MicroRNAs Team Up to Take Down Brain Cancer
MicroRNAs are small but powerful molecules in the cell. Containing only around 22 nucleotides, these small RNA molecules are responsible for binding to and silencing messenger RNA sequences before they can be translated into proteins, and thus are incredibly important regulators of gene expression. MicroRNAs (miRs) often regulate the expression of epigenetic regulators that when disrupted, can lead to cancer. For example, a protein called EZH2 (enhancer of zeste homologue 2) is upregulated in human malignancies such as the aggressive brain cancer, glioblastoma. Downregulating EZH2 decreased the ability of glioblastoma cancer stem cells to divide continuously as well as their ability to initiate the formation of tumors .
A new study by Bhaskaran et al found that increasing expression of a group of three miRs together leads to decreased expression of a group of misregulated epigenetic factors in glioblastoma, thus pointing to a possible new miR-based therapeutic for glioblastoma.
Searching through The Cancer Genome Atlas for information on brains of healthy people versus those with glioblastoma, the researchers identified a number of differentially expressed miRs. When they monitored the expression of these miRs during differentiation from neural progenitor cells into neurons or astrocytes, they found that three miRs: miR-124, miR-128, and miR-137, had increased expression in neurons. Interestingly, if they looked at the predicted targets of each of these three miRs, many of them had been previously implicated in glioblastoma. MiR-124 targets EZH2 , which works with BMI1, another chromatin repressor . MiR-128 targets BMI1 , and miR-137 targets LSD1, an epigenetic regulator that is involved in maintaining glioblastoma  that also works with EZH2 .
In fact, when the authors measured the expression of these three miRs and their protein targets, they saw that in patients with glioblastoma, all three miRs were downregulated and their target proteins were all upregulated when compared to healthy patient brains. These results suggest that in healthy brains these three miRs may function together to downregulate their specific chromatin repressor target genes, but this regulation becomes altered in glioblastoma.
It has been observed that after treatment with the drug temozolomide (TMZ) or radiation, patients with recurrent tumors show increased expression of EZH2 . Indeed, when the authors measured the expression of EZH2, BMI1, and LSD1 in tumor samples from glioblastoma patients before and after treatment, they saw expression of all three proteins increase after the treatment. When the researchers injected mice with glioblastoma cells and then either treated them with TMZ, radiation therapy, or no treatment, mice that had received any kind of treatment had higher levels of the epigenetic target proteins and decreased expression of all three miRs (miR-124, miR-128, and miR-137), indicating that this effect is likely a consequence of TMZ and radiation treatment.
So, if all three miRs have decreased expression in glioblastoma, what happens if they are all re-expressed together in glioblastoma cells? Expressing the three miRs together in a single RNA sequence they called Cluster 3, the authors created a glioblastoma cell line with increased miR-124, miR-128, and miR-137 activity. When they expressed Cluster 3 in glioblastoma cells, the three epigenetic target proteins and other proteins that depend on them but not on the miR’s expression, all showed decreased expression levels. Upregulation of the three miRs also caused an anti-cancer effect, as the glioblastoma cells showed much less proliferation. When they injected glioblastoma cells with or without Cluster 3 into mice, the researchers saw that the mice with Cluster 3 survived significantly longer and had smaller tumors than those without it. However, in the end, the mice that had received Cluster 3 still died from their tumors, likely because the miRs in Cluster 3 lost expression over time.
This loss in expression of the miRs in Cluster 3 suggests that this method might not make a very good therapeutic approach on its own. The researchers thought, however, that perhaps administering Cluster 3 in conjunction with treatment might prove more effective. In fact, they found that when glioblastoma cells were treated with either TMZ or radiation, cells with Cluster 3 showed no increase in EZH2, BMI1, or LSD1, and a greater number of these cells died. Intriguingly, if only one of the three miRs was present, with or without treatment, there was no significant increase in cell death, suggesting that all three miRs function together to have their anti-cancer effect. Finally, when the researchers injected glioblastoma cells with or without Cluster 3 into mice and treated the mice with TMZ, they saw that mice that had received both Cluster 3 and treatment survived 6 times longer than mice that had only received treatment. The combined effect of Cluster 3 plus treatment was incredibly effective.
The researchers knew that miRs are often transmitted to neighboring cells via extracellular vesicles (EVs), and they had seen that tumor cells next to ones with the Cluster 3 sequence, seemed to grow slower than other tumor cells. Using a trans-well set up where cells cannot come in physical contact with each other but EVs can be transmitted between cells, the authors showed that cells co-cultured with cells containing Cluster 3 had increased expression of the three miRs, decreased expression of EZH2, BMI1, and LSD1, and had impaired growth, suggesting that Cluster 3 is transferred to neighboring cells via EVs.
While the transfer of miRs occurred easily in cells in a petri dish, the researchers wondered if the same would happen in a live animal. They implanted mice with an equal number of red glioblastoma cells and green glioblastoma cells that either expressed a control or Cluster 3. After 12 days, they isolated the tumors from mice and separated the cells by color using fluorescence-activated cell sorting (FACs). They found that red cells that had been isolated from mice where green cells had Cluster 3, showed increased miRs expression and decreased expression of the repressor target proteins compared to controls, indicating that miRs can be transferred to neighboring tumor cells in a live animal.
Overall, this study shows how three miRs: miR-124, miR-128, and miR-137 can function together to repress their target epigenetic repressor proteins that promote cancer progression. Because miRs can be spread to neighboring tumor cells by EVs, not every cell in the tumor must be targeted in order to stop the growth of the cancer. Thus, this use of three miRs in conjunction with TMZ or radiation treatment may be an excellent new avenue in clinical trials for treatment of glioblastoma.
Original article: Bhaskaran V, Nowicki MO, Idriss M, Jimenez MA, Lugli G, Hayes JL, Mahmoud AB, Zane RE, Passaro C, Ligon KL, Haas-Kogan D, Bronisz A, Godlewski J, Lawler SE, Chiocca EA, Peruzzi P (2019). The functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma. Nat Commun, 10(1):442. doi: 10.1038/s41467-019-08390-z.
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