Protein in Embryonic Stem Cells Controls Malignant Tumor Cells

A protein that governs development of human embryonic stem cells (hESCs) also inhibits the growth and spread of malignant melanoma, the deadliest skin cancer, Northwestern University researchers have discovered. Metastatic melanoma, which develops from the transformation of skin pigment cells or melanocytes, has a death rate of more than 80 percent and a median survival of less than 7.5 months.

The Northwestern scientists, led by researcher Mary J. C. Hendrix, additionally found that the protein, called Lefty, prevents aggressive breast cancer cells from metastasizing. Death from metastatic breast cancer exceeded 40,000 in 2007, with over 180,000 new cases diagnosed in the United States.

Importantly, Lefty is secreted only in hESCs, and not in any other stem cell type tested – including stem cells isolated from amniotic fluid, cord blood or adult bone marrow – or placental cells.

Results of the study, described in an article in the March 3rd online version of The Proceedings of the National Academy of Sciences, build on an elegant body of research by the Hendrix lab to identify the genes and cellular pathways involved in cancer metastasis.

Hendrix is president and scientific director of the Children’s Memorial Research Center and professor in The Robert H. Lurie Comprehensive Cancer Center of Northwestern University and at Northwestern's Feinberg School of Medicine. Lynne-Marie Postovit, who was first author on the study and a post-doctoral trainee in the Hendrix lab, is currently an assistant professor at the University of Western Ontario, Canada.

Embryonic stem cells are pluripotent, meaning they can become any of 200-plus cell types in the adult body, depending on the signals they receive from their microenvironment (surrounding cells, tissues and vasculature). During cancer progression, malignant cells also receive and release signals from their microenvironment, cues that promote tumor growth and metastasis.

Groundbreaking work by Hendrix and colleagues is elucidating how, by becoming more like unspecialized stem cells, aggressive melanoma cells gain enhanced abilities to migrate, invade and metastasize while remaining virtually undetected by the immune system.

Hendrix and co-researchers previously demonstrated that a three-dimensional matrix conditioned by hESCs induced metastatic melanoma cells to revert to a normal, skin cell-like type with the ability to form colonies in the manner of hESCs (Postovit and Seftor et al, Stem Cells 24:501-505, 2006).

“This observation allowed us to appreciate the powerful influence of the hESC microenvironment on the reprogramming of metastatic melanoma cells,” Hendrix said.

In subsequent experiments, Hendrix, Postovit and co-researchers found that aggressive melanoma and breast cancer produce a “morphogenic” protein called Nodal, which is essential for human embryonic stem cell pluripotency (Topczewska et al, Nature Medicine 12:925-932, 2006). Other researchers have found that Nodal also is present in testicular cancer.

“Thus, Nodal may serve as a prognostic marker of aggressive behaviors in human cancers,” Hendrix said.

As described in the PNAS study, the Lefty protein inhibits production of Nodal and therefore plays a major role in embryonic cell differentiation and development – under normal circumstances.

Hendrix and colleagues discovered that metastatic tumor cells do not express Lefty, allowing them to overproduce Nodal in an unregulated manner.

However, when the group exposed metastatic tumor cells to the microenvironment of hESCs containing Lefty, they witnessed dramatically reduced Nodal expression (production) in these cancer cells together with decreased tumor cell growth and invasiveness and an increase in apoptosis, or programmed cell suicide.

Although exposure to a hESC microenvironment inhibited Nodal expression and tumor growth in both metastatic melanoma and breast cancer cells, the breast cancer cells underwent more complex reprogramming. Melanoma cells responded to the hESC-derived factors within three days, but breast cancer cells required two additional days to achieve the most significant reduction in Nodal.

This discrepancy is likely due to differences in signaling mechanisms between the two cell types. Yet, despite the inherent differences between melanoma cells and breast cancer cells, these divergent tumor types both underwent cell suicide following exposure to the hESC microenvironment.

“The remarkable similarity of the responses of the two tumor types is likely attributable to the commonality of plasticity (for example, the aberrant and unregulated expression of Nodal) that indiscriminately unifies highly aggressive cancer cells, regardless of their tissue of origin,” Hendrix said.

“Further, the tumor suppressive effects of the hECs microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored novel therapeutic modalities for cancer treatment,” Hendrix said.

However, while findings from the study suggest that hESC-derived Lefty may have potential to prevent metastasis, it is not the only tumor suppressive factor within the embryonic microenvironment.

Observations from the study highlight the potential utility of isolating factors within the hESC microenvironment responsible for influencing tumor cell fate and reversing the cancerous properties of metastatic tumor cells, such as melanoma and breast cancer.

Additional contributing authors on the study include N. Margaryan; E. Seftor; D. Kirschmann; D. Abbott; W. Wheaton; A. Lipavsky; and R. Seftor.

This study was supported by grants from the Illinois Regenerative Medicine Institute; the National Institutes of Health (CA50702 and CA121205); the Charlotte Geyer Foundation (to Dr. Hendrix); and a Canadian Institutes of Health Research Postdoctoral Fellowship (to Dr. Postovit).
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Human skin cells into embryonic stem cells

UCLA stem cell scientists have reprogrammed human skin cells into cells with the same unlimited properties as embryonic stem cells, without using embryos or eggs.

Led by scientists Kathrin Plath and William Lowry, UCLA researchers used genetic alteration to turn back the clock on human skin cells and create cells that are nearly identical to human embryonic stem cells, which have the ability to become every cell type found in the human body. Four regulator genes were used to create the cells, which are called induced pluripotent stem cells, or iPS cells.

The UCLA study confirms the work of researchers Shinya Yamanaka at Kyoto University and James Thomson at the University of Wisconsin, first reported in late November 2007. The UCLA research appears today in an early online edition of the journal Proceedings of the National Academy of the Sciences.

The implications for disease treatment could be significant. Reprogramming adult stem cells into embryonic stem cells could generate a potentially limitless source of immune-compatible cells for tissue engineering and transplantation medicine. A patient's skin cells, for example, could be reprogrammed into embryonic stem cells, and those stem cells could be prodded into becoming various cells types — beta islet cells to treat diabetes, hematopoetic cells to create a new blood supply for a leukemia patient or motor neuron cells to treat Parkinson's disease.

"Our reprogrammed human skin cells were virtually indistinguishable from human embryonic stem cells," said lead author Plath, an assistant professor of biological chemistry and a researcher with UCLA's Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research. "Our findings are an important step towards manipulating differentiated human cells to generate an unlimited supply of patient-specific pluripotent stem cells. We are very excited about the potential implications."

The UCLA work was completed at about the same time the Yamanaka and Thomson reports were published. Taken together, the studies demonstrate that human iPS cells can be easily created by different laboratories and are likely to mark a milestone in stem cell-based regenerative medicine, Plath said.

These new techniques to develop stem cells could potentially replace a controversial cell-reprogramming method known as somatic cell nuclear transfer (SCNT), which is sometimes referred to as therapeutic cloning. To date, therapeutic cloning has not been successful in humans. However, top stem cell scientists worldwide stress that further research comparing these reprogrammed iPS cells with stem cells derived from embryos — considered the gold standard — is necessary. Additionally, many technical problems, such as the use of viruses to deliver the four genes for reprogramming, need to be overcome to produce safe iPS cells that can be used in the clinic.

"Reprogramming normal human cells into cells with identical properties to those in embryonic stem cells without SCNT may have important therapeutic ramifications and provide us with another valuable method to develop human stem cell lines," said first author Lowry, an assistant professor of molecular, cell and developmental biology and a Broad Stem Cell Center researcher. "It is important to remember that our research does not eliminate the need for embryo-based human embryonic stem cell research but rather provides another avenue of worthwhile investigation."

The four genes used in combination to reprogram the skin cells regulate expression of downstream genes and either activate or silence their expression. The reprogrammed cells were not just functionally identical to human embryonic stem cells — they also had an identical biological structure, expressed the same genes and could be coaxed into giving rise to the same types of cells.

The UCLA research team included four young scientists recruited to UCLA's new stem cell center following the passage of California's Proposition 71 in 2004, which created $3 billion in funding for embryonic stem cell research. The scientists were drawn to UCLA in part because of California's stem cell research-friendly atmosphere and the funding opportunities created by the initiative. In addition to Plath and Lowry, the team included Amander Clarke, assistant professor of molecular, cell and developmental biology, and April Pyle, assistant professor of microbiology, immunology and molecular genetics.

The creation of the human iPS cells is an extension of Plath's work on mouse stem cell reprogramming. Plath headed one of three research teams that were able to successfully reprogram mouse skin cells into mouse embryonic stem cells. That work appeared in the inaugural June 2007 issue of the journal Cell Stem Cell.

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