An inactivating mutation in HDAC2 leads to dysregulation of apoptosis mediated by APAF1.
OBJECTIVE
Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing tumor suppressor genes. We examined mutation of HDAC2 and examined consequences of HDAC2 loss.


METHODS
Colon cancer cell lines and primary cancers were examined for mutations in HDAC2 by direct sequencing and capillary electrophoresis. Promoter methylation was determined using methylation-specific polymerase chain reaction in primary tumors. Sensitivity to HDAC inhibitors and resistance in vitro used colon cancer cell lines.


RESULTS
HDAC2 mutations in the poly(A) tract in exon 1 result in a frameshift and premature stop codon. These were found in microsatellite instability (MSI) cell lines and 43% of MSI colon cancers, but only 7% of microsatellite stable cancers. Mutation was associated with complete or regional tumor specific loss of HDAC2 protein. Inactivation of HDAC2 was not associated with large-scale changes in promoter region methylation, but rather is a consequence of epigenetic MLH1 inactivation leading to MSI. HDAC2 mutation results in apoptotic resistance to HDAC inhibitors (trichostatin A or suberoylanilide hydroxamic acid), despite induction of global histone acetylation. Differential induction of apoptosis by HDAC inhibitors is mediated by the proapoptotic gene APAF1, as shown by specific APAF1 induction only in cell lines with functional HDAC2, HDAC2 protein localization to the APAF1 promoter by chromatin immunoprecipitation, siRNA knockdown of HDAC2 leading to up-regulation of APAF1, and stable knockdown of APAF1 reducing apoptotic response to HDAC inhibitors.


CONCLUSIONS
Frequent HDAC2 mutations are found in MSI tumors and HDAC2 plays a major role in mediating apoptotic response to HDAC inhibitors through direct regulation of APAF1.
Sidney Kimmel Comprehensive Cancer Center and Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
Christin L
CL
Hanigan
Manon
M
Van Engeland
Adriaan P
AP
De Bruine
Kim A
KA
Wouters
Matty P
MP
Weijenberg
James R
JR
Eshleman
James G
JG
Herman
D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005091:Exons; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D004273:DNA, Neoplasm; D012097:Repressor Proteins; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases