Discovery of a gene that regulates the formation of embryonic stem cell tumors

In experiments using mice, silencing the E2F2 gene significantly inhibited the formation of teratomas without diminishing pluripotency.

By Karina Toledo

Agência FAPESP – In tests conducted on animals, researchers at the University of São Paulo (USP) have shown that the specific silencing of one gene—E2F2—significantly reduces the risk that embryonic stem cells will induce the formation of tumors without diminishing the pluripotency (i.e., capacity to differentiate into any type of tissue) of these cells.

This discovery may help to overcome one of the main obstacles to the advancement of pluripotent stem cell therapies, which are considered to be the best hope for a cure for spinal cord injuries and macular degeneration as well as diseases such as diabetes, Parkinson’s disease, Alzheimer’s disease and muscular dystrophy.

The findings from the FAPESP funded project, “E2F2 transcription factor and expression of proto-oncogenes in human embryonic stem cells,” were reported in January in the journal Stem Cells and Development.

“Although they offer many advantages, such as the capacity to self-renew and generate any type of body tissue, embryonic stem cells have one disadvantage: the risk of causing teratoma type tumors,” said Oswaldo Keith Okamoto, a professor in the Genetics and Evolutionary Biology Department of the USP Biosciences Institute who also serves as the study coordinator.

These tumors are formed by a combination of various types of cells containing three germinative layers: endoderm, mesoderm and ectoderm. “Although most of them are benign, there are some malignant forms of teratomas. Together, they represent a risk to patients and pose an obstacle to conducting clinical trials,” said Okamoto.

In an article published in the journal Biochimica et Biophysica Acta in 2007, Okamoto and his colleagues observed an aberrant pattern in the expression of the E2F2 gene in tumor stem cell cultures.

“We had been studying samples of glioblastoma, the most aggressive form of brain cancer. We isolated the stem cells from these tumors and conducted a genetic analysis to determine which genes in these cells showed aberrant expression. These cells are very important from a clinical standpoint because they are more efficient at generating tumors and more resistant to chemotherapy and radiation treatments,” the researcher explained.

The group noted that a higher level of E2F2 expression was associated with a greater degree of tumor aggressiveness. “We then suspected that this gene in the embryonic stem cells might be related to tumorigenesis,” said Okamoto.

The hypothesis was originally tested in vitro, using human embryonic stem cell cultures. The E2F2 gene was silenced with the aid of a technique known as RNA interference, which involves the use of small, non-coding RNA molecules capable of binding with the messenger RNA of a specific gene and interrupting its expression.

“The silencing of E2F2 in vitro significantly inhibited the proliferation of embryonic stem cells and their capacity to generate new colonies,” Okamoto reported.

The next step was to test the effect of the procedure on immunosuppressed mice. To achieve this, the animals were separated into two groups: Half were inoculated with control embryonic stem cells, and the other half were inoculated with the modified cells.

In an examination conducted 30 days later, the group that received the control cells had developed tumors with an average volume of more than 50 cubic millimeters (mm³), while the group that received the cells with the silenced gene remained tumor-free.

In a second examination conducted 60 days after the inoculation, the tumors in the group that had received the control stem cells had grown to more than 2,000 mm³, while in the other group, the average tumor volume was only 60 mm³.

“We monitored the animals for up to 90 days after inoculation with the modified stem cells and observed that the tumor growth stabilized,” said Okamoto.

Another series of experiments was then conducted to ensure that the procedure had not affected the pluripotency of the embryonic stem cells.

“We stimulated the modified stem cells in vitro to differentiate into the cells of the three germinative layers, and we found that this occurred normally. In addition, the cells continued to express the so-called pluripotency factor that includes proteins such as Oct4, Nanog and Sox2,” Okamoto explained.

The analysis of the small tumors formed in the mice that had received the modified stem cells also revealed typical traces of teratoma. “Although the tumors were underdeveloped because of E2F2 gene silencing, they contained cells from the three germinative layers, and the formation of the teratoma is an indicator of pluripotency,” said the professor of the USP Biosciences Institute.

According to Okamoto, E2F2 is a key gene because aside from regulating oncogenes (i.e., genes related to the appearance of malignant or benign tumors), it also regulates a series of other genes involved in important processes such as cell proliferation and differentiation, DNA replication and apoptosis.

Induced pluripotent cells

In the near future, Okamoto’s group, under the auspices of the Human Genome and Stem Cell Research Center (HUG-CELL), which is one of the Research, Innovation and Dissemination Centers (RIDC) funded by FAPESP, plans to conduct experiments to confirm whether the silencing of the E2F2 gene has the same beneficial effect on induced pluripotent stem cells (IPS cells).

IPS cells can be obtained from common skin cells. For this to occur, certain proteins capable of reprogramming the cell genome, known as transcription factors, are inserted into an adult cell. These transcription factors activate genes related to the embryonic stage of the cell and switch off other genes that should be active after maturity.

“We believe that the same thing we observed in human embryonic stem cells will occur in the IPS cells. This would be very interesting from a therapeutic standpoint because this type of cell can be obtained from tissue belonging to the very patient who will be treated. Thus, these cells do not present the same risk of rejection as embryonic stem cells,” said Okamoto.