Volume 143, Issue 4, Pages 1017-1026.e9 (October 2012) Increased Levels of Survivin, via Association With Heat Shock Protein 90, in Mucosal T Cells From Patients With Crohn's Disease  Heitor S.P. de Souza, Gail A. West, Nancy Rebert, Carol de la Motte, Judy Drazba, Claudio Fiocchi  Gastroenterology  Volume 143, Issue 4, Pages 1017-1026.e9 (October 2012) DOI: 10.1053/j.gastro.2012.06.039 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Distribution and quantification of survivin-expressing cells in colonic mucosa and colon cancer. (A) Survivin-positive cells (dark brown) in the crypts of normal colonic epithelium but not the lamina propria; marked increase of survivin-positive cells (arrows) in CD lamina propria; modest increase in survivin-positive cells (arrows) in UC lamina propria. (B) Increased numbers of survivin-positive cells in inflamed CD, noninflamed CD, and inflamed UC mucosa compared with normal control mucosa. *P < .001 for inflamed CD compared with control and UC. **P < .01 for inflamed CD compared with noninflamed CD, and UC compared with normal mucosa. ***P < .004 for noninflamed CD compared with control (groups included 11 control, 10 inflamed CD, 10 noninflamed CD, and 10 UC). (C) From top to bottom: CD3-positive lymphocytes (red) in the lamina propria and survivin-positive crypt cells (green) in normal mucosa; abundant CD3 (red) and survivin (green) co-expressing lymphocytes infiltrating colonic CD; CD3-positive lymphocytes (red) in the lamina propria and survivin-positive crypt cells (green) in UC mucosa; CD3-positive (red), survivin-negative lymphocytes infiltrating survivin-positive neoplastic cells (green) in colon adenocarcinoma. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Representative of 10–14 samples for normal and IBD mucosa and 3 colon cancers. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Levels of survivin protein in fresh, activated, and rested LPTs. (A) Increased survivin and p-survivin in freshly isolated CD compared with control and UC LPTs (P < .001 and P < .04, respectively). (B) Increased nuclear p-survivin in freshly isolated UC and CD compared with control LPTs (P < .05). (C) Up-regulation of total and p-survivin in control, CD, and UC LPTs upon exposure to IL-2 (P < .02–.001 compared with day 0). (D) Subcellular distribution of survivin in IL-2–activated LPTs. Increased survivin in activated UC and CD LPT cells compared with control LPTs. (E) Up-regulation of survivin in control, CD, and UC LPTs upon activation with anti-CD3/CD28 or CD2/CD28 (P < .05 and P < .04 compared with day 0, respectively). (F) Spontaneous increase of survivin in fresh CD LPTs and disappearance of survivin in control and UC LPTs cultured in the absence of IL-2 (P < .03 and P < .02, respectively). GAPDH used as loading control for total and cytoplasmic survivin, and lamin B1 for the nuclear fraction. Immunoblots are representative of 12–18 samples in each control, CD, and UC group; bands represent a loading of 10 μg protein lysate/lane regardless of the total (T), cytoplasmic (C), or nuclear (N) origin of survivin. P values were calculated using the ratio of survivin to GAPDH or lamin B1 obtained by densitometric analysis and comparing the ratio values among control CD and UC. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Effect of survivin and HSP90 silencing on protein levels, proliferation, and apoptosis of LPTs. (A) Immunoblot showing the effect of survivin and scrambled siRNA on protein levels of IL-2–stimulated control, CD, and UC LPTs. Bcl-2 was used as specificity control and GAPDH was used as loading control (figure is representative of 4 experiments). (B) Upper panel: time-dependent suppression of anti-CD3/CD28 plus IL-2–induced LPT proliferation upon treatment with survivin siRNA (each column indicates 1 control, 1 CD, and 1 UC LPT isolate). Lower panel: lack of significant effect on anti-CD3/CD28 plus IL-2–induced LPT proliferation by treatment with HSP90 siRNA (each column indicates 1 control, 1 CD, and 1 UC LPT isolate). Percentage of suppression compared with that of scrambled siRNA. (C) Increased LPT apoptosis induced by survivin, but not HSP90, siRNA compared with scrambled siRNA (*P < .012; n = 3 for control, CD and UC, respectively, for both survivin and HSP90 siRNA). (D) Flow cytometric analysis of increased LPT apoptosis induced by survivin siRNA as assessed by annexin-V/7-Aminoactinomycin D. Red indicates double-negative cells and green indicates annexin-V–positive cells (representative of 3 experiments). Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Effect of inhibiting proteasomal activity on time-dependent expression of survivin in LPTs. Western blots of IL-2–activated LPT extracts showing results with 3 different anti-survivin antibodies recognizing distinct survivin epitopes (anti–survivin-1, -2, and -3), one antibody against Bcl-2, and reciprocal immunoprecipitation of ubiquitin and survivin probed with antibodies against survivin (anti-survivin 3) and ubiquitin, respectively. Anti-survivin 1 is a 6E4 mouse monoclonal antibody, anti-survivin 2 is a mouse monoclonal antibody clone 60.11, and anti-survivin 3 used for immunoprecipitation (IP) is a rabbit polyclonal antibody (Supplementary Materials). Antibodies directed at distinct survivin epitopes ensured that all detected bands did contain survivin. Results displayed derive from a CD LPT isolate. Dimethyl sulfoxide (DMSO) is the solvent for the proteasomal inhibitor MG132, and E64 is a protease inhibitor used as control for MG132. GAPDH was used as loading control. Figure is representative of 1 control, 2 CD, and 2 UC LPTs. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Proteasomal activity of LPT and survivin sensitivity to proteolysis. (A) Immunoprecipitation of survivin in fresh control, CD, and UC LPT lysates followed by immunoblotting with an anti-ubiquitin antibody. Figure is representative of 6–8 samples in each group. (B) Differential levels of proteasomal activity in fresh control, CD, and UC LPTs. *P < .04 for CD compared with control LPT activity, and for degree of lactacystin-induced inhibition in CD compared with UC LPT (n = 17 for control, CD, and UC, respectively). (C and D) Effect of proteinase K activity on survivin levels in cytosolic and nuclear fractions of IL-2–stimulated LPTs. Immunoblots showing a dose-dependent decrease of survivin in the cytosolic fraction of control and UC, but not CD LPT lysates (*P < .02, **P < .004, by densitometry of the survivin:lamin B1, survivin:β-actin, and Bcl-2:β-actin ratios, respectively), and a dose-dependent decrease of survivin in the nuclear fraction of the same lysates. Immunoblots of the same lysates show a dose-dependent decrease in the cytosolic fraction of Bcl-2, which was used as control. Figure is representative of 4 experiments. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Association of survivin with HSP90 in LPTs and dissociating effect of 17-AAG. (A) Reciprocal immunoprecipitation of survivin and HSP90 in fresh control, CD, and UC LPT lysates. Immunoblots showing immunoprecipitated survivin probed with an anti-HSP90 antibody, and immunoprecipitated HSP90 probed with an anti-survivin antibody. Figure is representative of 6 experiments. (B) Effect of heat shock on expression of survivin, HSP90, and Bcl-2 by LPTs. Immunoblots showing time-dependent increase of survivin and HSP90 and a decrease of Bcl-2 in fresh control, CD, and UC LPTs. GAPDH was used as loading control. Figure is representative of 4 control, CD, and UC LPTs, respectively. (C) Effect of inhibition of HSP90 chaperone activity on LPT survivin levels. Immunoblots showing the dose-dependent effect of 17-AAG on survivin levels of IL-2–stimulated control, CD, and UC LPTs. MG132 was used as control to block survivin degradation through the proteasomal pathway, and dimethyl sulfoxide (DMSO) was used as solvent control for both 17-AAG and MG132. β-actin was used as a loading control. Figure is representative of 3 control, CD, and UC LPTs, respectively. (D) Dose-dependent increase of control, CD, and UC LPT apoptosis induced by 17-AAG (n = 3 control, CD, and UC LPTs). Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Association of survivin with HSP90 in LPTs. Confocal microscopy of LPT preparations showing co-localization of survivin (green) and HSP90 (red) in fresh, heat-shocked (6 hours), IL-2–stimulated (7–10 days), and alone (absence of IL-2 for 7 days) control, CD, and UC LPTs. Co-localization of survivin and HSP90 is indicated by the orange color generated by the combination of green (survivin) and red (HSP90) colors. Micrograph panel is representative of 5–10 isolates in each group. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) High magnification of an inflamed CD mucosa showing the lymphocyte-like morphology of survivin-positive cells (dark brown) in the lamina propria (arrows). (B) Mouse IgG isotype control for survivin in CD mucosa. (C) Rabbit IgG isotype control for CD3 in CD mucosa. (D) Survivin-positive cells (dark brown) in the crypts and lamina propria of UC mucosa. (E) Rabbit IgG isotype control of a sequential section of the same UC mucosa shown in panel D. (D and E) Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Expression of survivin mRNA by isolated LPT. (A) Variable levels of survivin mRNA in freshly isolated, IL-2–activated, and rested (alone) control, CD and UC LPT (4 samples each). (B) Progressive increase of survivin mRNA after culture with IL-2 (*P < .05, **P < .02, ***P < .005 compared with day 0). (C) Decline in LPT cultured in the absence of IL-2 (*P < .01 at day 7 compared with day 0). GAPDH was used as a loading control. (A) Representative of 15–20 samples in each group; (B and C) obtained by densitometric analysis of the same samples used for panel A. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Time-dependent changes in cell numbers and corresponding survivin levels in LPT cultures. (A) Significant increase of CD compared with control and UC LPT numbers in the presence of IL-2 (*P < .02, **P < .003, ***P < .002). (B) Significantly greater decrease of control and UC compared with CD LPT numbers in the absence of IL-2 (*P < .04, **P < .01). (C) Progressive increase of survivin protein levels in the same cultures in the presence of IL-2. (D) Progressive increase of survivin in CD compared with control and UC cultures (*P < .03) in the absence of IL-2. (A–D) Representative of 15–20 samples in each group. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Distribution and levels of survivin in freshly isolated, activated, and rested LPT. Confocal microscopy of LPT cytospin preparations showing the relative nuclear and cytoplasmic distribution and levels of survivin in freshly isolated, IL-2–stimulated (7–10 days), anti-CD3/CD28-activated, and alone (absence of IL-2 for 7 days) control, CD, and UC LPT. Micrograph panel is representative of 6–9 experiments in each group. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Effect of protein synthesis, proteasomal activity, and nuclear export inhibition on survivin level and distribution in LPT. (A) Time-dependent decrease of survivin levels in freshly isolated LPT induced by cycloheximide, and stabilization of survivin by addition of lactacystin or leptomycin B in the presence of cycloheximide. Dimethyl sulfoxide (DMSO) and methanol were used as solvents for lactacystin and leptomycin B, respectively, and were present in all cultures. GAPDH was used as a loading control. (B) Confocal microscopy of freshly isolated LPT cytospin preparations showing changes in nuclear and cytoplasmic level and distribution of survivin induced by inhibition of protein synthesis by cycloheximide, proteasomal activity by lactacystin, and nuclear export by leptomycin B. Similar results were obtained with control, CD, and UC LPT. Micrograph panel is representative of 6–10 isolates in each group. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 (A) Reciprocal immunoprecipitation of survivin and ubiquitin in freshly isolated control, CD, and UC LPT lysates. Immunoblot showing immunoprecipitated survivin probed with an anti-ubiquitin antibody, and immunoprecipitated ubiquitin probed with an anti-survivin antibody. Figure is representative of 6–8 samples in each group. (B) Reciprocal immunoprecipitation of survivin and HSP90 in freshly isolated control, CD, and UC LPT lysates. Immunoblot showing immunoprecipitated survivin probed with an anti-HSP90 antibody, and immunoprecipitated HSP90 probed with an anti-survivin antibody. Figure is representative of 6 separate experiments. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 7 Two-color confocal microscopy of IL-2–activated LPT showing that survivin (green) in the cytoplasm is associated with HSP90 (red), as shown by the orange color generated by the combination of green (survivin) and red (HSP90) colors. In the nucleus, survivin (green) is not associated with HSP90. Figure is representative of 5–10 control, CD, and UC LPT. Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 8 Effect of HSP90 silencing on protein levels and apoptosis of LPT. (A) Immunoblot showing the effect of HSP90 siRNA and scrambled siRNA on protein levels of IL-2–stimulated control, CD, and UC LPT. GAPDH was used as a loading control (figure is representative of 3 experiments). (B) Flow cytometric analysis showing lack of apoptosis-inducing effect of HSP90 siRNA compared with scrambled siRNA as assessed by annexin-V/7-Aminoactinomycin D. Red indicates double-negative cells and green indicates annexin-V/7-Aminoactinomycin D–double-positive cells (representative of 3 experiments). Gastroenterology 2012 143, 1017-1026.e9DOI: (10.1053/j.gastro.2012.06.039) Copyright © 2012 AGA Institute Terms and Conditions