NEUROBLASTOMA STEM CELLS
SCIENTIFIC SUMMARY

Introduction
Neuroblastomas account for 97% of neuroblastic tumors; neuroblastic tumors include neuroblastomas, ganglioneuromas, and ganglioneuroblastomas [1]. Neuroblastomas occur in infants and young children; it is rarely found in children over the age of 10 years. The development of neuroblastoma is thought to be derived from precursor cells of the sympathetic nervous system [2]. Neuroblastoma usually develops in the adrenal gland above the kidney. However, it may also develop in other locations and may metastasize throughout the body. (Figure 1) Most occurrences of neuroblastoma are found in children under the age of 14 [3]. Neuroblastoma has shown varied clinical behavior. There are four stages of neuroblastoma ranging from stage I being the least developed to stage IV being the most developed with possible metastasis [3]. Table 1 indicates the stages as defined by the International Neuroblastoma Staging System [4].  The most developed aggressive form of neuroblastoma is called “neuroblastoma proper”. In this form of neuroblastoma there is severe damage to the sympathetic nervous system and a tumor can usually be found in the adrenal gland [2]. The second less aggressive form of neuroblastoma which comprises both tumor and non-tumor cells is called “ganglioneuroblastoma”. Finally there is a benign form or neuroblastoma termed ganglioneuroma [1]. Approximately half the population of children that have neuroblastoma have the more aggressive form with metastasis and the survival rate for these children is low [5].

Treatment and Prognosis of Neuroblastoma
Neuroblastomas have diverse clinical behavior possibilities, which can include spontaneous regression (neuroblastoma in infants has been seen to disappear on its own without any treatment), metastasis, and evolvement into a ganglioneuroblastoma or a benign ganglioneuroma. Various factors are involved in determining prognosis and treatment options, including but not limited to metastasis extent, age of onset/diagnosis, and molecular markers of the tumor.

Evidence of Cancer Stem Cells in Neuroblastoma
Cancer stem cells are defined as cells that become biologically deregulated and gain the ability to proliferate uncontrollably through genetic or microenvironmental changes [6]. Like the typical stem cell, these cells have the ability to self-renew. However, cancer stem cells are not under proper regulatory control.  They are thought to compose only a small portion of the actual “cancer” or “tumor” [6]. It is still unclear whether cancer stem cells are actually derivatives of normal stem cells that have become mutated or if they are progenitors that have acquired mutations which give them the ability to act as stem cells [7].

The ability of neuroblastoma to persist in young children had been studied for many years, however new findings suggest that this ability to relapse after chemotherapeutic treatment may be due to cancer stem cells. One of the most important aspects of neuroblastoma that supports the presence of stem cells is the aspect of cellular heterogeneity within the tumor [4]. To be specific, a single tumor consists of many subcellular phenotypes, including neuroblasts, non-neuronal cells, and/or melanocytes. Tumors can also be stroma-rich and stroma poor.  Cell-lines derived from neuroblastoma tumors can also show cellular heterogeneity [8].

Studies on the different types of cells in neuroblastoma have revealed that there are three phenotypically distinct types of cells. These cells can be termed N cells, S cells and the most recently discovered I cells. N cells, sympathoadrenal neuroblasts, are embryonic neuronal precursors containing many of the same enzymatic and biosynthetic properties of a neuron. [9] N cells aggregate with short neuritic processes and show uncontrollable growth, forming slowly growing tumors in mice [8]. S cells, substrate adherent cells, show contact inhibition of cell growth. S cells can form melanocytes, Schwann cells, and smooth muscle cells, [10] but are not tumorgenic in athymic mice. [8, 9] This leads to the third distinct cell type, I cells. These cells were termed I, intermediate, because they appear to be a cross of N and S cells. I cells appear to be a more primitive precursor to S and N cells, as they are capable of self renewal and bidirectional differentiation. [8] When I cells are treated with factors that allow for differentiation among the N or S cell lines they show the ability to differentiate among both lineages. This ability is not present in N or S cells. This gives rise to the thought that I cells are the cancer stem cells and that N and S cells are cells that are derived from I cells. One important finding of I type cells is that only I cells not N or S cells express stem cell markers CD133 and c-kit. [4] It has also been observed in culture that I cells possess the greatest malignant ability as opposed to N cells or S cells [4]. I-type cell lines had the greatest potentials for tumor-forming capabilities [8]. This important finding indicates that I cells may be cancer stem cells and are important targets in a therapeutic approach to destroy neuroblastoma tumors. (Figure 2) It is clear that additional studies of I-type cell lines are essential to understanding the biology of malignant neuroblastoma stem cells.

Walton et al confirms that I-type cells are malignant stem cells, capable of self-renewal and proliferation as well as further differentiation. Patients with progressive neuroblastoma disease have shown greater frequency of I-type cells than those patients without recurrence of disease [8].

Another strong piece of evidence that neuroblastoma comprises cancer stem cells is the discovery of side population cells among the tumor. One study looked at cells from patients who had a relapse and whose tumor was undifferentiated [10]. Approximately 66% of the patients had side population cells within their tumor and these cells ranged in amount present among the tumor [4]. They further went on to look at cell markers of the side population cells to further prove these side population cells as cancer stem cells. Some of the markers that were used are c-kit/CD117, AC133/CD133, CD71 and CD56. Studies have also shown the ability of neuroblastoma cells to expand many times in culture [11]. This multipotent ability is a defining characteristic of stem cells further indicating the presence of stem cells among the neuroblastoma cell population.

As mentioned above, neuroblastoma can take on a very aggressive form in some children.  This aggressive form of neuroblastoma has been treated with multiple combined approaches such as chemotherapy, surgery and bone marrow stem cell transplants [12]. However, there is still a low rate of survival among patients with stage IV neuroblastoma [5]. While a small portion of patients do better after receiving chemotherapy, surgery and bone marrow transplants, many patients will succumb to aggressive neuroblastoma. One of the most important aspects of a stem cell is the ability to self renew. This ability of neuroblastoma to persist among patients even though therapeutic agents such as chemotherapy and bone marrow transplants have been used, suggests that some cells remain and have the ability to proliferate indefinitely. This supports the idea that neuroblastoma cells are primitive and are comprised of cancer stem cells (Figure 3).

Potential Cell-Signaling Cascades and Cellular Markers Associated with Neuroblastoma Tumorgenesis
The regulation of stem cells is extremely important for them to function properly. When these cells are disrupted or disturbed they may be genetically altered and function improperly. This will result in a cancer stem cell. One important regulation factor in neuroblastoma is MYCN. MYCN, a probable transcription factor, is amplified in neuroblastoma tumors and is important for the growth and development of neuroblastoma cells. This has been found in a significant amount of neuroblastoma tumors and is considered a proto-oncogene. MYCN amplification has been linked to progressively poorer prognosis for patients. The amplification of MYCN is most prevalent in the more severe cases of neuroblastoma indicating its importance in the cancer. Individuals who have high amplification of MYCN have shown worse survival rates than those with less amplification [13].

Koppen, et al further investigated MYCN oncogene involvement in cell proliferation with regards to minichromosome maintnenance complex (MCM). MYCN was found to stimulate expression of the complete MCM complex. [14] MYCN is part of the Myc oncogene family. Cell-cycle inhibitory genes are repressed by Myc family proteins, and conversely cell-cycle activation cyclins are stimulated by the Myc family. This allows for research into the role of MYCN as an agonist of cell proliferation, particularly in neuroblastomas. Amplification of MYCN by Koppen et al was shown to induce activation of all MCM genes. The researchers concluded that induction of the MCM complex by MYCN may play an important role in the stimulation of G1 to S phase transition in the cell cycle and DNA replication, thus allowing for an increase in cell proliferation [14].

Schwab, et al has researched the role of B-MYB oncoprotein in neuroblastoma survival. B-MYB protein, when exposed to UV irradiation in Ewing sarcoma cells, is destroyed and eventually leads to apoptosis of the cell. However, exposing neuroblastoma cells to UV irradiation has no effect on B-MYB concentration levels.

It had been previously shown that increased B-MYB phosphorylation regulates the transcriptional activity of the protein, increasing its levels in the cell but also initiating the negative-feedback loop in promoting its destruction. Upon further testing of B-MYB in neuroblastomas, researchers found that it is hypophosphorylated in neuroblastoma cells. Impairing B-MYB phosphorylation by means of mutation does not cause inactivation of B-MYB function; instead, it facilitates resistance to UV-irradiation induced apoptosis [15].

Importance of Future Research of Therapeutic agents for Neuroblastoma
Neuroblastoma is one of the leading causes of cancer in young children today. It affects 1 out of 100,000 children each year with 50% of the children who are diagnosed die from the cancer [1, 13] As mentioned earlier this form of cancer primarily affects children and 90% of the cases diagnosed are in children under the age of 5 [13]. This form of cancer accounts for approximately 10% of all cancers seen in children [1]. A large portion, 40%, of children who have neuroblastoma have the more severe and malignant form of neuroblastoma [13].

While neuroblastoma has the highest incidence of regression of all childhood cancers in some forms of the disease it is still of major clinical importance for those that succumb to the disease. The extreme variability in clinical behavior has puzzled many clinicians and scientists thus challenging efficacious therapy.

Singh, et al published evidenced of a possibility to use indole ethyl isothiocyanate analogs as a potential therapeutic agent against neuroblastoma. The specific anti-tumor agent investigated was 7-Methyl-indole-3-ethyl isothiocyanate (7Me-IEITC), which was seen to be cytotoxic to a few neuroblastoma cell lines. 7Me-IEITC was used in neuroblastoma cell lines with primary lung fibroblasts used as a control (due to the fact that they maintain a high metabolism and growth rate similar to neuroblastomas). Effects on pro-survival cellular markers, pro-apoptotic signaling, and apoptotic responses were analyzed.
Pro-survival cellular marker Akt can initiate resistance of the actions of chemotherapy agents. Apoptotic-commencing cell pathways such as JNK and caspase-activation were enhanced by 7Me-IEITC, concluding that its cytotoxicity could be implemented as a potential treatment for neuroblastoma [14].

CPT-11 is a topoisomerase I inhibitor, found to be an anticancer drug useful in the treatment of neuroblastoma [16]. Researchers at St. Jude’s have found that more efficient activation of CPT-11 occurs with the aid of a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors than with human enzymes alone in a human fetal telencephalon-derived immortalized cell line. In their work, 90% of those mice treated with the rCE survived for 1 year without tumor detection [17].

Recent evidence has shown that stem cells play a significant role in cancer, especially in neuroblastoma. A suitable treatment to eradicate the tumor may indeed need to be to target these cancer stem cells. The first step towards this is to identify the cancer stem cells from the entire population of tumor cells. Once this can be successfully carried out then we can begin to understand the cancer stem cells and why they become oncogenic. Interpreting what factors cause these stem cells to become deregulated can allow us to target this specific population of cells. With this knowledge in combination with already mentioned techniques such as chemotherapy there is the possibility of eradicating the cancer entirely.

Figure 1

Figure 2

Figure 3
Table 1: International Neuroblastoma Staging System [18]

REFERENCES

Acknowledgements
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:
Babak Baseri, Jennifer Brady, Nadia Senmartin, Fall 2006 (in alphabetical order)
Teaching Assistant: Kathy Trzaska
Updated by Aysha Mirza, Fall 2007.