Epigenetic regulators that modify factors involved in switching genes on and off represent attractive anticancer targets, but whether this strategy will work in vivo hasn’t yet been proven. Studies in mice by researchers headed by a Boston Children’s Hospital team now suggest that blocking one epigenetic regulator—a histone methyltransferase known as G9a—which was thought to represent a promising target for lung cancer actually had the opposite effect, and boosted the numbers of cancer stem cells, or tumor propagating cells (TPCs) that drive cancer progression. Their studies also found that inhibiting G9a-regulated genes repressed TPCs in mice, resulting in fewer lung tumors.
“People had looked at cell lines from lung tumors and found that they are sensitive to drugs inhibiting G9a,” said Samuel Rowbotham, Ph.D., first author of the researchers’ published paper in Nature Communications. “In general tumor cell populations, these drugs would slow down growth or even kill the cells. But we found that these drugs were also making the surviving tumor cells more stem-like. We predicted that this would advance disease progression, and this is what we saw.”
The team’s paper is titled, “H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression.”
Epigenetic regulators are chromatin-modifying or chromatin-interacting proteins that play key roles in governing the behavior of stem or progenitor cells, and delineating cell fate, the authors explain. Such proteins are often amplified, mutated, or misregulated in cancer and play key roles in the development of tumors. G9a (Ehmt2) and Glp (Ehmt1) are histone methyltransferases (HMTs) that add methyl groups onto histone H3 lysine 9 (H3K9) and which are known to be involved in regulating embryonic stem cell differentiation. Studies have also linked G9a with cancer, and the enzyme is mutated in various tumor types. “The majority of studies describe G9a as an oncogene, finding G9a to have pro-proliferative and pro-epithelial-mesenchymal transition (pro-EMT) functions,” the team noted. “These results have spurred the development of chemical inhibitors of G9a/Glp in the hope that they could be used as anticancer therapeutics.”
Studies by the Boston Children’s Hospital team now suggest that blocking G9a may have the opposite effect expected and actually promote cancer growth. Initial studies in adenocarcinoma cell lines showed that treatment with G9a led to cells becoming more stem-like.
The team then transplanted cancer stem cells into mice and followed tumor growth. They found that animals receiving cells in which the G9a gene had been knocked down were more likely to develop lung tumors and metastases than animals receiving transplants of cells with functional G9a. Further studies showed that lack of G9a was associated with increased numbers of TPCs. “These results suggest that G9a loss drives lung adenocarcinoma progression and metastasis by increasing the proportion of TPCs within the tumor,” the researchers wrote.
G9a isn’t commonly mutated in human lung adenocarcinomas, but the team’s analysis of data from hundreds of adenocarcinoma patients found that higher G9a expression correlated with much better 10-year survival. Interestingly, they also found that high expression levels of KDM3A, a lysine demethylase (KDM) that acts to demethylate H3K9, was linked with worse 10 years survival.
When they then silenced the KDM3A gene in mouse adenocarcinoma cells and transplanted these cells into mice, the animals developed fewer tumors than the control animals developed. Treating adenocarcinoma-carrying mice with an inhibitor of KDM3A also effectively reduced tumor growth compared with control mice, “suggesting that targeting TPCs through H3K9me KDM inhibition could be an effective therapy for lung adenocarcinoma.”
Although cancer stem cells haven’t been identified in human adenocarcinomas, research lead Carla Kim, Ph.D., believes that the findings are worth pursuing, especially given a line of evidence from a 2017 study suggesting that demethylase inhibitors could kill chemotherapy-resistant cells from patient tumors.
“Even if we can’t pinpoint cancer stem cells in human patients, Rowbotham’s work shows you can start by studying a cancer stem cell in a mouse model and identify targets that could be clinically important,” she commented. “It shows the importance of finding the right molecule the cancer is sensitive to. In adenocarcinoma, a demethylase inhibitor is likelier to be more useful than methyltransferase inhibitor.”
The team envisages a treatment strategy for adenocarcinoma that would initially target the bulk of the cancer cells, and then use a treatment to specifically target cancer stem cells.
“Our data suggest that down-regulation of G9a promotes TPCs, leading to disease progression, metastasis, and worse clinical outcome, thus raising caution for the use of G9a inhibitors, particularly for lung cancer treatment,” the authors concluded. “By studying the epigenetic dependencies of TPCs that represent a minority of tumor cells, our work has revealed a potential new therapeutic intervention for advanced lung cancer.”
They suggest that the reason their results are at odds with those of previous studies may be because prior work has been carried out primarily in cell lines. “Earlier studies couldn’t see that cancer stem cells were still around, and there’s more of them when you treat with these drugs,” said Kim. “Because they’re such a small fraction of the tumor, anything that affects them can easily be missed.”
The results also highlight the importance of choosing the best cell models when looking for new anticancer therapeutics, the researchers continued. “Studies primarily based on cell lines or whole tumors have suggested that G9a is oncogenic and a good target for epigenetic therapy, but our approach, beginning with analyzing different tumor populations, led to a startlingly different conclusion. We propose that analyzing the effects of new therapeutics on different tumor populations, especially the most tumorigenic stem-like cells, would be greatly beneficial. This may increase the success rate of clinical trials and thus reduce the cost and time taken to bring new cancer therapeutics to the patient.”