WO2016056995A1 - Profilage et/ou thérapie d'un carcinome hépatocellulaire - Google Patents

Profilage et/ou thérapie d'un carcinome hépatocellulaire Download PDF

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WO2016056995A1
WO2016056995A1 PCT/SG2015/050371 SG2015050371W WO2016056995A1 WO 2016056995 A1 WO2016056995 A1 WO 2016056995A1 SG 2015050371 W SG2015050371 W SG 2015050371W WO 2016056995 A1 WO2016056995 A1 WO 2016056995A1
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ect2
inhibitor
hcc
subject
rho
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Kam Man Hui
Jianxiang CHEN
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Singapore Health Services Pte Ltd
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of profiling (in particular molecular profiling) and/or therapy of tumour.
  • Hepatocellular carcinoma is a highly lethal malignancy (Farazi et al., 2006; Hoshida, 2009; El-Serag, 201 1 and Marquadi et al., 2014).
  • Early HCC recurrence is mainly responsible for poor patient survival and is the major obstacle to improving prognosis (Hoshida R, 2009 and El-Serag, 201 1 ).
  • Tumours in most HCC patients are resistant to many conventional chemotherapeutic drugs (Maluccio and Covey, 2012).
  • the present invention provides a method for profiling a liver sample from a hepatocellular carcinoma subject comprising:
  • the present invention provides a method for monitoring a HCC subject comprising determining ECT2 expression levels and/or ECT2/RHO/ERK signalling activity of (a) at least one liver sample taken from the subject at a first time point before the subject has been administered at least one Epithelial cell transforming sequence 2 inhibitor and (b) at least one liver sample separately taken from the subject taken at various subsequent time points after the subject has been administered the at least one Epithelial cell transforming sequence 2 inhibitor, wherein:
  • the present invention further provides a method for treating HCC in a subject comprising administering at least one Epithelial cell transforming sequence 2 inhibitor to the subject.
  • the present invention further provides a method for treating HCC comprising:
  • Fig. 1 Up-regulation of ECT2 in HCC tissues is associated with early tumour recurrence.
  • A Microarray analysis of ECT2 gene expression in histologically normal liver tissues from patients with colorectal metastases (NN), matched normal livers of HCC patients (MN-HCC), recurrent tumours (R-HCC), and nonrecurrent tumours (NR-HCC).
  • B The expression of ECT2 and GAPDH was compared by Western blotting in 14 pairs of HCC patient tissues (T, tumour; N, matched normal liver) and in 7 R and 7 NR tumours.
  • C ECT2 expression in HCC cell lines by Western blotting.
  • D Representative images of ECT2 expression in HCC patients' tissues by IHC.
  • E Expression of ECT2 was associated with disease-free survival (DFS) in HCC patients.
  • DFS disease-free survival
  • Fig. 2 Expression of ECT2 is associated with cell growth, oncogenesis, tumourigenesis and metastasis of HCC cells.
  • A The knockdown effect of ECT2 by siRNA was analysed and live cells observed after trypan blue staining were counted on different days post-treatment.
  • B Caspase-related apoptosis was analysed at 72 h post-treatment by immunoblotting.
  • E Stable ECT2 knockdown HCCLM3 cells were inoculated subcutaneously to monitor tumour development. At 5 weeks after inoculation, the mice and tumours were photographed and the changes in tumour volume were analysed.
  • F Representative images showing the effect of siRNA treatment on cell migration and invasion 1 day after seeding in vitro.
  • G Representative images of wound regions following a scratch wound healing assay to demonstrate the effect of stable ECT2 knockdown on HCC cell migration. Three independent experiments were performed for the in vitro cell migration and invasion assay and the wound healing assay, respectively.
  • ECT2 regulates cell proliferation and migration via the RhoA/ERK signalling axis.
  • A Pull-down and immunoblotting assays of the GTPase activity of Cdc42, Rac1 , and Rho after different treatments. GTPyS added to siScramble lysate acted as a positive control.
  • B Human Phospho-Kinase antibody analysis of HCCLM3 cell lysates obtained following transfection with siRNAs specific for ECT2 and Scramble control. p-ERK1/2 was significantly decreased while HSP60 was up-regulated markedly after ECT2 gene knockdown (circled by black boxes). The "R" indicate reference control dots.
  • RhoA activity was prepared in scrambled or ECT2 siRNA-treated HCCLM3 cells and the activity of downstream signalling molecules was analysed by immunoblotting. GAPDH was used as a loading control.
  • D Diagram depicting upstream and downstream signals of ECT2 that might be contributing to HCC recurrence. ⁇ indicates direct activation, -J indicates direct inhibition and a dotted arrow indicates possible indirect activation.
  • E RhoA expression level and ERK activation were analysed by immunoblotting at day 2 after ERK- or RhoA-siRNA treatment. In vitro cell migration of cells was studied.
  • Rho inhibitor I and ERK inhibitor (VX-1 1 e) were added to HCCLM3 cells at the indicated dose. After 24 h, the active forms of RhoA and ERK were detected by immunoblotting and the ability of the cells to migrate was also studied.
  • G ERK activation and ECT2 level were detected by immunoblotting in two tumours generated after subcutaneous inoculation of stable ECT2 knockdown cells in mice.
  • H Representative images of IHC staining showing the expression of ECT2 and p-ERK1/2. Representative images of three HCC patients' tumours with high (I) or low (J) ECT2 and p- ERK1/2 expression are shown. Twenty-eight of 43 samples showed high ECT2 or p-ERK1/2 levels.
  • ECT2 functionally and physically interacts with RACGAP1 to promote RhoA-GTP exchange, cell migration and protein stability of RACGAP1 in HCCLM3.
  • A I PA analysis to identify potential molecules that can interact with ECT2 in HCC cells. The interaction between ECT2 and RACGAP1 is highlighted in blue. The red coloured nodes indicate that the genes are up- regulated in HCC samples from patients with early recurrent disease.
  • B Endogenous co-IP was used to determine the interaction between ECT2 and RACGAP1 in HCCLM3 cells. 1 % of the lysate employed for immunoprecipitation was used for input detection.
  • D In vitro Transwell migration assays. The cells that migrated through the membrane were stained and counted.
  • E Representative confocal images showing the localisation of ECT2 and RacGAPI (red) at different phases in the cell cycle. Images were analysed at 63X magnification following immunostaining.
  • G Representative confocal images of results obtained to study ECT2 and RacGAPI interaction by immunoprecipitation and Duolink protein-protein interaction assays. The rabbit and mouse IgG antibodies were used for the controls.
  • H Correlation analysis of the expression of ECT2 and RACGAP1 using our previously established microarray database consisting of 70 HCC patients [19]. R>0.5 indicates a significant correlation.
  • I Western blot assay of ECT2 and RACGAP1 in cells at 72 h post treatment. The protein density was determined by Image-J software (NIH, USA) and the protein density ratio of ECT2 and RacGAPI to GAPDH was calculated. The protein density of the siScamble treatment group was regarded as 1.
  • J A CHX chase experiment was used to analyse the stability of RACGAP1 protein after siRNA treatment in cells. The relative protein density ratio of RACGAP1 to ⁇ -actin was calculated at 0, 1 , and 2 h post-CHX treatment (100 g/ml). P ⁇ 0.05 was considered statistically significant. Representative data from three independent experiments were employed for quantitation.
  • Fig. 5 Validation and growth curve of stable ECT2 knock down HCC cells.
  • A The knockdown of ECT2 by stable transfection of the pLKO.I-shvector, pLKO.I-shScramble control and pLK0.1-shECT2 in different cells was validated by immunoblotting, and
  • B-D the growth of live stable cells (trypan blue staining) was monitored on days 1 , 3, and 5 post-seeding.
  • ECT2 The expression and function of ECT2 is negatively regulated by p53 in wild-type p53 HCC cells.
  • the protein levels of ECT2, p21 and p53 were analysed by immunoblotting after p53 siRNA treatment on day 2 and day 3 in SK-Hep1 (A) and HepG2 (B).
  • ECT2 promoter luciferase reporter activity was also analysed after p53 siRNA treatment in SKHepl (C) and HepG2 (D).
  • the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of.
  • the variations of the word “comprising”, such as”comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
  • a subject with HCC is one diagnosed with HCC based on the typical diagnostic criteria for HCC.
  • a healthy individual not suffering from HCC is assessed as HCC negative based on the typical diagnostic criteria for HCC.
  • HCC Hepatocellular carcinoma
  • ECT2 Epithelial cell transforming sequence 2 oncogene
  • GEF guanine nucleotide exchange factors
  • Rho GEFs Rho family guanine nucleotide exchange factors
  • ERK extracellular-signal-regulated kinase
  • siRNA small interfering RNAs
  • Rho GEF Rho factor related guanine exchange factor
  • NN normal liver tissues
  • MN (N) matched normal tissues from HCC patients
  • T HCC tumours
  • R recurrent HCC
  • NR non-recurrent HCC
  • AKT protein kinase B (PKB)
  • FAK focal adhesion kinase
  • Ser serine
  • Tyr tyrosine
  • RacGAPI RacGTPase activating protein 1
  • IHC immunohistochemistry
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling
  • H2AX H2
  • the present invention provides a method for profiling a liver sample from a hepatocellular carcinoma subject comprising:
  • the present invention provides a method for monitoring a HCC subject comprising determining ECT2 expression levels and/or ECT2/RhoA/ERK signalling activity of (a) at least one liver sample taken from the subject at a first time point before the subject has been administered at least one Epithelial cell transforming sequence 2 inhibitor and (b) at least one liver sample separately taken from the subject taken at various subsequent time points after the subject has been administered the at least one Epithelial cell transforming sequence 2 inhibitor, wherein:
  • the liver sample from the HCC subject may be diseased liver tissue from the subject. It will be appreciated that the diseased liver tissue comprises transformed cells. It will be further appreciated that the liver sample may be taken from a liver neoplasm.
  • control comprises at least one normal liver tissue sample.
  • a normal liver tissue sample may be from a healthy individual not suffering from HCC.
  • a normal liver tissue sample may also be from non-diseased liver tissue from a subject with HCC.
  • Non-diseased liver tissue refers to liver tissue with non-transformed cells.
  • non-diseased liver tissue and/or cells can be easily distinguished from the HCC tissue, for example morphologically. It will be appreciated that non- diseased liver tissue and/or cells also do not show any manifestation of any other liver conditions, including but not limited to liver cirrhosis, hepatic steatosis and hepatitis, for example.
  • control HCC subjects may be diagnosed as early stage, intermediate stage or advanced stage HCC.
  • the method further comprises identifying a subject with higher expression of ECT2 compared to the control as likely to have a higher recurrence of hepatocellular carcinoma.
  • said recurrence of hepatocellular carcinoma comprises early recurrence of hepatocellular carcinoma.
  • the method for profiling a liver sample from a HCC subject or the method for monitoring a HCC subject as described herein may be an in vitro method.
