Bioelectricity has fascinated humans since the ancient Egyptians first felt the shocks of electric fish swimming in the Nile River. Science has since connected bioelectricity to many functions, from neural signaling to the direction of development and regeneration. More recent research has associated abnormal ion channels, called channelopathies, with diseases including migraines, epilepsy, and more recently, metastatic cancer.
In new work, Forsyth Institute researchers have discovered a precise role for electrical signals in the control of embryonic stem cell proliferation and migration. The work adds to an increasing body of evidence that signals in the cellular microenvironment may regulate behaviors that are common to both stem cells and cancer cells. By identifying a novel bioelectrical switch that triggers these behaviors, this work could lead to new therapeutic approaches to modulating them.
Michael Levin and Junji Morokuma began this work, published in the Oct. 28 Proceedings of the National Academy of Sciences, by investigating the role of ion channels in embryonic development. They aimed to understand how these channels help form left–right asymmetries, such as the shape of the liver or the location of the heart.
Levin, HSDM associate professor of developmental biology, performed a targeted screen of his own design to identify which ion transporters are involved in left–right development. The researchers then methodically knocked down the activity of each channel in frog embryos.
Morokuma, first author and HSDM research associate, knocked down one ion channel, the KCNQ1 potassium channel, by damping it with an accessory subunit protein called KCNE1. He injected KCNE1 mRNA into one-celled embryos, thereby giving the cells tools to block the KCNQ1 channels. They later observed left–right asymmetry defects in about 30 percent of the injected tadpoles. These results appeared in the May 2008 Cellular Physiology and Biochemistry.
Morokuma realized, however, that this channelopathy might have broader implications when he looked into the incubator and saw something unexpected. “The dish was swarming with black tadpoles,” he said.
Though only a fraction of the injected tadpoles exhibited such hyper-pigmentation, the researchers did not see the phenomenon at all in control animals injected with other ion channel constructs. Also, pigment cells come from the neural crest, a key stem cell population in embryos, suggesting the possibility of a novel control mechanism for embryonic stem cell function.
Why the tadpoles turned black was not clear at first. “We didn’t know whether we were causing other tissues in the embryo to become pigment cells or whether the original set of pigment cells was multiplying or crawling around more,” said Levin.
They answered these questions by counting; the hyper-pigmented tadpoles had twice the number of pigment cells as the control tadpoles. They also found these pigment cells, melanocytes, packed in places they did not belong, such as inside internal organs and blood vessels. Individual pigment cells also appeared more dendritic than normal.
Though melanocytes were the only cells to exhibit this neoplastic phenotype, few of them received the injected RNA themselves. Rather, the researchers de-termined that the invasive behavior arose through a cas-cade of cell-to-cell signaling.
The transmission begins when the cells in the embryo that did contain the injected RNA depolarize. This change in membrane potential acts as an electrical signal in the microenvironment, triggering nearby melanocytes to overexpress two genes, Sox10 and Slug. These genes have been associated with proliferation and migration in many cell types. Levin confirmed the overexpression of these genes using in situ hybridization, but also said, “we can’t rule out that there may be other genes affected.” Aside from the invasive melanoma-like phenotype the tadpoles exhibited, the animals otherwise appear normal, suggesting that this effect is not a broad misregulation of gene expression.
Levin’s lab is still working to understand how close the depolarized cells must be to the melanocytes to have this effect. His lab also aims to determine how the bioelectric signals in the microenvironment around the depolarized cells are translated into molecular signals in the melanocytes.
By identifying a bioelectrical switch that confers a phenotype in embryonic stem cells that resembles metastatic cancer, these findings strengthen the connections between stem cell biology and cancer biology.
“This phenotype bears all three of the most important properties of metastatic cells—overgrowth, invasiveness, and shape change; moreover, it is mediated by two well-known tumor markers,” said Levin. Unlike melanoma, which this phenotype most closely resembles, the black tadpoles have no primary tumor generating the metastatic cells. The phenotype may more closely resemble cancer stem cells, which are believed by many to give rise to cancer.
“These findings justify further research,” said Mustafa Djamgoz, professor of cancer biology at Imperial College London. “The role of ion channels in cancer has had a major pat on the back with this paper and, less directly, so has the stem cell hypothesis of cancer, though it doesn’t actually prove it.” The work is particularly relevant, he said, because it was done in an in vivo model system with cells in their “native, natural, and juicy microenvironment.”
Following up on this study, Levin’s lab will try to “tame” tumors by hyperpolarizing them; the idea is that if depolarization stimulates cancerous behaviors, polarization may normalize them. In addition, he will continue to explore the ways that electrical signals control cell behavior, particularly in terms of how these signals can be applied to limb and tissue regeneration (see Focus, March 9, 2007, Regenerative Biology).
While this work will likely influence both cancer research and stem cell biology, another intriguing aspect for Levin is the integration of biophysical signals into existing, but so far largely biochemically defined, signaling pathways. “Here is an example of a new and very specific information-bearing signal that is electrical in nature and ties into well-characterized genetic pathways,” he said. “This work may induce more and more people to think out of the box of purely chemical signals.”
For Students: Contact Michael Levin at mlevin@forsyth.org for more information on this and other lab projects.
Conflict Disclosure: The researchers report no conflicts.
Funding Sources: The National Institutes of Health, the American Heart Association, the National Highway Traffic Safety Administration, and the March of Dimes.
