Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

BACKGROUND: Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention. METHODS: For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function. RESULTS: We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential. CONCLUSIONS: We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well-documented, microscopically detectable hallmarks of cancer

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

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

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

<p><b>A)</b> 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 <i>L_d</i> in an individual nucleus. <b>B)</b> Percent difference in combined nuclear <i>L_d</i> over experimental repeats in HT-29 and CSK knockdown cells. Nuclear <i>L_d</i> mostly decreased following VPA treatment in each cell line and to a greater extent in the CSK constructs. <b>C)</b> 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 <i>L_d</i> differences between the cell lines.</p

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

<p><b>A)</b> mRNA expression of HDAC1, HDAC2, HDAC3, HDAC5, HDAC7 in human field carcinogenesis from (n = 86, patients with adenomas vs. controls). <b>B)</b> Representative TEM images of nuclei in histologically normal rectal cells from patients with or without adenomas elsewhere in the colon. <b>C)</b> Up-regulation of HDAC2 in field carcinogenesis was confirmed in human resection samples by qRT-PCR methods (n = 12, patients with adenomas vs. controls). <b>D)</b> Representative TEM images of saline-injected or azoxymethane-injected (AOM) nuclei obtained from the distal colon at a premalignant time point. <b>E)</b> 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 *<i>p</i><0.05.</p

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

<p><b>A)</b> TEM micrographs of chromatin structure in the HT-29 colon cancer cell line genetic variants, HT-29 control and CSK knockdown. <b>B)</b> HDAC2 expression is up-regulated in the CSK knockdown cell lines. <b>C)</b> MNase assay on HT-29 (H) and CSK knockdown (C) cells also indicate a more compact chromatin structure present in the CSK constructs. <b>D)</b> 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 *<i>p</i><0.05.</p