  • the present invention further provides a method for treating HCC in a subject comprising administering at least one Epithelial cell transforming sequence 2 to the subject.
  • the present invention further provides a method for treating HCC comprising:
  • ECT2 expression level(s) may be determined at the transcription and/or translation level(s).
  • any suitable method may be employed for determining ECT2 expression level(s) at the transcription level, including but not limited to Northern blot analysis, microarray analysis and/or reverse-transcription PCR.
  • the reverse-transcription PCR may be quantitative reverse-transcription PCR (RT- qPCR).
  • Any suitable method may also be used to determine ECT2 expression level at the translation level, including but not limited to Western blot, immunohistochemistry (IHC) and/or enzyme-linked enzyme-linked immunosorbent assay (ELISA) analysis.
  • IHC immunohistochemistry
  • ELISA enzyme-linked enzyme-linked immunosorbent assay
  • the present invention relates to an Epithelial cell transforming sequence 2 inhibitor for use in treating HCC in a subject.
  • the present invention also relates to use of an Epithelial cell transforming sequence 2 inhibitor in the preparation of a medicament for treating HCC in a subject.
  • a HCC sample from said subject has higher ECT2 expression compared to at least one normal liver tissue sample and further, it will be appreciated that said subject will be profiled by a method as described herein.
  • the Epithelial cell transforming sequence 2 inhibitor may inhibit ECT2 expression.
  • the Epithelial cell transforming sequence 2 inhibitor comprises at least one RNA interfering agent targeting ECT2 mRNA.
  • the RNA interfering agent may comprise a small interfering RNA (siRNA).
  • the Epithelial cell transforming sequence 2 inhibitor may inhibit ECT2 protein activity.
  • the Epithelial cell transforming sequence 2 inhibitor may inhibit the GTP exchange in Rho-GTPases. Inhibition of ECT2 protein activity by the Epithelial cell transforming sequence 2 inhibitor may lead to suppression of phosphorylated ERK levels.
  • the Epithelial cell transforming sequence 2 inhibitor may be at least one antibody and/or a functional fragment thereof.
  • the at least one antibody may be a monoclonal antibody or polyclonal antibodies. Monoclonal and polyclonal antibodies may be produced by standard methods.
  • a further example of an Epithelial cell transforming sequence 2 inhibitor includes a peptide.
  • the present invention is suitable for personalised treatment, for example a subject is profiled for ECT2 expression levels and to assess response to Epithelial cell transforming sequence 2 inhibitor before administration of treatment.
  • tumour and adjacent normal liver tissue from HCC patients was approved by our Institutional Review Board (IRB) and all tissues studied were provided by the Tissue Repository of the National Cancer Centre Singapore (NCCS). Written informed consent was obtained from participating patients and relevant clinical and histopathological data provided to the researchers were anonymised.
  • IRS Institutional Review Board
  • NCCS National Cancer Centre Singapore
  • mice were approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). Tumour volumes (V) were monitored every week and calculated according to the formula V 0.52 ⁇ length xwidth 2 , as previously described (Chen et al., 2011 ). Stably transfected HCCLM3 cells were resuspended in PBS and implanted into the left and right flanks (5 ⁇ 10 6 cells per flank) of male BALB/c nude mice by subcutaneous injection. Student's t test was used to evaluate differences between the tumour sizes in the shScramble- and shECT2-transfected groups.
  • the DFS analysis was performed according to the Kaplan-Meier method and the log-rank test. The median of the log-transformed ECT2 expression level was used as the cut-off value.
  • Kaplan-Meier disease free survival analysis was performed on 67 HCC patients based on high and low ECT2 expression. The patients with low ECT2 expression had a significantly higher disease-free survival rate compared with the patients with high ECT2 expression.
  • CHX was from Sigma-Aldrich (St. Louis, MO); VX-1 1 e was from ChemieTek (Indianapolis, IN, USA); cell- permeable C3 transferase Rho inhibitor I was from Cytoskeleton, Inc. (Denver, USA).
  • Antibodies for Western blotting were: rabbit anti-ECT2 (C-20), mouse anti-p53 (DO-1 ), mouse anti-caspase-9, mouse anti-HSP70, and mouse anti- PARP-1 (Santa Cruz, CA, USA); rabbit anti-GAPDH, anti-p-ERK1/2 (p-p44/42), anti-total-ERK1/2, anti-FAK, anti-p-FAK (Thr925), anti-p-MEK1/2, anti-total MEK1/2, anti-p-AKT (Ser308), anti-p-AKT (Ser473), anti-total AKT, anti-p-H2AX (Ser139), anti-caspase-3 and anti-p21 (Cell Signaling Technology, Danvers, MA, USA); and mouse anti-RacGAP1 (1 G6) (Abnova, Taipei City, Taiwan).
  • Antibodies for immunofluorescence were: rabbit anti-ECT2 (Abnova); and mouse anti-a-tubulin (ab7750), rabbit anti-RacGAP1 (ab61 192), rabbit IgG control, and mouse IgG control (Sigma-Aldrich, St. Louis, Missouri, USA ). siRNA, Western blotting, and Immunofluorescence
  • siRNA mix targeting luciferase was purchased from Sigma-Aldrich (St. Louis, MO, USA). The sequences were as follows; luciferase sense: 5'- CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 1 ), anti-sense: 5'- UCGAAGUAUUCCGCGUACG-3" (SEQ ID NO: 2).
  • Scramble siRNA (ON- TARGETplus Non-Targeting Pool, D-001810-10-05), ECT2 siRNA (ON- TARGETplus SMARTpool, L-006450-00-0005) and RacGAPI siRNA (ON- TARGETplus SMARTpool, L-008650-00-0005) were purchased from Thermo Scientific Dharmacon (UK).
