Volume 135, Issue 5, Pages 1654-1664.e2 (November 2008) An Inactivating Mutation in HDAC2 Leads to Dysregulation of Apoptosis Mediated by APAF1  Christin L. Hanigan, Manon van Engeland, Adriaan P. De Bruine, Kim A. Wouters, Matty P. Weijenberg, James R. Eshleman, James G. Herman  Gastroenterology  Volume 135, Issue 5, Pages 1654-1664.e2 (November 2008) DOI: 10.1053/j.gastro.2008.07.078 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 HDAC2 mRNA is expressed in all colon cancer cell lines, but RKO lack HDAC2 protein. (A) Reverse-transcription PCR demonstrates HDAC2 expression in several colon cancer cell lines. (B) HDAC2 mRNA expression, normalized to GAPDH, in colon cancer cell lines, measured via real-time PCR and quantitated as a fold increase relative to expression in RKO cells. Western blots of whole-cell protein extracts with antibodies specific for HDAC2 (C) and HDAC1 (D). Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Mutations in the poly(A) tract of HDAC2 occur in MSI colorectal cancer. (A) Direct sequencing chromatographs revealing a heterozygous T deletion on the antisense strand in HCT116 and a homozygous deletion shown on the sense strand in RKO. Fluorescently labeled PCR products from primary tumor samples with both WT allele and mutant HDAC2 alleles with either an insertion (B) or deletion (C) in the poly(A) tract. Direct sequencing chromatographs are inserted with respective capillary trace. Capillary analysis distinguishes tumors with minimal “mutant” HDAC2 owing to polymerase stutter (D) from tumors with a deletion mutation (E), despite similar sequencing traces. Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 HDAC2 mutations in primary colorectal samples correlate with loss of expression measured by immunohistochemistry. Typical patterns of immunohistochemical staining of HDAC2 show strong nuclear expression in most cells, including epithelial, stromal, and inflammatory cells (all original magnification ×20 unless otherwise noted). (A) Homogeneous nuclear staining in crypts of normal colonic mucosa. (B) Homogeneous nuclear staining in moderately differentiated adenocarcinoma (HDAC2 WT). (C) Complete loss of HDAC2 expression in tumor nuclei in poorly differentiated adenocarcinoma (HDAC2 mutant, original magnification, ×10), whereas interstitial inflammatory and stromal cells retain expression. Nuclear staining is absent only in specific regions of the tumor (HDAC2 mutant) in moderately (D, E) and poorly differentiated (F) adenocarcinomas, suggesting clonal gene inactivation. Note the homogeneous staining in adjacent normal crypts (E). Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Differential apoptotic response is associated with HDAC2 loss, but not mediated by reduced global histone acetylation. (A) Apoptosis in HCT116, LOVO (both with intact HDAC2), and RKO (HDAC2 mutant) via annexin V staining 24 hours after treatment with SAHA. Shift of cell population to the right and upper right quadrants indicates cell death. A decrease of apoptosis is observed in the HDAC2-deficient cell line, RKO, compared with HCT116 and LOVO. (B) Quantitation of annexin V staining from C demonstrates decreased apoptotic response to SAHA only in the HDAC2 deficient cell line, RKO. (C) Global acetylation at H3K9 was similar in Western analysis of whole cell extracts of HCT116, LOVO (both with intact HDAC2), and RKO (HDAC2 mutant) after treatment with 2.5 μmol/L SAHA before treatment (U) and after 2 or 24 hours. (D) Increased acetylation at H3K9 after HDAC inhibitor treatment is dependent on cell confluence. H3K9 acetylation in HCT116 (HDAC2 WT) and RKO (HDAC2 MT) after treatment with 250 nmol/L TSA for 24 hours, demonstrates increased H3K9 acetylation only at lower confluencies (30%–50%). (E) FAS-mediated apoptosis remains intact in RKO. Cell death detected via trypan blue exclusion in cell lines after treatment with FasL for 24 hours demonstrates cell death is induced in RKO similar to other colorectal cancer cell lines. Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 APAF1 is selectively up-regulated in cell lines with an intact HDAC2 undergoing cell death mediated by HDACi. mRNA expression of the proapoptotic genes (FasR, APAF1, BAX, SURVIVIN) and the control gene p21 was determined using real-time PCR, normalized to GAPDH, at baseline and following treatment with 2.5 μmol/L SAHA. Results are presented as fold induction relative to vehicle treated cells. APAF1 is the only apoptotic gene up-regulated by HDAC inhibitors. Induction of p21 in RKO occurred, but was reduced compared with HCT116 and LOVO. Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 HDAC2 directly represses APAF1. (A) ChIP demonstrates HDAC2 is localized to the proximal promoter of APAF1 in HCT116 cells using 2 different primer sets (Supplementary Table 1). (B) Real-time PCR of APAF1 mRNA in HCT116, LoVo, and RKO, normalized to GAPDH and relative to HCT116 (with lowest APAF1 expression). Baseline expression of APAF1 is highest in RKO, which lacks HDAC2. (C) Quantitative ChIP analysis of H3K9Ac at the APAF1 promoter in HCT116 and RKO, normalized to H3K9Ac at an active gene, the GAPDH promoter. H3K9Ac is enriched at the APAF1 promoter in RKO cells relative to HCT116. (D) Transient knockdown of HDAC2 in HCT116 and LoVo. HCT116 and LoVo cells were untreated (U), or treated with siRNA specific to HDAC2 (HD2) or nontarget sequence (NT) for 48, 72, and 96 hours. (E) APAF1 mRNA is induced in these cells as HDAC2 is reduced. Real- time PCR of APAF1 after HDAC2 knockdown for 48, 72, and 96 hours. Fold induction is normalized to GAPDH relative to each corresponding NT siRNA control. Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Stable knockdown of APAF1 prevents HDACi induced apoptosis. (A) APAF1 and NT shRNA were expressed in HCT116 cells; stable clones were selected for apoptotic study, with AKD21 and AKD30 having markedly reduced APAF1 protein. (B) Apoptosis in HCT116 (transfected with nontarget shRNA or APAF1 shRNA) via Annexin V as in Figure 3C after 24 hours' treatment with SAHA. A decrease of apoptosis in APAF1 KD clones is observed. (C) Cell death based on annexin V staining in parental HCT116, HCT116, with NT control is greater than in 2 clones of APAF1 knockdown (AKD21, AKD30), or RKO. Gastroenterology 2008 135, 1654-1664.e2DOI: (10.1053/j.gastro.2008.07.078) Copyright © 2008 AGA Institute Terms and Conditions