A simple twist of cell fate

Tapping an embryonic program, adult cells gain properties of stem cells

Human mammary epithelial cells that undergo an epithelial-mesenchymal transition (EMT) may grow in suspension into structures called mammospheres—a trait of stem cells. Image: Sendurai Mani

Suddenly the pieces fit.

For years, scientists in Robert Weinberg’s lab had been patiently examining two puzzles in the vast jigsaw of cancer research.

One was the ability of some cancer cells to migrate through the body by reactivating biological programs that have lain dormant since the embryo stage.

The second was the role of cancer stem cells, the self-renewing, tumor-seeding cells that have been found in a number of solid tumors by several labs in the past five years.

For years, these efforts proceeded in parallel. But in a startling paper published in May, researchers reported that these two areas overlap—cancer cells that are induced to follow one of these pathways may gain properties of adult stem cells. Moreover, this mechanism seems to work in normal cells as well.

Top: In mammospheres, some cells present surface proteins indicating that they can develop into more than one type of mammary cell (the cells in yellow have the potential to differentiate into two types). Bottom: When further cultured, mammospheres start to develop complex secondary structures, with inner layer cells and outer layer cells similar to those in normal mammary ducts. Images: Wenjun Guo

The finding may offer major implications for cancer treatment and for regenerative medicine.

“This for us is a very exciting discovery, not only because of its unexpectedness but because it offers a route by which one could in principle generate unlimited numbers of stem cells committed to creating a specific cell type,” says Weinberg, a Whitehead Member and professor of biology at Massachusetts Institute of Technology. “One could imagine, for example, that if one takes skin cells and induces them to follow this pathway, they could become skin stem cells.”

Switching cell states

In a paper published in Cell, former Weinberg lab postdoctoral researcher Sendurai Mani and his colleagues demonstrated in mice and in human cells that cells that have undergone an “epithelial-mesenchymal transition” (EMT) acquire several important characteristics of stem cells, including the ability to self-renew.

Conversely, the researchers also showed that naturally existing normal stem cells as well as tumor-seeding cancer stem cells present characteristics of the post-EMT cells, including the acquisition of mesenchymal cell traits, which are usually associated with connective tissue cells.

Epithelial cells, which make up most of the human body, bind together in sheet-like structures. In embryonic development, the EMT process breaks up cell-cell adhesion in the epithelial layer and converts epithelial cells into more loosely associated mesenchymal cells. In the context of cancer development, some cells within a primary cancer may undergo an EMT, migrate through the body to their end destination, and there resume their epithelial form through a reverse process (the mesenchymal-to-epithelial transition).

Earlier, Mani and his colleagues had identified FOXC2, one of the key genes involved in invasion and metastasis. FOXC2 appears to program the metastatic ability of some breast cancers.

Mani knew that during embryonic development, FOXC2 expression is restricted to mesoderm (one of three layers in early embryos) and mesoderm-derived cells before they specialize into a differentiated state. FOXC2 expression disappears once these cells differentiate. Similarly, his experiments showed that epithelial cells that undergo EMT express FOXC2, but that expression is lost when they revert to an epithelial state.

A question of identity
In collaboration with Andrea Richardson and Jeffery Kutok, pathologists at Boston’s Brigham and Women’s Hospital, Mani went on to study FOXC2 expression in normal human breast tissue. It turned out that cells expressing this gene were located precisely where researchers expect to find mammary epithelial stem cells.

Sendurai Mani (far above), Mai-Jing Liao (above left) and Wenjun Guo (above right) collaborated to show that adult cells that reactivate long-dormant biological programs may acquire traits of stem cells—a finding with potentially major impact on cancer treatment and regenerative medicine. Photos: Rocky Kneten (Mani), Tim Gray (Guo), Ryan Donnell (Liao)

As he pondered these findings and the earlier results about FOXC2’s role in metastasis, Mani wondered: Just what were these cells generated by EMT that expressed FOXC2?

Were they simply fibroblasts, the most common cells in normal connective tissue?

Or were they actually stem cells?

“I asked Mai-Jing Liao, another postdoc in the Weinberg lab, to check whether cells generated by EMT would have any stem cell properties,” recalls Mani, now an assistant professor in the department of molecular pathology at the University of Texas’s M. D. Anderson Cancer Center in Houston. “He said, ‘You must be out of your mind, but it won’t take more than half an hour to check.’”

Much to Liao’s surprise, when he examined mammary epithelial cells that had undergo an EMT, his tests did highlight surface proteins that are known to be key markers for stem cells.

The researchers found that the cells that underwent the EMT process were mesenchymal-like in appearance and demonstrated stem-cell surface markers.

These cells also displayed an increased ability to grow in suspension, forming structures called mammospheres—another trait of mammary stem cells. Some cells in the resulting mammospheres showed, in turn, stem cell markers, indicating they could differentiate into two kinds of mammary cells. And cells in the mammospheres retained their stem cell properties even after the EMT induction process was stopped.

Furthermore, when the Weinberg lab scientists isolated stem-cell-like cells from cultured human mammary epithelial cells or from mouse breast tissue, their properties were very similar to the EMT-induced cells. Working with Kornelia Polyak of Dana-Farber Cancer Institute and Harvard Medical School, Mani found that this was also true with normal and tumor cells obtained from human patients.

Top, normal human mammary epithelial cells in culture. Bottom, the epithelial-mesenchymal transition (EMT) generates cells that are more loosely associated and offer some of the properties of adult stem cells. Images: Wenjun Guo

Creating cancer stem cells
Importantly, the researchers also demonstrated that inducing the EMT process can produce cells with many characteristics of cancer stem cells.

This finding could help to answer a key question about metastasis: When tumor cells spread into different sites, how do they multiply enough to form a dangerous new tumor?

“If you take a population of human cancer cells that normally form a tumor very inefficiently and induce an EMT, their tumor-initiating abilities increase by about a hundred-fold, so that it takes about 10,000 cells rather than a million cells to form a tumor,” says Wenjun Guo, co-lead author on the paper and postdoctoral researcher in the Weinberg lab.

“This suggests cancer stem cells are using pre-existing normal stem cell machinery to propagate their own self-renewal and therefore their tumor-initiating ability,” he emphasizes.

Guo and other researchers in the Weinberg lab are now investigating the EMT process with other cell lines. They also will attempt to give final proof in mice that the process creates completely defined stem cells, doing so by taking cells from mouse mammary fat pads, inducing an EMT for some of the cells, returning the resulting cells to the fat pad, and seeing if they can regenerate the mammary gland.

Mani, meanwhile, is continuing his own research on the EMT/stem cell connection and its role in cancer metastasis at the M. D. Anderson Cancer Center. Putting together this scientific puzzle took him no fewer than eight years as a postdoc. “But I have no complaints,” he remarks. “This is enormously exciting.”