  • Human ERK1/2 siRNA was from Cell Signaling Technology (Danvers, Massachusetts, USA).
  • Human p53 siRNA (ID: s605) was from Ambion, Thermo Fisher Scientific (Waltham, Massachusetts, USA) .
  • Cell lysates were prepared in RIPA buffer with an added protease inhibitor cocktail, phosphatase inhibitor cocktail, DTT (Dithiothreitol), and Benzonase (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was calculated using the BCA Protein assay kit (Thermo Scientific Pierce, UK). Western blotting was performed using a standard method.
  • the cells were incubated with primary antibody overnight at 4 °C and then incubated with Alexa Fluor® 594 goat anti-rabbit or Alexa Fluor® 488 goat anti-mouse (Molecular Probes, Thermo Fisher Scientific, Waltham, Massachusetts, USA).
  • the slides were counterstained with Hoechst 33342 (Molecular Probes, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and imaged using a LSM510 Meta confocal laser-scanning microscope (Carl Zeiss AG, Oberkochen, Germany).
  • Stable cells and vectors pLK0.1 -shECT2 (validated) and pLKO.I -shScramble clone vectors were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lentivirus was prepared following the instructions of Sigma Mission Lentivirus packaging and then used to infect cells three times. The cells were subcultured to 10% confluence in a medium containing puromycin (Sigma-Aldrich, St. Louis, MO, USA; Hep3B: 1.5 Mg/ml, HuH-7: 1.5-2.0 Mg/ml, and HCCLM3: 2.5 Mg/ml). Antibiotic-resistant clones were picked and passaged in medium containing puromycin.
  • ECT2 promoter luciferase assay the previously described (Scoumanne and Chen 2006). Human ECT2 gene promoter region (-151 1/+78) was amplified from 293T genome DNA and cloned into pGL3-Basic (Promega, Fitchburg, Wisconsin, USA).
  • Migration and invasion assays were carried out in 24-well plates using Boyden chambers using a PET membrane with an 8-mm pore size (Falcon). Briefly, cells were transfected with ECT2, P53, RHOA, and ERK1/2 siRNAs; 24 h post transfection, cells were counted and processed for transwell assay as described before (Wang et ai, 201 1 ). After overnight or 4-h incubation, any non-invasive cells on the upper surface of the Matrigel membrane were gently removed using a cotton-tipped swab. The invasive cells were fixed in 100% methanol and stained with 1 % toluidine blue. The stained invasive cells that had passed through to the lower surface of the membrane were photographed under an inverted light microscope with a 40x objective and quantified by manual counting in three randomly selected areas. Results were obtained from three independent experiments performed in duplicate.
  • the wound healing assay was done using stable ECT2 knockdown or scramble HCCLM3 lines because stable cell lines are easier to grow to over-confluence, which is needed in the wound healing assay. Briefly, when the stable cells were grown to confluence, we used a 100 ⁇ tip to make a scratch in the middle of the well. After this, photos of different wound regions were taken, the size of the wound was calculated by image-J software (NIH, Bethesda, Maryland, USA) and the relative wound closure was analysed.
  • Stable knockdown cells were counted and seeded in different numbers (for Hep3B and HuH-7, 8,000 cells were seeded; for HCCLM3, 1 ,000 cells were seeded) in six-well plates in growth medium containing 0.7% agar (2 ml per well) on top of a layer of growth medium containing 1.4% agar (1 ml per well). Growth medium (1 ml) with 10% FBS (Fetal bovine serum, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added on top of the agar. The cell suspension was plated and cultured in a 37 °C incubator for around 20 days. After that, the colonies were fixed with 4% PFA (Sigma-Aldrich, St.
  • Transcriptional activity assays were performed using the Luciferase Assay System (Promega, Fitchburg, Wisconsin, USA) according to the manufacturer's instructions.
  • SK-Hep1 , HepG2, and HLE cells were transfected with the ECT2- Promoter Luc reporter plasmid and vector control plasmid 24 h post P53 siRNA or control siRNA treatment. Twenty-four hours after transfection of the plasmids, the cells were lysed and luciferase activity was measured using a Dual- Luciferase Reporter (DLR) Assay Kit (Promega, Fitchburg, Wisconsin, USA) as instructed by the supplier. Firefly and Renilla luciferase activities were measured for normalisation using a luminometer (Lumat LB9507, Berthold, Bad Wildbad, Germany).
  • DLR Dual- Luciferase Reporter
  • HCCLM3 cells were cultured in a 15-cm dish and then transfected with ECT2 siRNAs and scrambled siRNAs. After 48 h, the cells were lysed with the buffer in the kit, which contained protease inhibitors, and the protein concentration was measured as described above. In total, 1 mg of protein was prepared from each sample and, in the case of the scrambled treatment lysate, GTPyS and the related buffer were added and incubated following the protocol of the kit. This was used as the positive control. The following pull-down assay was done as described before (Wang et a/., 201 1 ).
  • Protein G Dynabeads (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were pre-cleaned and incubated with rabbit anti-ECT2 (C-20), mouse anti- RacGAPI (1 G6), rabbit anti-PRC1 (Protein regulator of cytokinesis 1 ) (H-70), or their respective rabbit or mouse IgG control antibodies (Sigma) for 6 h in 5% BSA IP buffer. After that, the beads were washed with IP buffer (20 mM Tris pH8, 10% glycerol, 150 mM NaCI, 0.1 % NP-40, 0.1 mM EDTA) (Sigma-Aldrich, St. Louis, Missouri, USA) five times on a magnet, where the beads migrate to the side of the tube facing the magnet.
  • IP buffer 20 mM Tris pH8, 10% glycerol, 150 mM NaCI, 0.1 % NP-40, 0.1 mM EDTA
  • HCCLM3 cells were grown in a 15-cm dish to a density of 70-90% and then lysed on ice with IP buffer for 30 min. Protein concentration was measured and 500 ⁇ g of protein was added to the beads and incubated for 2 h at 4 °C. After that, the beads were washed six times. Finally, the proteins on the beads were boiled with 1 ⁇ SDS loading buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) on a 95 °C heat block for 10 min. The samples were checked by Western blotting and finally detected by anti-mouse (ab99697, Abeam, Cambridge, UK) or anti-rabbit (ab99617, Abeam, Cambridge, UK) secondary antibodies specifically recognising the light chain.
  • Duolink (PLA) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was performed to image protein-protein interactions using microscopy.
  • oligonucleotide-conjugated secondary antibodies were directed against primary antibodies raised against cell surface receptors. Annealing of the oligonucleotides conjugated to the secondary antibodies occurred only when the target proteins were in close proximity and initiated the amplification of a Texas red reporter signal.
  • Hep3B and HCCLM3 were fixed with 4% PFA (Sigma-Aldrich, St. Louis, Missouri, USA), permeabilised with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, Missouri, USA) for 10 min, blocked with PBS (Sigma- Aldrich, St.
  • a ligation solution was added together with ligase to join the two hybridised oligonucleotides to form a closed circle
  • the samples were subjected to several cycles of rolling-circle amplification (RCA) using the ligated circle as a template and generating a concatemeric product extending from the oligonucleotide arm of the PLA probe.
  • RCA rolling-circle amplification
  • a detection solution consisting of fluorescently labelled oligonucleotides was added and the labelled oligonucleotides were hybridised to the RCA product.
  • the signal was visible as a distinct fluorescent dot in the Texas red channel and analysed by fluorescence microscopy. Hoechst (Sigma-Aldrich, St. Louis, Missouri, USA) was included in the detection solution.
  • Negative controls consisted of wild-type samples treated as described except for IgG control antibodies (Sigma-Aldrich, St. Louis, Missouri, USA)
  • Cells were treated with scrambled or ECT2 siRNAs. Forty-eight hours post treatment, cells were either lysed for Western blot analysis or fixed in 80% ethanol at -20 °C for flow cytometry analysis. The cells were resuspended in PBS (Sigma-Aldrich, St. Louis, Missouri, USA containing 50 g/ml propidium iodide (Sigma-Aldrich, St. Louis, Missouri, USA) , 20 pg/ml RNaseA, and 0.1 % Triton X-100 (Sigma-Aldrich, St. Louis, Missouri, USA), and incubated for 30 min at 37 °C.
  • PBS Sigma-Aldrich, St. Louis, Missouri, USA
  • PBS propidium iodide
  • RNaseA pg/ml RNaseA
  • Triton X-100 Sigma-Aldrich, St. Louis, Missouri, USA
  • the DNA content was monitored by a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA). Cells were seeded on coverslips at 30% density. After the second day, siRNAs were added to the cells. Forty-eight hours post transfection, cells were fixed by 4% PFA (Sigma- Aldrich, St. Louis, Missouri, USA) and processed for TUNEL assay using the DeadEcdTM Fluorometric TUNEL System (Promega, Fitchburg, Wisconsin, USA) .
  • HCCLM3 cells were resuspended in PBS (Sigma-Aldrich, St. Louis, Missouri, USA) and implanted into the left and right flanks (5x106 cells per flank) of male BALB/c nude mice (InVivos, Singapore, Singapore) by subcutaneous injection. Student's t test was used to evaluate the difference between tumour sizes of the shScramble- and shEC72-transfected groups.
  • Paraffin-embedded tissue samples from consenting patients or mouse HCCLM3 xenografts were cut into 5-pm sections and placed on poly-lysine-coated slides (Sigma-Aldrich, St. Louis, Missouri, USA). Then, the samples were deparaffinised in xylene (Sigma-Aldrich, St. Louis, Missouri, USA) and rehydrated using a series of graded alcohols. Antigen retrieval was performed by heat mediation in citrate buffer (pH 6; Dako, Carpinteria, CA, USA). Samples were blocked with 10% goat serum (Sigma-Aldrich, St. Louis, Missouri, USA) before incubation with primary antibody.
  • the samples were incubated overnight using a primary antibody, rabbit anti-ECT2 (C-20; 1 :75) (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), rabbit-anti-p-ERK1/2 (p-p44/42; Cell Signaling Technology, Danvers, MA, USA; 1 :50), or an isotype-matched IgG as a negative control in a humidified container at 4 °C.
  • Immunohistochemical staining was performed with the Dako Envision Plus System (Dako, Carpinteria, CA, USA) according to the manufacturer's instructions. The intensity of staining was evaluated by three individual researchers on a scale of 0 to 4 according to the percentage of positive tumours.
  • the expression of ECT2 or p-ERK1/2 was scored by two pathologists and the average scores were used for the Kaplan- Meier survival analysis.
  • Hep3B and HCCLM3 cells were transfected with scrambled or ECT2 si ' RNAs; 48 h post treatment, the cells were incubated with 10% FBS DMEM (Sigma- Aldrich, St. Louis, Missouri, USA) containing 100 g/ml CHX. Then, at 0, 1 , and 2 h post CHX treatment, the cells were collected by trypsin treatment and lysates were prepared for Western blot assay.
