Histone Variant H2A.X Regulates Colon Cancer EMT


Hypothetical model for the role of H2A.X in EMT and metastasis

To spread to distant organs, cancer cells sever contacts with surrounding cells, disrupt their internal polarity, and adopt spindle-like protrusions. This process, known as epithelial-mesenchymal transition (EMT), involves decreasing expression of epithelial genes and inducing mesenchymal ones. Establishing tumors at distant sites, however, may require at least partial re-expression of epithelial genes. Changes in chromatin structure have been implicated in EMT, and alterations in levels of histone H2A family members are associated with cancer. William Bonner, Ph.D., of CCR’s Developmental Therapeutics Branch and his colleagues observed that loss of family member H2A.X via small interfering RNA in a colon cancer cell line enhanced the cells’ migration and invasion abilities, leading them to hypothesize that decreased levels of H2A.X may induce gene expression necessary for EMT.

The researchers then compared genome-wide gene expression between H2A.X-deficient and parental cancer cells. Of those genes differentially expressed, 13 mesenchymal-related genes increased in cells with reduced H2A.X while five epithelial genes decreased. These changes were not due to general disruption of chromatin structure since loss of other H2A family members or overexpression of H2A.X had little effect on them. Two genes particularly upregulated in H2A.X-deficient cells were the transcription factors Slug and ZEB1. Inhibiting expression of both factors in these cells restored the epithelial phenotype, indicating that Slug and ZEB1 are essential for mediating H2A.X loss-induced EMT. Studies of H2A.X knockout cells, generated using the CRISPR/Cas9 system, confirmed these results. Increased expression of Slug and ZEB1 in H2A.X-null cells were due to enhanced promoter activation, though the investigators were uncertain why it occurred specifically at these promoters.

Since loss of H2A.X promotes EMT, the scientists wondered whether reintroducing it would reverse the process. Expressing H2A.X in knockout cells restored epithelial proteins to levels similar to parental cells, but expression of mesenchymal markers remained high. Similarly, the morphology of cells re-expressing H2A.X was intermediate between that of parental and knockout cells. In contrast, the H2A.X expressing cells had a growth rate similar to parental cells, which was faster than the knockouts.

The researchers then tested the abilities of parental, knockout, and H2A.X re-expressing cells to establish lung metastases in immunocompromised mice. While the parental and knockout cells formed similar numbers of lung nodules in the mice four weeks after injection, the H2A.X re-expressing cells formed 2.5 times as many. By reverting to an epithelial state but retaining mesenchymal characteristics, H2A.X re-expressing cells were better able to survive environmental stresses and thus establish enhanced distant metastases. The investigators supported this assertion by demonstrating that H2A.X re-expressing cells had higher DNA repair capacity and survival when exposed to ionizing radiation than knockout cells and that these abilities depend on the serine 139 residue of H2A.X.

To determine whether these observations are relevant to human disease, the scientists examined gene expression in 18 primary colorectal tumors and matched liver metastases. They found similar levels of Slug, ZEB1, and H2A.X in both locations, suggesting that EMT markers are similar in metastases and their primary tumors. Further evaluation of human colon cancer samples from The Cancer Genome Atlas and cancer cell lines from the NCI60 dataset supported a strong negative correlation between H2A.X expression and that of Slug or ZEB1.

These studies demonstrated that loss of H2A.X activated EMT in human colon cancer cell lines, promoting their invasiveness. However, cells lacking H2A.X failed to show enhanced metastases perhaps because of a reduced capacity to survive and/or proliferate in a new location. Restoring H2A.X partially reversed EMT, allowing cells greater metastatic ability, which may explain the lack of difference in H2A.X expression in patient primary tumors and matched liver metastases.

Summary Posted: 02/2016

Reference

Weyemi U, Redon CE, Choudhuri R, Aziz T, Maeda D, Boufraqech M, Parekh PR, Sethi TK, Kasoji M, Abrams N, Merchant A, Rajapakse VN, and Bonner WM. The Histone Variant H2A.X Is a Regulator of Epithelial-Mesenchymal Transition. Nature Communications. February 15, 2016. PubMed Link