A myriad of cells contribute to the normal function and maintenance of adult tissues. Some cells, such as goblet cells that produce mucus in the colon, play functional roles in specific tissues. These altruistic cells are terminally differentiated and will die serving the tissue. Other rare and undifferentiated cells, called stem cells, are responsible for replenishing the pool of differentiated cells while maintaining an adequate supply of themselves through the process of self-renewal. Stem cells can divide asymmetrically to produce one daughter cell that is more committed to a specific cell lineage, a transit-amplifying (TA) cell, and one that retains stem-ness. TA cells have a limited life span and self-renewal potential while repopulating the differentiated cells of the tissue.

The colon is organized in this hierarchical fashion and its epithelium is renewed every 5 days in humans 11. In this very dynamic process lies a complex collection of epithelial cell lineages along with an intricate set of molecular mechanisms to maintain order. To preserve tissue function, the colon is structurally organized in an elegant network of invaginations, termed crypts, which aid in the absorption of water and vitamins. Stem cells of the colon are located at the base of these crypts and produce epithelial cells that are committed to three different cell lineages. These differentiated cells are the absorptive enterocytes, mucus-secreting goblet cells, and hormonal enteroendocrine cells that will migrate up the crypt wall to form the colon (Figure 2).

Since one of the hallmarks of stem cells is their ability to self-renew and differentiate, investigators were prompted to explore the similarities and differences that exist between normal stem cell maintenance of tissues and organs and the uncontrolled proliferation of cancer 2. In essence, tumors can be viewed as small aberrant organs containing a hierarchy of progenitor cells and differentiated cells (Figure 2, for example). Albeit dysfunc tional when compared to physiologically functioning organs, they maintain their own abnormal proliferation and survival mechanisms 7. If tumors are indeed aberrant organs, then there is a program by which the system is controlled, however loosely it might be. Therefore, in the cellular hierarchy of a tumor, there is a differentiation mechanism from tumor stem cell to tumor progenitor cell to mature tumor cell which ends in apoptosis and turnover 6. Abnormal cellular behavior in this tightly controlled system can occur via genetic alterations, such as tumor suppressor loss or gene destabilization, which result in incremental neoplastic gains and disruption of the homeo static system 12. With these alterations, resulting in a loosely controlled system, cancer will eventually prevail. If the alterations can be restricted and coerced to force cells to terminally differentiate and die, the invading neoplastic tissue will cease to exist.

To determine what makes a normal stem cell misbehave, it must first be determined what makes them behave normally. Wnt signaling is one of the driving forces in crypt formation and maintenance of the colon 13. When Wnt proteins bind Frizzled/LRP receptors, the canonical Wnt pathway ensues. This involves the disruption of the destruction complex that sequesters beta-catenin in the cytoplasm with axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK3) beta. This complex marks beta-catenin for degradation and leads to a decrease in Wnt targeted transcription. When the complex is disrupted, beta-catenin can enter the nucleus and, in concert with transcription factor 4 (TCF4), effect transcription of Wnt targets 14. The dysregulation of the Wnt pathway and its role in self-renewal as well as differentiation have been shown to be required for the development of cancer 13. Deviance in any of these factors will tip the balance toward cancer. Furthermore, recent in vivo studies in rodents have indicated that colonic crypts are derived from Lgr5⁺ (leucine-rich repeat-containing G protein-coupled receptor 5-positive) crypt base columnar cells. Lineage-tracing assays using Lgr5-lacZ mice indicated that the rare crypt base columnar cells represent the stem cells of the colon 15. The Lgr5 gene is a Wnt target and is repressed upon inhibition of the pathway 15. Therefore, if aberrantly active, the Wnt pathway could result in an expanded progenitor population and colon cancer (Figure 1).

One of the most commonly mutated genes in human colorectal cancer is Apc, which has been implicated in both the sporadic and inherited forms of this cancer 16. As discussed above, this is one of the players involved in the destruction of beta-catenin. Upon disruption of this protein interaction, beta-catenin is free to roam into the nucleus, where it upsets gene transcription via interactions with transcription factors and subsequently disrupts homeostasis. This concept was nicely demonstrated with Lgr5⁺ colon stem cells when Lgr5-EGFP mice with floxed Apc were treated with tamoxifen. The resulting deletion of the Apc gene in the stem cell compartment supported the idea that stem cell-specific loss of Apc will result in a progressive neoplasia and eventually cancer 17. Since some of these transformed cells had retained their Lgr5 expression, Lgr5⁺ tumor cells could also be considered true CSCs and the Lgr5 epitope as a possible CSC marker (Figure 2).

Parallel to the studies investigating Lgr5 and its role as a stem cell marker of the intestine was the investigation of Bmi1. Bmi1 is a transcriptional receptor in the polycomb group (PcG) gene family and is involved in stem cell maintenance, proliferation, differentiation to more committed cells, malignant transformation, and biologic aggressiveness of human carcinomas 1819. It is a member of the PcG proteins and, more specifically, the polycomb-repressing complex 1 (PRC1), indicating a role in maintaining chromatin silencing 20. As expected, any factor involved in gene silencing has the capacity to exponentially misbehave. If the genes affected are involved in maintaining control, their silencing could perturb the structured environment of a tissue. Bmi1 transcriptionally regulates the INK4a locus encoding p16^INK4a and p19^ARF tumor suppressors 21. By suppressing a tumor suppressor, a double-negative results in a positive for cancer growth as well as for the expansion of cells with stem cell properties. Therefore, Bmi1 became an interesting endeavor for the cancer stem cell biologist. As with Lgr5, a lacZ reporter assay identified Bmi1 as the source for all differentiated cells of the mouse intestine. In addition, its ablation led to crypt loss and adenoma formation upon stable beta-catenin expression in these cells 22. The relation of Bmi1 to cancer was further enhanced by expression profiles, which showed that increased Bmi1 expression correlates with poor clinical survival in patients with colon cancer 23.

Bmi1 also gives a good example of how epigenetics can influence the initiation or propagation of cancer or both. Epigenetic modifications can result in altered signaling and function, thus contributing to the formation or progression of cancer or both 24. Being a PcG protein, Bmi1 coordinates the regulation of histone modification and methylation, adding a layer of epigenetic modification to its already existing regulatory role. As seen in breast cancer, Bmi1 overexpression can block terminal differentiation, leading to an expansion of cells capable of self-renewing 19. Normally, Bmi1 is lowly expressed in differentiated cells and highly expressed in stem cells. The increased expression of PcG proteins in metastatic breast and prostate cancer occurs in a pool of cancer cells with stem cell-like properties, indicating that this alteration may occur in the CSC population 25.

Although the studies on Lgr5 and Bmi1 suggest that the transformation of stem cells from obedient to deviant cells may lead to tumorigenesis, complete molecular mechanisms have yet to be resolved. Understanding the steps by which stem cell homeostasis is lost and cancer is initiated would pave the way for possible cancer treatments. The Hedgehog-Gli (HH-Gli) signaling pathway is proposed as a mechanism through which normal stem cell maintenance can be dysregulated, leading to colon cancer 26. In an active HH pathway, Patched is blocked, releasing Smoothened to activate Gli. Although colon cancers and their stem cells of all stages have active HH-Gli signaling, an increase in HH-Gli signaling is seen in metastases. This may indicate that while APC/betacatenin signaling disruption may be the first of many hits toward colon cancer, HH-Gli may be an added push toward deviance 27.

In addition to these pathways, other signaling mechanisms involved in normal stem cell function and homeostasis may contribute to the misbehavior evident in cancer and its stem cells. These include the phosphatase and tensin homolog (PTEN) and Notch pathways 28. PTEN is a tumor suppressor whereas the Notch pathway regulates cell fate decisions 2930. PTEN not only has been implicated in the signaling of CSCs themselves but also has been found in stromal cells of the surrounding tumor microenvironment. The genetic inactivation of Pten in stromal fibroblasts was shown to accelerate the initiation, progression, and malignant transformation of mammary epithelial tumors in mice, a phenotype that was correlated with a specific PTEN signature in patients with breast cancer 31.