  • FBS DMEM Sigma- Aldrich, St. Louis, Missouri, USA
  • ECT2 is significantly up-regulated in HCC and a high level of expression of ECT2 correlates with a short time to recurrence in patients with HCC.
  • liver tissues 10 histologically normal liver tissues from patients with colorectal cancer that metastasized to the liver (NN), 41 histologically normal adjacent liver tissues from HCC patients (MN) and 70 HCC tumour tissues including 34 non-recurrent (NR) HCC (with no recurrent disease observed in >5 years) and 36 recurrent (R) HCC (with recurrent disease detected in ⁇ 2 years) samples were compared and analysed as previously described (Wang et ai, 2011 ). It was observed that ECT2 expression was significantly up-regulated in the tumour samples compared to both NN and MN tissues (PO.001 ; Fig. 1A).
  • ECT2 expression was markedly up- regulated in the early recurrent tissues compared to non-recurrent HCC tissues (PO.001 ; Fig. 1A).
  • ECT2 protein expression was significantly increased in the HCC tumour samples (T) compared to the matched normal liver tissues (MN; Fig. 1 B) and ECT2 protein expression was also markedly elevated in early recurrent HCC samples compared to nonrecurrent samples (Fig. 1 B).
  • ECT2 was markedly up-regulated in 10 out of the 1 1 HCC cell lines that was studied (Fig. 1 C). IHC staining also confirmed that ECT2 expression was elevated in HCC compared to histologically normal liver tissues (Fig. 1 D). Importantly, it was demonstrated that patients with high ECT2 expression (expression level above the median expression of all the HCC tissues studied) had a significantly shorter disease- free duration compared to patients with low ECT2 expression (expression level below the median expression of all the HCC tissues studied) (P ⁇ 0.001 ; Fig. 1 E and Supplementary Table 1 ). Corroborating these results, univariate and multivariate analyses showed that ECT2 could be an independent predictor of poor prognosis for patients with HCC (Table 1 ).
  • n.s. is not significant; P ⁇ 0.05 is significant.
  • Univariate and multivariate Cox regression analysis was performed to determine whether ECT2 expression could serve as an independent survival prognostic factor for HCC.
  • AJCC stage Tumour venous Infiltration, Child's Grade, Tumour size and ECT2 expression were all significantly associated with the recurrence of HCC.
  • Multivariate survival analysis using the Cox's regression model also showed that ECT2 expression, Child's Grade, and Tumour size were statistically significant factors for identifying HCC recurrence.
  • ECT2 expression had a P- value of 0.000, suggesting that it could serve as an independent survival prognostic factor for HCC.
  • ECT2 promotes the proliferative, oncogenic and tumourigenic activities of HOC
  • ECT2 strongly increased the level of apoptosis-induced cleaved Caspase-3 as well as p-Ser139 H2AX (a marker for DNA damage), and decreased levels of pro-caspase-9 and PARP-1 compared to incubation with siScramble or siLuciferase control at 72 h post-siRNA transfection (Fig. 2B).
  • the percentage of TUNEL-positive cells was also markedly increased after silencing ECT2 (P ⁇ 0.001 ) (Fig. 2C).
  • ECT2 knockdown HuH- 7, Hep3B, and HCCLM3 cell lines was generated (Fig. 5A).
  • the soft agar colony formation assay was employed to evaluate the oncogenic potential of these cells. It was found that stable ECT2 knockdown clones showed significantly attenuated colony formation ability compared to siScramble knockdown clones in the HCC cell lines studied (Fig. 2D). In addition, stable ECT2 knockdown cells also exhibited delayed cell growth kinetics compared to the controls (Fig. 5B-D).
  • tumours developed in mice with clones derived from stable ECT2-knockdown cell lines were significantly smaller than from corresponding clones derived from the control siScramble-knockdown cell lines (Fig. 2E), indicating that ECT2 can function as an oncogenic driver in HCC.
  • ECT2 has been reported to be an important cofactor for Rho GEF and to regulate cell migration (Morita et ai, 2005).
  • siRNA specific for ECT2 Fig. 2A
  • silencing the expression of ECT2 in both Hep3B and HCCLM3 significantly suppressed their ability to migrate and invade P ⁇ 0.001 , Fig. 2F.
  • the migratory ability of clones derived from stable ECT2 knockdown HCCLM3 cells was significantly attenuated when studied by scratch wound healing assays (P ⁇ 0.05, Figs. 2G). Knockdown of ECT2 impairs the activation of Rho/ERK signals
  • ECT2 in HCCLM3 cells impaired the activity of Cdc42, Rac1 , and Rho when compared to siScramble- and GTPvS-treated siScramble controls (Fig. 3A).
  • Fig. 3A To identify potential downstream signalling targets of ECT2 and Rho, we screened human phospho-kinase antibody arrays following treatment of HCCLM3 cells with siRNA specific for ECT2.
  • P-ERK1/2 was markedly suppressed compared to the reference control (R) after silencing ECT2 (Fig. 3B), suggesting that ERK kinase might be an important downstream target of ECT2-Rho in HCC.
  • the mouse orthotopic xenograft tumour model with HCCLM3 cells was employed. Western blot and IHC assays were performed. Stable depletion of ECT2 was detected in the shECT2 tumours (Figs. 3G, H). Moreover, the depletion of ECT2 resulted in a reduction in p-ERK1/2 expression in the HCCLM3-derived tumours compared with the shScramble controls (Figs. 3G, H).
  • ECT2 may promote metastasis by regulating RhoA activation, and to identify its potential interacting transcription co-factors.
  • RACGAP1 was identified as a potential cofactor that interacted with ECT2 (Fig. 4A).
  • HCCLM3 cells it was demonstrated that ECT2 and RACGAP1 could be precipitated together as one complex (Fig. 4B). While silencing ECT2 expression alone could suppress the GTP-binding activity of RhoA (P ⁇ 0.01 ; Fig. 4C), silencing RACGAP1 alone did not suppress the GTP-binding activity of RhoA.
  • ECT2 and RACGAP1 could be localized to the mitotic spindle and midbody of HCC cells during mitosis (Fig. 4E).
  • ECT2 and RACGAP1 were mainly localised in the nucleus (Fig. 4E).
  • Fig. 4F the expression of ECT2 colocalized with RACGAP1 in both interphase and mitosis
  • Duolink assays provide precise detection and quantification of protein-protein complex interactions.
  • many Duolink + complexes could be detected in interphase and mitotic cells, confirming the physical interaction of ECT2 and RACGAP1 (Fig. 4G).
  • the detection of Duolink + complexes in mitotic cells P ⁇ 0.001 ; Fig. 4G) would suggest that ECT2 and RACGAP1 function as a single complex throughout the cell cycle.
  • ECT2 functionally interacts with RACGAP1 and regulates the stability of RACGAP1
  • ECT2 Epithelial cell transforming sequence 2
  • GEF guanine nucleotide exchange
  • ECT2 has been described in several human cancers including lung, oesophageal and glioblastoma (Tatsumoto et al., 1999; Cook et al., 2013; Hirata et al., 2009; Justilien and Fields, 2009 and Roversi et al., 2006).
  • ECT2 has also been shown to be negatively regulated by wild-type p53 expression via protein methyltransferases in human non-small cell lung carcinoma H1299 and breast cancer MCF7 cells (Scoumanne et al., 2006).
  • ECT2 In non-small-cell lung cancer (NSCLC), ECT2 was shown to be associated with Rac1 activation leading to ECT2-dependent NSCLC anchorage-independent growth and invasion in vitro (Justilien and Fields, 2009). In addition, ECT2 has also been reported to interact with RacGAPI in cytokinesis (Matthews et al., 2012 and Yuce et al., 2005) and to be involved in epithelial cell polarity and migration (Cook et al., 2013; Liu et al., 2004 and Morita er a/., 2005.
  • ECT2 Up-regulation of ECT2 has been reported in several types of human cancer (Hirata et al., 2009; Justilien and Fields, 2009 and Roversi et al., 2006). However, little is known about the molecular role of ECT2 in HCC.
  • Rho GTPases are some of the most upstream regulators of the assembly of the contractile ring in the context of cytokinesis that takes place as part of a cell cycle. Rho GTPases can be activated by GEFs, and ECT2 is one of the most important factors catalyzing GEFs (Tatsumoto et al., 1999). These results showed that ECT2 could activate Rho, Cdc42, and Rac1 while silencing ECT2 markedly suppressed the activation of ERK-AKT and FAK-Tyr925 phosphorylation, which might induce Ras-dependent activation of the MAP kinase pathway in HCC cells.
  • RACGAP1 can be an independent predictor for early recurrent HCC disease (Wang et al., 201 1 ). It is therefore interesting to observe that the expression of ECT2 was significantly correlated with RACGAP1 expression. Phosphorylation of RACGAP1 recruits ECT2 to the central spindle to promote Rho-dependent cytokinesis (Yuce et al., 2005 and Wolfe et al., 2009). In this study, it was demonstrated the physical and functional interactions between ECT2 and RACGAP1 to promote ECT2- mediated HCC tumour cell metastasis. It was also subsequently demonstrated that ECT2 can modulate the expression of RACGAP1 by regulating its protein stability. It is likely that ECT2-GEF function could be dependent on additional factors working in the fashion of an interactome to promote early recurrent HCC disease.
  • ECT2 was significantly up-regulated in early recurrent HCC tumours. It has been reported that a high frequency of p53 mutations was associated with poorly or moderately differentiated tumours with a high rate of recurrence (Kan et a/., 2013). It has also been reported that p53 is frequently mutated in HCC and that p53 overexpression could decrease ECT2 expression in breast cancer cells Scoumanne et a/., 2006 and Kan er a/., 2013).
  • the HCC cell lines SK-Hep1 and HepG2 express wild-type p53 endogenously (Puisieux et a/., 1993 and Hsu et al., 1993).
  • ECT2 can interact with different cellular molecules to promote early recurrent HCC disease and could therefore serve as a potential candidate for designing novel therapeutic strategies in the clinical management of HCC. Summary of example
  • HCC recurrence is mainly responsible for the poor overall survival of patients with HCC and is the major obstacle to improving prognosis. It is therefore desirable to identify molecules that contribute to HCC recurrence and could provide potential targets for novel therapeutic strategies for the clinical management of HCC.
  • ECT2 Gene and protein expression profiles of ECT2 were analyzed by microarrays, immnoblotting and immunohistochemistry in human HCC samples. siRNA and lentivirus-based gene knockdown were employed to dissect the molecular functions of ECT2 in vitro and in vivo.
  • ECT2 up-regulation of ECT2 is significantly associated with early recurrent HCC disease and poor survival. Knockdown of ECT2 markedly suppressed the activation of Rho GTPases and induced apoptosis, attenuated oncogenicity and reduced the ability of HCC cells to metastasize. Moreover, knockdown of ECT2 or RHO also suppressed ERK activation while the silencing of RHO or ERK could lead to a marked reduction in cell migration. Stable knockdown of ECT2 in vivo resulted in significant retardation of tumour growth and the suppression of ERK activation. A high-level of gene expression of ECT2 correlated with high ERK phosphorylation and poor survival of HCC patients.
