HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure

Abstract

Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

Figure 1. HDAC2 expression is up-regulated in human field carcinogenesis and early carcinogenesis.

A) mRNA expression of HDAC1, HDAC2, HDAC3, HDAC5, HDAC7 in human field carcinogenesis from (n = 86, patients with adenomas vs. controls). B) Representative TEM images of nuclei in histologically normal rectal cells from patients with or without adenomas elsewhere in the colon. C) Up-regulation of HDAC2 in field carcinogenesis was confirmed in human resection samples by qRT-PCR methods (n = 12, patients with adenomas vs. controls). D) Representative TEM images of saline-injected or azoxymethane-injected (AOM) nuclei obtained from the distal colon at a premalignant time point. E) HDAC2 expression is also up-regulated in the AOM (azoxymethane-injected) rat model for early colorectal carcinogenesis (n = 12 animals). Standard error bars shown with *p<0.05.

Figure 2. HDAC inhibition differentially affects cell viability in colon cancer cell line variants.

A) TEM micrographs of chromatin structure in the HT-29 colon cancer cell line genetic variants, HT-29 control and CSK knockdown. B) HDAC2 expression is up-regulated in the CSK knockdown cell lines. C) MNase assay on HT-29 (H) and CSK knockdown (C) cells also indicate a more compact chromatin structure present in the CSK constructs. D) HT-29 and CSK knockdown cells in 96-well plates were treated with increasing concentrations of VPA for 24 h and then assayed for proliferation using standard WST-1 assay. Absorbance was measured after 20 min at 37°C. VPA treatment reduced cell viability in both cell lines, while the effect was greater in the CSK constructs. Standard error bars shown with *p<0.05.

Figure 3. HDAC inhibition increases chromatin accessibility in colon cancer cell line variants.

MNase assays of A) HT-29 cells and B) CSK knockdown cell lines treated with increasing concentrations of VPA for 24 h. C) Western blot analysis probing with antibodies against acetyl-histone H3 and apoptotic marker with cleavage of PARP (poly ADP ribose polymerase) in VPA treated HT-29 and CSK knockdown cells. β-actin is shown as a protein loading control. D) Representative TEM images showing altered chromatin distribution in the VPA treated samples compared to the control cells.

Figure 4. Changes in nuclear disorder strength (L_d) following VPA treatment.

A) Representative pseudocolor PWS images from nuclei of HT-29 and CSK constructs untreated or treated with 0.5 mM VPA. Color shows the magnitude of the L_d in an individual nucleus. B) Percent difference in combined nuclear L_d over experimental repeats in HT-29 and CSK knockdown cells. Nuclear L_d mostly decreased following VPA treatment in each cell line and to a greater extent in the CSK constructs. C) Percent difference in nuclear disorder strength between HT-29 and CSK constructs after each treatment. Treatment with higher concentrations of VPA (0.5 mM and 1.5 mM) nullified the nuclear L_d differences between the cell lines.