RRM2B inhibits cell migration and spreading by Egr-1-mediated PTEN/Akt1

Transcription

1 RRM2B inhibits cell migration and spreading by Egr-1-mediated PTEN/Akt1 pathway in hepatocellular carcinoma Hua Tian 1, Chao Ge 1, Hong Li 1, Fangyu Zhao 1, Helei Hou 1, Taoyang Chen 2,5, Guoping Jiang 3,5, Haiyang Xie 3,5, Ying Cui 4,5, Ming Yao 1, Jinjun Li 1*. 1 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2 Qi Dong Liver Cancer Institute, Qi Dong, Jiangsu Province, China 3 Department of General Surgery, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 4 Cancer Institute of Guangxi, Nanning, China 5 These authors contributed equally to this work. * Correspondence: jjli@shsci.org Supporting information Supporting Materials and Methods Immunohistochemistry All of the tumors were confirmed by compatible histopathology. None of the patients underwent chemotherapy or radiotherapy before surgery. All of the patients gave consent for the use of their tumor tissues in the study. The University Ethical Committee approved the collection of fresh tumor tissue samples for clinical analysis. 1

2 Immunohistochemistry, signal evaluation, and statistical data analysis were performed as described previously. HCC tissues were sectioned in 4-μm-thick slices and were dewaxed in xylene and rehydrated using 95% ethanol. The endogenous peroxidase activity was quenched by immersing the slides in a 0.3% H 2 O 2 solution for 30 min at room temperature. Sections were incubated for 1 h at 37 C with primary antibodies. Negative controls were prepared by using the same isotype IgG instead of the primary antibodies. The horseradish peroxidase (HRP)-conjugated secondary antibody was applied at 37 C for 30 min and incubated with diaminobenzidine solution, and nuclei were then counterstained with Mayer s hematoxylin. In addition, incomplete data is due to various combined factors in the collection process (1 of 236 patients' information on age was missing. 7 of 236 patients' information on tumor size was missing. 4 of 236 patients' information on AFP was missing. 7 of 236 patients' information on HBV was missing.). Thus, these cases were excluded in our Table 1 and supporting Table 4. Immunostaining of each HCC case in the tissue microarray was compared to the matched adjacent liver tissue by semiquantitative scores. Immunohistochemical scores were obtained as follows. The intensity of staining was categorized into 0, 1 or 2, denoting negative or weakly stained, moderate, and strong staining, respectively. The extent of immunostaining was categorized into 0, 1 or 2, denoting less than 5% scope expression, 6-50% scope expression, and more than 50% scope expression, respectively. The scores were evaluated with three random microscope fields per 2

3 tissue. The sum of the products of intensity and extent of staining was used as score of the expression level. Cell lines and transfections Huh7 cells were obtained from Riken Cell Bank (Tsukuba, Japan). SMMC-7721 cells were obtained from the cell bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). MHCC-97L cells were provided by the Liver Cancer Institute of Zhongshan Hospital of Fudan University (Shanghai, China). SMMC-7721, SMMC-97L and Huh-7 were maintained in Dulbecco s modified Eagle s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 C in 5% CO 2. Human primary HCC-LY10 cell lines established in our laboratory were cultured in Williams' Medium E supplemented with 10% FBS. Standard transient transfections for all cell lines were conducted using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to manufacturer s directions. Vector constructs To generate stable RRM2B-overexpressing cell lines, the RRM2B ORF (open reading frame) sequence was PCR amplified using specific primers (forward: 5'- CGACGCGTATGGGCGACCCGGAAAGG-3', and reverse: 5'- GGAATTCCATATGTTAAAAATCTGCATCCAAGG-3') and cloned into the lentiviral expression vector pwpxl (Addgene). To generate stable knockdown cell lines, RRM2B, Akt1 and N-cadherin shrna vectors were constructed. Two target 3

4 sequences of RRM2B were selected: 5 -GAACAAGCTTAAAGCAGAT -3 ; and 5 - TGAGTTTGTAGCTGACAGA-3. Two target sequences of Akt1 were selected: 5 -CGAGGGGAGTACATCAAGA -3 ; and 5 - AGATGACAGCATGGAGTGT-3. Two target sequences of N-cadherin were selected: 5 - GCTACAGACATGGAAGGCA -3 ; and 5 -GACTGGATTTCCTGAAGAT-3. A shrna with a non-targeting sequence (scrambled sequence) was used as a negative control (shnc) in our experiment. The sequences of scrambled were TTCTCCGAACGTGTCACGTT. These sequences were synthesized and inserted into the plvthm vector (Addgene) in accordance with the manufacturer's instructions. The PTEN and RRM2B promoter were generated by PCR and cloned into the luciferase reporter gene vector, pgl3-enhancer (Promega, Madison WI). The fidelities of the constructs were confirmed by sequencing. RRM2B promoter bearing Egr-1 binding sites was mutated using the mutagenic primers which consist of the substitutions at the core sequence of Egr-1 binding sites by site directed mutagenesis. The sequences of mutagenic primers were CCCTTAGGCCGCAGGCGTTAAGGCTTGGCTGGCCGAAGTTAG. The colonies obtained were verified by sequencing for desired mutations. Lentivirus production and cell transduction Viral packaging was performed in HEK 293T cells after co-transfection of the pwpxl-rrm2b vector or the plvthm-shrrm2b vector with the packaging plasmid pspax2 and the envelope plasmid pmd2.g (Addgene) using Lipofectamine 4

5 2000 (Invitrogen).Viruses were harvested 72 h after transfection, and viral titers were determined. Target cells, including Huh7, SMMC-7721, HCC-LY10 and MHCC-97L cells, were infected with recombinant lentivirus-transducing units in the presence of 6 μg/ml polybrene (Sigma). Cell growth assay Cell proliferation was determined by the MTT assay. Briefly, cells were plated in 96-well plates at 5,000 cells per well. After 24 h in culture, 10 μl of MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/ml, Sigma) was added to each well and incubated for another 4 h at 37 C. Cells were then incubated for 15 minutes at 37 C, followed by the addition of 100 μl DMSO to each well. The absorbance was measured at 570 nm using an ELISA microplate reader. Colony formation assay Two thousand cells were placed in a fresh six-well plate and maintained in DMEM containing 10% FBS for 2 weeks. Colonies were fixed with 4% phosphate-buffered formalin (ph 7.4) and Giemsa stained for 15 min. Immunofluorescent confocal imaging Cells were seeded onto glass slides for 24 h, fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 15 minutes. The slides were then incubated with primary antibody in blocking solution overnight at 4 C in a humidified chamber. 5

6 The glass slides were then washed three times in PBS and incubated in Alexa 594-conjugated secondary antibody and 4 6-diamino-2-phenylindole (DAPI) in blocking solution for 60 minutes at 37 C in a humidified chamber. Images were obtained with a confocal laser microscope (IX-70, Olympus 60M, Olympus, London, UK). 6

7 Supporting Figures and Figure legends Supporting Figure 1 Specificity of RRM2B antibody in IHC. RRM2B overexpression SMMC-7721 cells were incubated with RRM2B antibody (ab8105) (A) or the same isotype IgG (B) and detected by immunocytochemical staining. HCC tissue was incubated with RRM2B antibody (sc-10840) (C) or the same isotype IgG (D) detected by immunohistochemistry. 7

8 Supporting Figure 2 The expression of RRM2B and RRM2 in HCC tissues. (A) RRM2B protein expression in 236 pairs of non-tumor and tumor liver tissues of human HCC based on the results of immunohistochemistry. Of the 236 pairs, 138 (58.5%) had higher RRM2 protein expression in non-tumor tissues than in tumor liver tissues (I), 75 (31.8%) had similar expression (II), whereas only 23 (9.7%) had lower expression (III). Therefore, RRM2B expression was frequently down-regulated in HCC. (B) Representative images of IHC staining for RRM2B in HCC tissue. (C) Representative images of IHC staining for RRM2 in noncancerous liver tissue and HCC tissue, as detected by immunohistochemistry. (D) The expression of RRM2 mrna in noncancerous liver tissue (N) and cancer tissue (T) were detected by qrt-pcr in 15 cases tissue samples. 8

9 Supporting Figure 3 The expression of p53 and RRM2B in HCC cell lines was detected by Western blotting. 9

10 Supporting Figure 4 Representative image of p53 gene point mutation in HCC tissues. The exon 7 of p53 gene is the mutation hotspot in HCC. A rough estimate of the p53 mutation is detected in the exon 7 of p53 gene by sequencing. Therefore, we detected the exon 7 of p53 gene in HCC by sequencing. The results may represent the state of the p53 gene in HCC. The mutation of p53 gene exon 7 was detected by sequencing in 68 cases of HCC tissue samples. Sequence analysis confirmed that there was a AGG AGT point mutation (Arg Ser) at codon 249 of the p53 gene exon 7 in HCC tissues. The results showed the frequency of p53 mutations was approximately 29.4% (20/68) in HCC. Furthermore, the results of DNA sequence showed that the positive immunohistochemical staining of p53 was derived from mutant p53 rather than wild-type p53. 10

11 Supporting Figure 5 Functional effects of RRM2B on HCC cell migration and invasion in vitro. (A) The in vitro migration and invasion potentials of SMMC-7721, HCC-LY10 and MHCC-97L cells transfected with shrrm2b-1 or shrrm2b-2 were detected and analyzed. The migrated (B) and invaded (C) MHCC-97L cells were stained, counted, and analyzed using a Student's t-test. *, p<0.01 (D) The in vitro migration and invasion potentials of Huh7, SMMC-7721 and MHCC-97L cells stably transfected with RRM2B were detected and analyzed. Original magnification

12 Supporting Figure 6 RRM2B inhibits the proliferation of Huh7 cells. (A) Huh7 cells were stably transfected with RRM2B constructs as indicated and the expression of RRM2B was detected by Western blotting. (B) Huh7 cell lines stably expressing RRM2B constructs were seeded into 6-well plates. Cell proliferation was determined by colony formation assay 12

13 Supporting Figure 7 RRM2B regulates the expression of E-cadherin, N-cadherin and slug in MHCC-97L cells. (A) The expression of RRM2B, E-cadherin, N-cadherin and slug was detected by Western Blotting in MHCC-97L cells stably transfected with shrrm2b-1, shrrm2b-1 or shnc.(b) The expression of RRM2B, E-cadherin, N-cadherin and slug was detected by Western Blotting in MHCC-97L cells stably transfected with RRM2B or control vector. (C) E-cadherin and N-cadherin protein expression and subcellular localization were determined by immunofluorescence in SMMC-7721, MHCC-97L and Huh7 stably transfected with shrrm2b-1, shnc, RRM2B or control vector. Red staining indicates E-cadherin or N-cadherin. DAPI staining (blue) indicates the nuclei. 13

14 Supporting Figure 8 RRM2B inhibit CD44 expression in HCC cells. (A) The relative mrna expression of CD44, CD133, Notch1, Nanog and Oct4 was detected by qrt-pcr in SMMC-7721 and Huh7 cells stably transfected with RRM2B or shrrm2b. (B) The expression of CD44 and CD133 was detected by Western blotting in SMMC-7721 and Huh7 cells stably transfected with RRM2B or shrrm2b. 14

15 Supporting Figure 9 LY and wortmannin inhibit Akt1 activition and reduce cell migration and invasion in vitro. The expression of Akt1, p-akt, N-cadherin and E-cadherin was measured using Western blotting in SMMC-7721 (A) and HCC-LY10 (B) cells treated with 10 μm LY (LY), 1 μm wortmannin (Wort) or DMSO only for 12 h. SMMC-7721 (C) and HCC-LY10 (D) cells were treated with 10 μm LY (LY), 1 μm wortmannin (Wort) or DMSO and then migration and invasion of cells were measured by transwell/matrigel-coated transwell assays.*, p<

16 Supporting Figure 10 The Knockdown of N-cadherin expression reduces cell migration and invasion in SMMC-7721-shRRM2B cells. (A) SMMC-7721-shRRM2B cells were transfected with N-cadherin shrna as indicated, the expression of p-akt1, Akt1 and N-cadherin was detected by Western blotting. (B) SMMC-7721 cells were treated with N-cadherin shrna-1 or shrna-2 and expression of N-cadherin and RRM2B was detected by Western blotting. (C) SMMC-7721-shRRM2B cells were treated with N-cadherin shrna-1 or shrna-2 and migration and invasion of cells were measured with transwell and Matrigel-coated transwell assays. Representative Giemsa s staining images were shown (C). Original magnification 200. (D) The data are presented as the mean ± SDs (from five random 200 magnification fields). *P<

17 Supporting Figure 11 p65, HSF, GATA-3, VDR, c/ebpb and GATA-2 don t inhibit RRM2B promoter activity. (A) SMMC-7721 and Huh7 cells were transfected with luciferase reporter vectors containing different truncations of the RRM2B promoter:-1181 bp, bp,-896 bp,-763 bp, -375 bp, -234 bp or -163 bp relatively to the ATG. Corresponding relative luciferase activities were determined by reporter gene assay. (B) SMMC-7721 cells were cotransfected with luciferase reporter vectors containing RRM2B promoter (-1181/-5 bp) and p65, HSF, GATA-3, VDR, c/ebpb and GATA-2 or control. Corresponding relative luciferase activities were determined by reporter gene assay. (C) SMMC-7721 cells were transfected with p65, HSF, GATA-3, VDR, c/ebpb and GATA-2 or control vector, the expression of RRM2B was detected by Western blotting. (D) Sequencing results of RRM2B luciferase reporter vector (mutant Egr-1 binding sites, -375/-5). The Egr-1 binding sites were mutated at GGGGAGGCGGGGC, where underlined nucleotides represent point mutations. 17

18 Supporting tables Supporting Table 1 Antibodies used in this study Antibody Clone, host Dilution Company For Western blotting RRM2B N-16, goat polyclonal 1:500 Santa Cruz E-cadherin H-108, rabbit polyclonal 1:400 Santa Cruz N-cadherin H-63, rabbit polyclonal 1:400 Santa Cruz slug C19G7, rabbit mab 1:400 CST p-akt1 Ser473, rabbit polyclonal 1:500 CST Akt1 C67E7, rabbit mab 1:500 CST PTEN 138G6, rabbit mab 1:500 CST Egr-1 C19,rabbit polyclonal 1:400 Santa Cruz CD44 DF1485, mouse IgG1 1:300 Santa Cruz CD133 W6B3C1,mouse IgG1 1:100 MACS β-actin AC-15, mouse mab 1:20000 Sigma Secondary antibody HRP conjugated goat anti-rabbit 1:4000 Sigma IgG Secondary antibody HRP conjugated goat anti-mouse IgG 1:4000 Sigma For Immunohistochemistry RRM2B Ab8105, rabbit polyclonal 1:100 Abcam RRM2 SS-22 1:50 Santa Cruz p53 PAb 1801, mouse mab 1:50 Thermo E-cadherin NCH-38, mouse mab 1:50 DAKO N-cadherin 6G11, mouse mab 1:25 DAKO Secondary antibody Envision kit (HRP, rabbit/mouse, DAB+) Ready-touse DAKO For Immunofluorescence staining E-cadherin H-108, rabbit polyclonal antibody 1:50 Santa Cruz N-cadherin H-63, rabbit polyclonal antibody 1:50 Santa Cruz Secondary antibody Alexa Fluor 594 anti-rabbit IgG (Cat No. A-21207) 1:50 Invitrogen 18