  • ECT2 enhances the expression and stability of RACGAP1 , accelerating ECT2-mediated Rho activation to promote metastasis.
  • ECT2 is closely associated with activation of the Rho/ERK signalling axis to promote early HCC recurrence.
  • ECT2 can crosstalk with RACGAP1 to catalyse the GTP exchange involved in Rho signalling to further regulate tumour initiation and metastasis.
  • Ect2 links the PKCi-Par6a complex to Racl activation and cellular transformation.
  • Polo-like kinase 1 directs assembly of HsCyk-4 RhoGAP/Ect2 RhoGEF complex to initiate cleavage furrow formation.
  • MicroRNA-216a/217-induced epithelialmesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer.

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Abstract

La présente invention concerne un procédé permettant de profiler un carcinome hépatocellulaire comprenant la classification de l'expression génétique Ieveis de séquence 2 de transformation de cellules épithéliales (ECT2) et/ou l'activité de signalisation de ECT2/RHO/ERK dans des échantillons de foie de CHC par rapport à un témoin. Un sujet présentant de plus grands niveaux d'expression de ECT2 et/ou une plus grande activité de signalisation de ECT2/RHO/ERK est susceptible d'avoir une plus grande récurrence du CHC. La présente invention consiste également à surveiller et à traiter un CHC avec des inhibiteurs de séquence 2 de transformation de cellules épithéliales tels que l'ARNsi ou des anticorps, en particulier chez des sujets présentant une grande expression de ECT2 par rapport à un tissu hépatique normal.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018107011A1 (fr) * 2016-12-08 2018-06-14 City Of Hope Vaccins ciblant p53 et inhibiteurs de la voie pd -1 et leurs procédés d'utilisation
CN110741098A (zh) * 2017-05-29 2020-01-31 朱拉蓬基金会 预测患有肝细胞癌(hcc)或胆管癌(cca)的受试者的生存结果的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004108964A1 (fr) * 2003-06-04 2004-12-16 Agency For Science, Technology And Research Genes de carcinome hepatocellulaire a regulation differentielle et leurs utilisations
US20090181394A1 (en) * 2008-01-15 2009-07-16 Catholic University Industry Academy Cooperation Foundation Diagnostic methods and kits for hepatocellular carcinoma using comparative genomic hybridization
US20090181091A1 (en) * 2007-08-27 2009-07-16 Banerjee Abhijit G Product and process to regulate a gene network involved in constitutive inflammation and early cancer progression
WO2010007791A1 (fr) * 2008-07-16 2010-01-21 Oncotherapy Science, Inc. Oncogène ect2 comme cible thérapeutique et indicateur de pronostic pour le cancer du poumon et de l'œsophage
WO2011062563A1 (fr) * 2009-11-23 2011-05-26 Singapore Health Services Pte Ltd Modulateurs de progression de cycle cellulaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004108964A1 (fr) * 2003-06-04 2004-12-16 Agency For Science, Technology And Research Genes de carcinome hepatocellulaire a regulation differentielle et leurs utilisations
US20090181091A1 (en) * 2007-08-27 2009-07-16 Banerjee Abhijit G Product and process to regulate a gene network involved in constitutive inflammation and early cancer progression
US20090181394A1 (en) * 2008-01-15 2009-07-16 Catholic University Industry Academy Cooperation Foundation Diagnostic methods and kits for hepatocellular carcinoma using comparative genomic hybridization
WO2010007791A1 (fr) * 2008-07-16 2010-01-21 Oncotherapy Science, Inc. Oncogène ect2 comme cible thérapeutique et indicateur de pronostic pour le cancer du poumon et de l'œsophage
WO2011062563A1 (fr) * 2009-11-23 2011-05-26 Singapore Health Services Pte Ltd Modulateurs de progression de cycle cellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN J. ET AL.: "ECT2 Regulates The Rho/ERK Signalling Axis To Promote Early Recurrence In Human Hepatocellular Carcinoma.", JOUMAL OF HEPATOLOGY, vol. 62, no. 6, 21 January 2015 (2015-01-21), pages 1287 - 1295, XP029173074, [retrieved on 20151123], DOI: doi:10.1016/j.jhep.2015.01.014 *
FIELDS A.P. ET AL.: "The Guanine Nucleotide Exchange Factor (GEF) Ect2 Is An Oncogene In Human Cancer.", ADV. ENZYME REGUL., vol. 50, no. 1, 2010, pages 190 - 200, XP027035214, [retrieved on 20151123] *
WANG S.M. ET AL.: "Upregulation of Rac GTPase-activating Protein 1 Is Significantly Associated With The Early Recurrence Of Human Hepatocellular Carcinoma.", CLINICAL CANCER RESEARCH, vol. 17, no. 18, 8 August 2011 (2011-08-08), pages 6040 - 6051, [retrieved on 20151123] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018107011A1 (fr) * 2016-12-08 2018-06-14 City Of Hope Vaccins ciblant p53 et inhibiteurs de la voie pd -1 et leurs procédés d'utilisation
US11602554B2 (en) 2016-12-08 2023-03-14 City Of Hope P53-targeting vaccines and pd-1 pathway inhibitors and methods of use thereof
CN110741098A (zh) * 2017-05-29 2020-01-31 朱拉蓬基金会 预测患有肝细胞癌(hcc)或胆管癌(cca)的受试者的生存结果的方法

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