Volume 130, Issue 7, Pages 2130-2144 (June 2006) SULF1 Inhibits Tumor Growth and Potentiates the Effects of Histone Deacetylase Inhibitors in Hepatocellular Carcinoma  Jin–Ping Lai, Chunrong Yu, Catherine D. Moser, Ileana Aderca, Tao Han, Thomas D. Garvey, Linda M. Murphy, Megan M. Garrity–Park, Viji Shridhar, Alex A. Adjei, Lewis R. Roberts  Gastroenterology  Volume 130, Issue 7, Pages 2130-2144 (June 2006) DOI: 10.1053/j.gastro.2006.02.056 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Forced expression of SULF1 significantly delays growth of HCC xenografts in nude mice. (A and B) High level of SULF1 expression was detected in Huh7 and Hep3B clones stably transfected with SULF1 (Huh7 SULF1-5 and Hep3B SULF1-5), using real time RT-PCR. (C and D) Two million Huh7 (C) or Hep3B (D) cells stably transfected with empty plasmid vector (vector, injected into the left side of the mouse in C and the right side of the mouse in D) or with a plasmid expressing the full-length SULF1 mRNA (SULF1, injected into the right side of the mouse in C and the left side of the mouse in D) were inoculated subcutaneously into the flanks of nude mice and the resulting xenografts photographed on the 30th day (Huh7) and 70th day (Hep3B) after inoculation. The graphs show the results from xenografts implanted into 10 nude mice in each case; there was profound inhibition of growth of both Huh7 and Hep3B xenografts induced by expression of SULF1. (E and F) To allow longer observation of nude mice bearing tumors expressing SULF1, which grow more slowly than tumors from vector-transfected cells, cells stably transfected with vector or SULF1 expressing plasmids were inoculated into the right flanks of separate sets of nude mice and the tumors were measured every 3 days. Mice were censored when TV reached 2,000 mm3. Differences between the Kaplan–Meier curves were analyzed using the log-rank test. (G) Immunohistochemistry was performed to confirm human SULF1 protein expression in the xenografts from cell lines stably transfected with SULF1 using a rabbit polyclonal antibody against SULF1. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Forced expression of SULF1 increases the acetylation of histone H4 in HCC cells. (A) The levels of both acetylated histone H4 and H3 were detected in 6 HCC cell lines by Western immunoblotting. There is a low level of acetylated histone H4 in 5 of the 6 HCC cell lines. Acetylated histone H4 and H3 levels in Huh7 cells treated with TSA are shown as positive controls. (B) Compared to cells stably transfected with the empty vector, Huh7 and Hep3B cells stably transfected with SULF1 expressing plasmids express higher levels of SULF1 mRNA by RT-PCR and also show increased acetylated histone H4 levels by Western immunoblotting. GAPDH (for RT-PCR) and Actin (for Westerns) are shown as controls for equal loading. (C) Transient transfection of a plasmid expressing SULF1 shRNA into a cell line stably transfected with a SULF1 plasmid decreases SULF1 expression and down-regulates acetylation of histone H4. (D) Huh7 cells transfected either with a Flag-tagged vector or Flag-tagged SULF1 expressing plasmid were immunostained with both anti-Flag and anti-acetylated histone H4 antibodies and imaged by confocal microscopy. SULF1 expression leads to increased acetylated histone H4 in the cell nuclei. Nuclei are shown counterstained with the DNA binding fluorescent dye, DAPI. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 (A) SULF1 enhances apicidin-induced acetylation of histone H4. Stably transfected clones of Huh7 and Hep3B cells with or without SULF1 expression were treated with 0 or 2.5 μmol of apicidin for 24 hours. Whole cell lysates were used for Western immunoblotting using antibodies recognizing acetylated histone H4 and H3; total histone H4 and actin were used as loading controls. (B and C) HDAC activity was measured in Huh7 and Hep3B clones. Apicidin decreases HDAC activity in both Huh7 and Hep3B cells (P < .01). SULF1 expression decreases HDAC activity in Hep3B cells (P < .05) but does not change HDAC activity in Huh7 cells. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 SULF1 increases HAT expression and activity in Huh7 cells. (A) Whole cell lysates from Huh7 vector and Huh7 SULF1-5 cells were used in Western immunoblotting using antibodies recognizing p300 and CBP; actin was used as a loading control. (B) HAT activity was measured in Huh7 cells with or without SULF1 expression. SULF1 increased HAT activity in Huh7 cells (P < .05). (C) Huh7 SULF1-5 cells were transiently transfected with either SULF1 shRNA or its empty vector pSS-H1p. SULF1 shRNA abolished the SULF1-induced increase in HAT activity in Huh7 cells (P < .05). Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 SULF1 enhances HDAC inhibitor-induced apoptosis in HCC cells. Apoptotic cells were identified by fluorescence microscopy after staining with DAPI (A and B) or annexin V (C and D). Quantitation of apoptosis 48 hours after treatment with 2.5 μmol apicidin or 2 μL 10% DMSO was recorded in the indicated vector or SULF1-transfected Huh7 (A and C) and Hep3B clones (B and D). SULF1 increased apicidin-induced apoptosis in both Huh7 SULF1 clones (P < .05) and Hep3B SULF1 clones (P < .05) as compared with the corresponding vector clones. (E and F) Five thousand cells per well of Huh7 vector or Huh7 SULF1-5 clones (E) and Hep3B vector or Hep3B SULF1-5 clones (F) were plated into 96-well plates and treated with either 0.1% DMSO diluent or 2.5 μmol apicidin. Combined caspase 3 and 7 activity expressed as relative fluorescence units (RFU) was measured 24 hours after treatment. SULF1 enhanced apicidin-induced caspase 3 and 7 activity in both Huh7 (P < .001) and Hep3B cells (P < .05). (G and H) Huh7 vector and Huh7 SULF1-5 cells were treated with or without 1.0 μg/mL scriptaid for 24 hours. Apoptosis was evaluated after DAPI staining (P < .05). Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Knockdown of SULF1 up-regulates phospho-AKT and MAPK kinase and attenuates apicidin-induced apoptosis. Hep3B SULF1-5 cells were transiently transfected with either SULF1 shRNA or its corresponding empty vector. Cells were treated with 0 or 2.5 μmol apicidin for 48 hours. Whole cell lysates were used in Western immunoblotting against phospho-MEK, -ERK, and -AKT; total ERK and AKT were used as loading controls (A). In parallel experiments, apicidin-induced apoptosis was quantitated for the Hep3B (B; P < .001) and Huh7 clones (C; P < .05). Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Inactivation of ERK and PI3K/AKT occurs downstream of histone H4 acetylation. Huh7 cell were treated with 0.5 μmol of apicidin, 20 μmol of U0126, 20 μmol of LY294002 alone and in combination as indicated. Whole cell lysates (20 μg/lane) were loaded for Western immunoblotting for acetylated histone H4, phospho-ERK and phospho-AKT; total ERK was used as the loading control. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 SULF1 enhances apicidin-induced inhibition of HCC cell migration. Hep3B and Huh7 clones with or without SULF1 expression were treated with 2.5 μmol apicidin, and their migration was evaluated using a wound assay. The wounds were recorded when they were made and at 24 hours thereafter. Both apicidin and SULF1 decreased migration in Hep3B (A and B) and Huh7 cells (C). SULF1 expression enhanced the effect of apicidin on HCC cell migration (P < .05 for both cell lines). Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 9 SULF1 enhances the efficacy of apicidin in growth inhibition of HCC xenografts. Both Huh7 and Hep3B xenografts with or without SULF1 expression were generated. When the volume of xenografts reached 400–800 mm3, the nude mice were grouped randomly and treated with 100 μL 10% DMSO for controls and 100 μL of apicidin dissolved in 10% DMSO at a total dose of 2.5 mg/kg body weight. The mice were treated IP daily for 1 week, every other day for 1 week, and every 3 days for 2 more weeks. Tumor size was measured with calipers every 3 days and the mice were sacrificed when their TV reached 4,000 mm3. The combination of SULF1 and apicidin inhibits tumor growth in mice bearing Huh7 (P < .001) (A and C) and Hep3B (P < .05) (B and D) xenografts. The in vivo experiments were repeated and the mice were sacrificed at the end of the fourth week’s treatment. Different gross appearances of the resected tumors from each group were photographed (E). The tumors were fixed in 10% formalin and embedded in paraffin. After staining with hematoxylin and eosin (F), the vessel area (filled with red blood cells) as a percent of total tissue area was quantitated (G). A large number of vessels were present in control vector xenografts treated with DMSO; fewer vessels were seen in samples of either SULF1-expressing xenografts or vector xenografts treated with apicidin. The combination of SULF1 and apicidin resulted in necrosis and in very few vessels visible in the xenografts. T, tumor; V, vessel; N, necrosis. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 10 Working model for the effect of SULF1 and HDAC inhibitors on histone acetylation and downstream pathways. Forced expression of SULF1 disrupts the balance between HDACs and HATs by inhibiting HDACs in some cell lines and by up-regulating HAT activity in other cell lines. This leads to an increase in the acetylation of histone H4 and to subsequent down-regulation of AKT and MAPK kinase pathways. Therefore, SULF1, potentiates the effects of HDAC inhibitors both in vitro and in vivo. Gastroenterology 2006 130, 2130-2144DOI: (10.1053/j.gastro.2006.02.056) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions