After analyzing the properties of cancer stem cells, scientists from Ohio State University Medical Center, L. Chen et al., reported in the March 15th online edition of PLoS their results on identifying and characterizing a precancerous stem cell population (pCSCs) associated with leukemias. Cloning cells with a dendritic cell-like phenotype from the spleen of leukemic mice, the investigators identified a subpopulation of precancerous stem cells with the CD45^- c-kit^- Sca1^- Lin^- CD44^high (CD45KSL- CD44^high) phenotype. These pCSCs had multiple cytogenetic alterations and were phenotypically distinct from differentiated cancer cells in the same host. Using a colony-forming assay for measuring hemapoietic multipotency, in vitro experimental results demonstrated that the pCSCs had lost some but not all of their potential for hemapoietic differentiation. In competitive engraftment experiments, pCSC-derived cells were found circulating in the blood 8 weeks after transplantation of both host's hematopoietic progenitors and pCSCs in a lethally irradiated host. However, the cells disappeared from circulation 13 weeks post transplantation. The authors surmised that the pCSCs had undergone differentiation-induced cell death (DICD) as a protective mechanism that prevents the formation of malignant cells. The experimental data also demonstrated long-term repopulating activity in the bone marrow of the recipients. Engrafted pCSCs were also found in the liver, kidney, small intestine, and adipose tissues of the host. In immunodeficient mice, the pCSCs had progressed to cancer concomitant with phenotypic and genetic changes of the cells. Analysis of the tumor cells in vitro showed up-regulation of CD45 and lineage markers (CD3e,CD11b, B220, Ter-119 and Gr-1). From the data, the investigators propose pCSCs progressing to cancer cells in the following manner: CD45^- c-kit^- Sca1^- Lin⁺ through CD45^- c-kit⁺ Sca1⁺ Lin⁺ through CD45⁺ c-kit⁺ Sca1⁺ Lin⁺. The researchers also reported that ectopically expressed piwil2 gene regulates in vitro proliferation of pCSCs. It was also noted that the fate of pCSCs is determined by environmental cues in which they can differentiate into benign cells in an immunocompetent host or progress to cancer in an immunodeficient host. The authors speculated that their newly identified pCSCs may be an intermediary cell between committed progenitors and a cancer stem cell.

Nestin is believed to be a stem cell marker found in cells that are descendants of the ectoderm germ layer lineage. Researchers from the Dartmouth School of Medicine are now reporting experimental evidence for a nestin+ progenitor cell leading to pancreatic ductal adenocarcinoma (PDAC). Using mice that express the mutated oncogene Kras (KrasG12D), C. Carriere et al., published in the March 13th issue of PNAS their findings that specific targeting of the Kras mutation of the ductal cell compartment led to the formation of pancreatic intraepithelial neoplasia, a precursor to PDAC. Since this form of neoplasia was of the same grade and frequency to Kras mutation targeting the whole pancreas by Pdx1-driven Cre/lox recombination, the authors concluded that the nestin positive cells were highly responsive to the Kras oncogenic activation, and these cells potentially represent the putative precursors leading to PDAC. (Here we find indirect evidence suggesting the formation of cancer stem cells in the ductal epithelial of the pancreas by targeting specific genes in the stem cell compartment whose mutant forms represent oncogenes.)

In 2000, Susan Bonner-Weir and colleagues from the Joslin Institute at Harvard University published (July 2000, PNAS, 97 {14}: 7999-8004) their groundbreaking results demonstrating the ability to generate human islets through in vitro expansion of the ductal tissues of the pancreas. Although this phenomenon was interpreted as transdifferentiation of the ductal epithelium, the results could also be interpreted that a pool of primordial stem cells resides within the ductul epithelium of the pancreas which are being expanded and differentiating into β-cells. In delineating the plasticity of both β-cells and ductal cells in their ability to transdifferentiate, O. Strobel et al. from Massachusetts General Hospital and Harvard Medical School, conducted in vivo studies for analyzing the cells involved in both regeneration of normal tissues and the formation of tubular complexes (metaplastic ductal lesions/intra-epithelial neoplasia) following inflammatory injury of the pancreas. The authors published the results of their study in the March 13th online edition of PNAS. The study was based upon the observation that metaplastic ductal lesions are found in both chronic pancreatis and pancreatic cancer. The presence of these lesions may be the precursor to "inflammation-mediated carcinogenesis." However, the authors noted that the pancreatic cells giving rise to these tubular complexes is controversial based upon both in vitro and in vivo studies conducted by others; particularly, the in vitro studies in which the interpretation of the data are subject to ductal cell-associated contamination. With RIP-CreER Z/AP mice, the investigators conducted in vivo studies to trace the lineage of β-cells and their contribution to normal tissue repair and metaplasia following cerulein-induced pancreatitis. The authors concluded that the results of the study ruled out the possibility of transdifferentiating β-cells giving rise to normal acinar and ductual tissues. Lineage tracing also ruled out the suggestion that formation of metaplastic ductal lesions are of β-cell origin. (This study is particularly interesting since it merges the alternative interpretation of Bonner-Weir that a stem cell compartment may exist in the ductal region of the pancreas, and during chronic inflammation these stem cells could give rise to a cancer stem cell.)

