt-1, erb-2, MEK1,β-catenin, c-fos etc. were also downregulated by Stat3 ASO. These genes were perhaps involved in multiple pathways described above.To sum up, 2'-MOE modified Stat3 antisense oligonucleotides significantly inhibited the expression of Stat3 , which leaded to the disruption of Stat3 signaling pathway. As a result, the growth, invasion, and angiogenesis of HCC were impeded obviously. We can conclude that 2'-MOE modified Stat3 antisense oligonucleotide is an anticancer drug with therapeutic potential. On the other hand, Stat3 signling pathway plays an important role in the development of HCC.PartⅡInhibition of growth and metastasis of human hepatocellular carcinoma growing in nude mice by 2'-O-(2-Methoxyethyl)-Modified Antisense Stat3 OligonucleotidesThe purpose of the present study was to investigate the efficacy of 2'-O-(2-methoxyethyl)-modified antisense Stat3 oligonucleotides on growth, invasiveness of human high metastatic hepatocellular carcinoma by using in vivo nude mice xenograft models, and to evaluate it as an anti-cancer therapeutic drug.A highly metastatic xenograft model of human hepatocellular carcinoma (HCC) was established in nude mice via orthotopic implantation of HCCLM3 cells with a highly metastatic potential. 90 mice with transplanted tumours were randomly assigned to two large groups .Mice in each large group were then randomized into three experimental groups (n=15) as follows: (G1) i.p. injections of physiological physiological saline as a control; (G2) i.p. injections of 50mg/kg base-randomized control oligonucleotides (BRC oligon.); (G3) i.p. injections of 50mg/kg 2'-MOE modified antisense Stat3 oligonucleotides (Stat3 ASO, ISIS-113176-3) . After randomization, mice in two large group respectively received experimental antisense therapy in different schemes. In a first in vivo experiment (E1) , treatment of mice in each experimental group was started at the second day after tumor implantation. 50mg/kg of Stat3 ASO (group E1G3) , or BRC oligon. (group E1G2) , or 0．2ml of physiological saline (group E1G1) was injected i.p. once every three days for 9 times, and then injected i.p. once every two days for another 3 times. In a second in vivo experiment (E2) , treatment of mice in each experimental group was started at the tenth day after tumor implantation. 50mg/kg of Stat3 ASO (group E2G3) , or BRC oligon. (group E2G2) , or 0．2ml of physiological saline (group E2G1) was injected i.p. once every two days for 12 times. Three days after the last injection (i.e. 35 days after tumor implantation), randomly selected mice from each experimental group (n=9) were humanely sacrificed to observe the growth and matastasis of HCCLM3 tumors ,and tumors and lungs were harvested. Human Stat3 and mouse CD34 were assessed by immunohistochemistry and positive nuclei of Stat3 and microvessel density (MVD) labeled by CD34 were measured. Pulmonary metastases were detected by observing H.E. stained sequential sections of paraffin-embedded lung tissues. Blood samples were collected to allow for routine blood analysis and to determine AST, ALT in serum. The determination of VEGF, bFGF in serum was performed by ELISA. Another 6 mice in each group were maintained to evaluate life span.In the present study, we have demonstrated at least four significant results:1．Treatment with 2'-MOE modified Stat3 ASO effectively inhibited growth and metastasis of HCCLM3 tumors orthotopically implanted in nude mice, and obviously prolonged life span of mice with tumors.In two experiments, the expression of Stat3 protein in tumors was inhibited significantly and specifically by Stat3 ASO. As a result, the growth, local metastasis (intrahepatic, intra-celiac, diaphragmatic,etc.) ,and lung metastasis were impeded significantly. In the first experiment, treatment with Stat3 ASO (group E1G3) significantly decreased the mean tumor weight compared with control mice (group E1G1, E1G2) (0．93±0．I2g, 1．73±0．17g, 1．67±0．27g, respectively; P<0．01) . Lung metastasis was not found in group E1G3, however, incidence of lung metastasis in control group E1G1 and E1G2 was 100% (P<0．001) , 89% (P＜0．001) , respectively. Mice in group E1G3 obtained prolonged life span compared with control group E1G1(101±14．6 days vs 49．0±4．9 days, P<0．001) , or with group E1G2 (101±14．6 days vs 52．7±6．7 days, P<0．001) . In the second experiment, treatment with Stat3 ASO (group E2G3) also significantly decreased the mean tumor weight compared with control mice (group E2G1, E2G2) (1．18±0．11g, 1．89±0．24g, 1．77±0．39g respectively; P<0．01) . Lung metastasis in group E2G3 was also inhibited significantly compared with control E2G1 (22% vs 100% , P<0．01) or E2G2 (22% vs 100% , P<0．01) . Mice in group E2G3 obtained prolonged life span compared with control group E2G1(82．3±11．1days vs 46．2±4．3 days, P<0．001) , or with group E2G2 (82．3±11．1days vs 50．2±6．2 days, P<0．001) .2．Treatment with 2'-MOE modified Stat3 ASO effectively inhibited angiogenesis in HCCLM3 tumors.In both experiments, treatment with Stat3 ASO significantly inhibited microvessel density (MVD) compared control mice. MVD in group E1G3 was obviously decreased compared with control group E1G1 (3．3±1．5 vs.15．2±3．4; P<0．01) , or with group E1G2 (3．3±1．5 vs.13．8±4．0; P＜0．01) . MVD in group E2G3 was also obviously decreased compared with control group E2G1 (4．0±1．8 vs. 16．8±1．7; P<0．01) , or with group E2G2 (4．0±1．8 vs.14．5±4．0; P<0．01) . The results of ELISA assay showed that treatment of Stat3 ASO significantly decreased the secretion of VEGF and bFGF. These results indicated that downregulation of VEGF and bFGF is one of the mechanisms that Stat3 ASO inhibited the growth and metastasis of HCCLM3 tumor in vivo.3．Tumorous burden impacted on the efficacy of Stat3 ASO.Treatment with Stat3 ASO started at the second day after tumor implantation (group E1G3) showed more efficacy than treatment started at the tenth day after implantation (group E2G3) on tumor weight (0．93±0．12g vs. 1．18±0．11g; P<0．05) , incidence of lung metastasis (0 vs 22%; P<0．05 ), life span (101±14．6 days vs. 82．3±11．1days; P<0．05) . However, the difference between two groups had no statistic significance in the expression of Stat3, serum VEGF/bFGF level and MVD.4．Treatment with 2'-MOE modified Stat3 ASO had no obvious hepatic or blood toxicity.Thus, disruption of Stat3 by using antisense approach may serve as a novel therapeutic strategy for hepatocellular carcinoma. Our results provide a basis for further evaluation of 2'-MOE modified antisense Stat3 oligonucleotides as an anticancer drug.