A recent study of colon cancer examined FOLFOX (folinic acid, 5-fluorouracil plus oxaliplatin), a widely used treatment for colon cancer 47. In that study, initial treatment of colon cancer cells enriched the population for CD133⁺, CD44⁺, and/or CD166⁺ cells with increased levels of epidermal growth factor receptor-positive (EGFR⁺). Upon subsequent treatment of the surviving cells (the CSCs) with curcumin or curcumin plus FOLFOX, a reduction in CSCs was seen. Although the mechanism is not fully understood, it was shown to increase methylation of the EGFR promoter. This hypermethylation, via changes in the level of DNA methyltransferase 1, decreases the expression of EGFR, stabilizing the chromatin and preventing the binding of transcription factors 47. While this may be a desired effect, care must be taken to investigate possible detrimental effects of this type of treatment. Hypermethylation is also known to inactivate the transcription factor p16^INK4a 47. As discussed previously, increased Bmi1 expression leads to inactivation of p16^INK4a. Therefore, a therapy that may promote hypermethylation and gene silencing may cause additional imbalances in the already mischievous CSCs.

Recent drug screenings revealed salinomycin as a specific inhibitor of CSCs. This study focused on breast cancer and used CD44^high/CD24^low as the molecular profile of CSCs. They found that the number of these cells decreased with salinomycin treatment as compared with a current treatment of paclitaxel. Surprisingly, paclitaxel actually increased the number of CSCs. In addition, there was a decrease in tumor sphere formation as well as decreased metastases over paclitaxel 48. Although this study used breast cancer as a model, colon cancer has been shown to be resistant to paclitaxel. Modifications that include the use of a mitogen-activated protein kinase (MAPK) inhibitor in combination with paclitaxel to enhance apoptosis of colon cancer cells have been suggested 49. In light of the results in breast cancer, it may be beneficial to investigate the selection of colon CSCs by treatment with paclitaxel. Such a treatment may give a survival advantage that results in a more invasive and aggressive cancer. This is especially true given that salinomycin has been shown to induce apoptosis as well as overcome apoptotic resistance in breast cancer cells 50. There is a strong connection between colon and breast cancer because they are both epithelial cancers that resemble the tissue of origin, even at metastatic sites.

Drug screenings such as those mentioned above do not take into consideration or define the mechanisms by which the drug has affected the CSC population or the tumor as a whole. Several other studies have used what is known about stem cell and CSC signaling, discussed above, to target CSCs. The HH pathway is active in both bulk and stem cell populations of a tumor and therefore it may represent a mechanism that can be targeted for therapy. One study showed that cyclopamine, an HH inhibitor, could increase apoptosis and decrease proliferation 51. In addition, the manipulation of the Notch pathway via drug intervention may successfully target CSCs. Gamma-secretase inhibitors have been shown to reduce cancer growth, inhibit CSC self-renewal, and increase differentiation to goblet cells 5253. Similar results were found with small-molecule and RNA knockdown studies showing that the inhibition of Notch leads to increased apoptotsis, decreased self-renewal, and increased secretory cell lineage differentiation of CSCs in colon cancer 54. In yet another study focusing on the Notch pathway, inhibition of Delta-like ligand 4 (DLL4) inhibited the expression of Notch targets and reduced tumor-initiating cell frequency 55. These targeted therapies may enhance the efficacy of chemo therapeutic drugs when used in sequence or combination 46. However, one must realize that these are mechanisms shared by normal intestinal stem cells and may lead to toxicity.

While there are treatments that seem to reduce the number of CSCs, two problems remain. Just reducing the population of misbehaving cells does not solve the problem. As soon as the treatment ceases, these cells can again flourish. Unfortunately, thus far, there has been no demonstration that targeting CSCs will improve the outcome of patients with cancer.