19 Supporting Table 2. The sequences of gene-specific primers used for qrt-pcr and vector constructs Gene name Forward (5-3 ) Reverse (5-3 ) RRM1 TGGAGGAATTGGTGTTGCTG AAATGCCAAGGCTCCAGGT RRM2 CGCCGCTTTGTCATCTTCC CTATGCCATCGCTTGCTGC RRM2B AGGAGGTGCAGGTTCCAGAG CTGCTATCCATCGCAAGGC PTEN TTGTCATTATCCGCACGC ATACCAGGACCAGAGGAA ACC Egr-1 CTTCAACCCTCAGGCGGACA GGAAAAGCGGCCAGTATAG GT E-cadherin GCTCTTCCAGGAACCTCTGTG CAGCTTGAACCACCAGGGT A N-cadherin GAGCTTGTCAGGATCAGGTCT G AATGTCAATGGGGTTCTCC AC CD44 CCATCCCAGACGAAGACAGT GGTTGTGTTTGCTCCACCT T CD133 TGGCAACAGCGATCAAGGAGA C TCGGGGTGGCATGCCTGTC ATA Notch1 CCTGAGGGCTTCAAAGTGTC CGGAACTTCTTGGTCTCCA G Oct4 CTTGCTGCAGAAGTGGGTGGA GGAA CTGCAGTGTGGGTTTCGGG CA Nanog AATACCTCAGCCTCCAGCAGA TG TGCGTCACACCATTGCTAT TCTTC GAPDH AGAAGGCTGGGGCTCATTTG AGGGGCCATCCACAGTCTT C RRM2B (-1181/-5) GGTCATGGCAAGATCCCGTC CAGACTCCGCCGAAGCTAC G RRM2B (-1025/-5) CCCGCATTCACACACACA CAGACTCCGCCGAAGCTAC G RRM2B (-896/-5) TCCCACAGCAACAGCGCG CAGACTCCGCCGAAGCTAC G RRM2B (-763/-5) CGCACCAAGTGGCTAGAG CAGACTCCGCCGAAGCTAC G RRM2B (-375/-5) CAGGGATAATCCCTTAGGCC CAGACTCCGCCGAAGCTAC G RRM2B (-234/-5) AGTTAGGCGGAGCCCCGA CAGACTCCGCCGAAGCTAC G RRM2B (-163/-5) GAGAAAGCAGGACCGGCG CAGACTCCGCCGAAGCTAC G PTEN (-1123/-779) GCATGCTCAGTAGAGCCT CCTCGCCTCACAGCGGCTC 19

20 Supporting Table 3. The sequences of sirna duplexes Identifier Forward (5-3 ) Reverse (5-3 ) Negative UUCUCCGAACGUGUCA ACGUGACACGUUCGGAGAATT Control CGUTT Egr-1-1 Egr-1-2 GCAGACAAAAGUGUUG UGGTT CGACAGCAGUCCCAUU UACTT CCACAACACUUUUGUCUGCTT GUAAAUGGGACUGCUGUCGTT 20

21 Supporting Table 4 Correlation between the expression of RRM2 and clinicopathological features of HCC tissues Clinicopathological Features RRM2 expression (cancer) Cases Score 0 Score 1 Score 2 P Value Cases (%) Cases (%) Cases (%) Age (years) < (73.6) 33(65.3) 34(58.6) (26.4) 19(34.7) 24(41.4) Gender male (72.2) 45(86.5) 54(93.1) 0.002** female 46 35(27.8) 7(13.5) 4(6.9) Tumor size 5cm (50.4) 23(45.1) 28(50.9) >5cm (49.6) 28(54.9) 27(49.1) AFP (35.5) 10(19.6) 25(43.9) 0.026* > (64.4) 41(80.4) 32(56.1) HBV negative 42 19(15.7) 7(14) 16(27.6) positive (84.3) 43(86) 42(72.4) Cirrhosis absent 38 19(15.1) 8(15.4) 11(19) present (84.9) 44(84.6) 47(81) Edmondson s grade I, II (47.6) 24(46.2) 35(60.3) III, IV (52.4) 28(53.8) 23(39.7) Intrahepatic metastasis # absent (69) 34(65.4) 40(69) present 75 39(31) 18(34.6) 18(31) P value represents the probability from a Chi-square test for different immunohistochemical scores of RRM2 in HCC tissues. *: p <0.05; **:p<

22 Supporting Table 5 Correlation between expression of RRM2B with or without p53 expression, and intrahepatic metastasis in HCC tissues Clinicopathological Intrahepatic metastasis features positive negative P value Cases (%) Cases (%) p53(low)/rrm2b (Low) 27(46.6) 60(48.4) p53(low)/rrm2b (High) 31(53.4) 64(51.6) p53(high)/rrm2b (Low) 3(16.7) 14(37.8) p53(high)/rrm2b (High) 15(83.3) 23(62.2) 22