Identifying Tumor Progenitor Cells

A proposed model for Cripto-1 (CR-1) gene expression and tumor forming ability in embryonal carcinoma cells. High-CR-1 cells also express stem cell proteins Oct4 and Nanog and show reduced DNA methylation. Low-CR-1 cells, however, express differentiation markers and have increased DNA modification. The level of CR-1 expression correlates with tumor-forming ability.

A proposed model for Cripto-1 (CR-1) gene expression and tumor forming ability in embryonal carcinoma cells. High-CR-1 cells also express stem cell proteins Oct4 and Nanog and show reduced DNA methylation. Low-CR-1 cells, however, express differentiation markers and have increased DNA modification. The level of CR-1 expression correlates with tumor-forming ability.

All cells within a tumor are not identical. In fact, only a small subset appears to be capable of actually generating the tumor. These tumor-initiating cells tend to resemble normal stem cells, which have the unique ability to give rise to differentiated cells while simultaneously producing additional undifferentiated stem cells. Most chemotherapeutics affect the bulk of a tumor but spare the stem-like cells, allowing the tumor to re-grow once chemotherapy is stopped. If, however, the cancer-initiating cells could be successfully targeted, cancer recurrence could be prevented.

A number of specific signaling proteins expressed by stem cells have been linked to their capacity to self-renew and might be employed to identify cancer stem-like cells. Cripto-1 (CR-1) is a cell membrane-associated protein found on the surface of embryonic cells. As described in recent reviews by Caterina Bianco, M.D., Ph.D. and by Nadia Castro, Ph.D. and their associates, CR-1 interacts with a number of other well-known stem cell genes to establish the body plan of an embryo as well as the development of organs including the heart. In adult cells, CR-1 expression is usually low but is significantly increased in several types of carcinoma. This suggested to Kazuhide Watanabe, Ph.D. and his mentor David S. Salomon, Ph.D. of the Mammary Biology and Tumorigenesis Laboratory and their colleagues that CR-1 may be a good marker of tumor progenitor cells.

The researchers decided to investigate the function of CR-1 in embryonal carcinoma (EC) cells, which resemble embryonic stem cells except that they form tumors. A spectrum of CR-1 expression was observed on the surface of the EC cells, which would be expected if high CR-1 levels were indeed a marker of stem-like cells. The researchers then separated the high-CR-1 and low-CR-1 expressing cells to examine differences in the two populations.

The high-CR-1 cells also expressed a number of other stem-cell-related genes. In contrast, low-CR-1 cells were heterogeneous, displaying a higher expression of differentiated tissue-specific genes. While no difference in proliferation was observed under normal growth conditions, the CR-1 high cells formed significantly more tumor spheres in a nutrient-depleted environment, which is a measure of stem-like activity. Finally, the researchers evaluated the tumor-forming ability of high-CR-1 and low-CR-1 cells. Both cell populations did form tumors, but those from the high-CR-1 cells had a faster growth rate and a shorter tumor-free period. This was likely due to the ability of the low-CR-1 cells to spontaneously give rise to high-CR-1 expressers, though at a low rate. Together these results indicated that CR-1 expression correlates with a cancer-initiating phenotype.

Salomon and colleagues then wanted to understand how CR-1 expression is regulated in EC cells. Members of the Tumor Growth Factor β (TGFβ) family, like Activin and Nodal, are known to regulate embryonic stem cell self-renewal. Treatment of EC cells with these growth factors increased the proportion of high-CR-1 cells. Inhibition of their cell surface receptors, however, significantly reduced the high-CR-1 population. Likewise, treating the EC cells with BMP4, a growth factor that induces cell differentiation, decreased the &be number of high-CR-1 cells, further suggesting that CR-1 expression corresponds with stem-like cells.

The researchers next assessed whether Oct4 and Nanog, two stem-cell-related factors that regulate gene expression, could control CR-1. Reduction of either Oct4 or Nanog protein levels decreased the high-CR-1 population. In fact, loss of either, blocked the TGFβ-mediated stimulation of CR-1 expression, suggesting that Oct4 and Nanog are downstream of TGFβ signaling. The scientists also demonstrated that Oct4 and Nanog directly bind a specific region of the CR-1 promoter in EC cells. Inducing differentiation in EC cells led to reduced binding of the CR-1 promoter by Oct4 and Nanog, decreasing CR-1 expression.

Another method cells use to control gene expression is the direct modification of DNA. When DNA is methylated, gene expression is blocked. The researchers found that in differentiated EC cells and in low-CR-1 cells, the CR-1 gene was shut off by being highly methylated, whereas in high-CR-1 and undifferentiated EC cells, CR-1 DNA was unmodified. These results showed that CR-1 expression was regulated by known stem-cell self-renewal factors in concert with stem-cell-like activity. But does CR-1 actually control the stem-like function of EC cells? To address this important question, the researchers eliminated CR-1 expression. Loss of CR-1 failed to decrease the expression of known stem cell-related genes and was unable to prevent the formation of tumors.

These studies clearly demonstrated that, while CR-1 is not required for the tumor initiating ability of EC cells, its expression appears to be a reliable marker for cancer progenitor populations. Thus, CR-1 can be used to direct targeted therapies to cancer stem-like cells. In fact, these researchers are currently testing a CR-1-directed drug delivery system in a phase I clinical trial. This work is an important step forward, but more research is still needed to understand and target cancer stem-like cells.

Summary Posted: 07/2010

Reference

Stem Cells 2010 Jun 14 [Epub ahead of print] PubMed Link