Histone modifiers and marks define heterogeneous groups of colorectal carcinomas and affect responses to HDAC inhibitors in vitro.

Little is known about histone modifiers and histone marks in colorectal cancers (CRC). The present study therefore addressed the role of histone acetylation and histone deacetylases (HDAC) in CRCs in situ and in vitro. Immunohistochemistry of primary CRCs (n=47) revealed that selected histone marks were frequently present (H3K4me3: 100%; H3K9me3: 77%; H3K9ac: 75%), partially displayed intratumoral heterogeneity (H3K9me3; H3K9ac) and were significantly linked to higher pT category (H3K9me3: p=0.023; H3K9ac: p=0.028). Furthermore, also HDAC1 (62%), HDAC2 (100%) and HDAC3 (72%) expression was frequent, revealing four CRC types: cases expressing 1) HDAC1, HDAC2 and HDAC3 (49%), 2) HDAC2 and HDAC3 (30%), 3) HDAC1 and HDAC2 (10.5%) and 4) exclusively HDAC2 (10.5%). Correlation to clinico-pathological parameters (pT, pN, G, MSI status) revealed that heterogeneous HDAC1 expression correlated with lymph node status (p=0.012). HDAC expression in situ was partially reflected by six CRC cell lines, with similar expression of all three HDACs (DLD1, LS174T), preferential HDAC2 and HDAC3 expression (SW480, Caco2) or lower HDAC2 and HDAC3 expression (HCT116, HT29). HDAC activity was variably higher in HCT116, HT29, DLD1 and SW480 compared to LS174T and Caco2 cells. Treatment with broad (SAHA) and specific (MS-275; FK228) HDAC inhibitors (HDACi) caused loss of cell viability in predominantly MSIpositive CRC cells (HCT116, LS174T, DLD1; SAHA, MS-275 and in part FK228). In contrast, MSI-negative CRC cells (Caco2, HT29, SW480) were resistant, except for high doses of FK228 (Caco2, HT29). Cell viability patterns were not linked to different efficacies of HDACi on reduction of HDAC activity or histone acetylation, p21 expression and/or induction of DNA damage (γH2A-X levels). In summary, this study reveals inter- and intra-tumoral heterogeneity of histone marks and HDAC expression in CRCs. This is reflected by diverse HDACi responses in vitro, which do not follow known modes of action. Together, this implies further exploitation of histone alterations in CRC for molecular classification and/or novel treatment options.