Protein localization and Western blot analysis of NANOG in hESCs and testis -----
UCSF scientists have discovered that the activity of several embryonic stem cell genes is elevated in testicular and breast cancers, providing some of the first molecular evidence of a link between embryonic stem cells and cancer.
The finding, reported in the November issue of Cancer, suggests that the genes may play a role in the development of tumors or serve as valuable markers of tumor progression, the researchers say. As such, the genes ultimately could lead to new targets for therapy or markers for diagnosis.
A next step, according to the researchers, will be to “dig deeper” to determine the functions of the genes in the cells that make up the tumors.
Another step will be to explore whether the genes are expressed in other cancers, such as prostate cancer.
The genes are known as OCT 4, NANOG, STELLAR and GDF3.
The researchers focused on seminomas - tumors that account for 50 percent of the cases of testicular cancer, the most frequent cancer in Caucasian males ages 15-40—and breast cancers, as represented by a sample of breast cancer cells grown in the culture dish and tissue from a Stage 3 breast tumor.
Previously, the team had discovered that expression of the genes was elevated in two samples of seminomas (Stem Cells, March 2004). In the current study, they expanded their investigation, determining that the expression of the genes was elevated, at varying levels, in nine seminomas.
In the process, they identified the “window” during which seminomas begin to develop. Scientists have known that seminomas arise in sperm-producing germ cells, which are located in the testis and produce sperm in boys following puberty. Germ cells, like all cells of the body, develop from embryonic stem cells, and go through a multi-stage evolution in their structure and genetic activity before attaining their mature state.
The study revealed that the genetic misregulation leading to seminomas begins very early in the formation of sperm-producing germ cells.
Next, turning to cancerous tissue outside of the reproductive system, in this case breast tissue, the team determined, to their surprise, that the expression of the genes was active there, too.
“We thought this was going to be a phenomenon unique to germ cell tumors, says the senior author of the study,” Amander Clark, PhD, assistant research geneticist in the laboratory of co-author Renee Reijo Pera, PhD, associate professor of obstetrics, gynecology and reproductive sciences and co-director of the UCSF Human Embryonic Stem Cell Center.
“This is the first indication that these genes are markers of breast cancer. The finding might lead to information about where breast cancers come from,” Clark adds. “Are they derived from an original stem cell population, one that is expressing these genes, or do the cells in the tumor represent normal cells that have reverted to a more embryonic state that would express these genes?”
The suggestion that embryonic stem cell genes play a role in some cancers is not surprising. Embryonic stem cells, which emerge in the first five to seven days of the embryo’s development, undergo a series of steps in which they selectively “turn on,” or “express,” genes that cause the cells progressively to assume the characteristics of a particular cell type, such as that of the skin, heart or brain. Cancers, meanwhile, develop when particular genes that regulate cell growth become misregulated, leading a cell to begin proliferating excessively. Misregulated embryonic stem cell genes, scientists have reasoned, could cause or advance cancer.
Substantial evidence already suggests, in fact, that genes in adult stem cells - which reside in many tissues of the body - cause some leukemias, and that they may cause gliomas, the most common form of brain tumor ( UCSF -Nature, Feb. 19, 2004 ; UCSF - New England Journal of Medicine, Aug. 25, 2005 ).
The current study evolved from ongoing research in the Reijo Pera lab aimed at identifying the genes that lead human embryonic stem cells to evolve into early-stage germ cells, and ultimately to specialize as sperm-producing cells or eggs, or “ooctyes.” The goal of this research is to identify the genetic missteps that lead to birth defects and infertility.
As part of this research, the team had determined that the four genes - OCT 4, NANOG, STELLAR and GDF3—play a role in prompting human embryonic stem cells to develop into primordial, or earliest-stage, germ cells. Three of these genes—NANOG, STELLAR and GDF3 - are located on chromosome 12, at a site designated 12p13.
Because seminoma cells resemble these early-stage cells in structure, and have duplications in genetic material on chromosome 12p13 that are associated with invasive seminoma (though no genes have been linked to the malignancy), the team had subsequently investigated two samples of seminomas, to see if the genes were expressed at elevated levels. They were, as was OCT 4.
The current study demonstrated that the four genes had elevated expressions in the nine seminoma samples examined, when compared with normal testis tissue. GDF3 was elevated in 90 percent of cases, OCT4 was elevated in 56 percent of cases and NANOG and STELLAR expression were elevated in 33 percent of cases.
The team also examined the relative expression of these genes, and four other genes that play an important role in normal testis, in the nine samples. OCT 4 and NANOG were the highest expressed genes in each of the seminoma samples.
The variability of the genes’ expression within the very earliest stage of germ cell tumor progression, says Clark, “could tell us something about the potential for these tumors to metastasize.”
Finally, they examined breast cancer tissue and normal breast tissue. While normal tissue did not express detectable levels of the genes, both the breast carcinoma cell lines and stage 3 breast carcinoma expressed all four genes.
The scientists suspect that expression of the genes may be elevated in other non-reproductive cancers, as well, such as those of the prostate. Studies have shown, says Clark, that OCT 4, which works with NANOG to regulate the capacity of cells to specialize, causes dysplatic lesions in the intestines, skin and stomach. “It would be useful to look at NANOG in those places where we know over expression of OCT 4 results in tumor progression,” she says. “It may be playing a role there, too.”
Other co-authors of the study were Uche I. Ezeh, MD, UCSF clinical instructor of physiology, and Paul J. Turek, MD, UCSF associate professor of urology.
The study was funded by the Lance Armstrong Foundation and the National Institutes of Health.
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