In 1976, Peter C. Nowell proposed the "clonal evolution" hypothesis of cancers. Briefly, he argued that cancers evolved over time as a result of greater genetic instabilities in somatic cells. These abnormal cells eventually give rise to a heterogeneous population of mutant clones having diverse genetic variations within tumors from different individuals diagnosed with the same type of cancer. However, in 2003, Michael Clarke and Sean Morrison and their group from the University of Michigan published (M. Al-Hajj et al., 2003, PNAS 100: 3983-3988) their finding in which they isolated a very small population of CD44⁺CD24^low cells in breast cancer patients which were highly tumorigenic and of an adult stem cell-like phenotype. These rare "cancer stem cells" within a tumor were described as the cancer seeds for most cancers. However, researchers from the Dana-Farber Cancer Institute and Harvard Medical School, M. Shipitsin et al., reported in the March 13th issue of Cancer Cell the results of their study on examining the gene expression profiles of both cancerous and normal breast tissues. The investigators found both CD24+ and CD44+ cells in tumors that were clonally related, but not always identical. The genetic differences between the two cell populations seemed to support more the "clonal evolution" of cancer than the "cancer stem cell" hypothesis. The data suggest that cancers' stem-like progenitor cells arose from mutation of normal cells which gave rise to a genetically diverse subpopulation of cancer-forming cells. However, the researchers also found that the virulence of the cancer cells was associated with CD44+ cancer cells expressing many of the known stem cell markers (e.g. expressing genes such as Bmi1, Gili1 and Gili2). It was also found that the TGF-β1 pathway was upregulated in these CD44+ cells. By measuring the TGFBR2 mRNA, the investigators reported that the CD24+ cells were hypermethylated and epigenetically distinct from the CD44+ cells. Remarkably, inhibitors of the pathway induce the CD44+ cells to terminally differentiate into an epithelial phenotype. The data also revealed that high expression of TGF-β was associated with shorter metastasis-free survival and disease progression. Since the CD24+ cells were very similar to the CD44+ cells, but not always genetically identical, the authors concluded that the findings fit more closely with the clonal evolution model than the cancer stem cell model. (Although the data suggest the clonal evolution involving intratumoral heterogeneity as an alternative to the cancer stem cell hypothesis, it still does not rule out the possibility that a cancer stem cell can divide asymmetrically giving rise to a number of closely related progenies. These progenitor stem cells could produce clones of different genetic signatures while the one of the daughter cell from a dividing cancer stem cell maintains its capacity as an unit of self-renewal.)

M. Milyavsky et al. from the Weizmann Institute of Science reported in the February 13th issue of Cancer Cell the results of their study on the mechanism in which the repression of the regulatory transcription factor, myocardin, can lead to human malignant transformation. Myocardin expression regulates differentiation into smooth and cardiac muscle during tissue regeneration. Myocardin is also a transcriptional target of TGF-β during differentiation of human fibroblasts. The investigators also found that in vitro serum deprivation, intact contact inhibition response, and the p16 ink4a/Rb pathway contribute to the expression of myocardin and subsequent differentiation. More importantly, the study provides experimental evidence in which restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth. In fibroblast cultures, inactivation of myocardin results in a hyperproliferative fibroblast response. The authors also noted that myocardin expression is dramatically reduced in many forms of human tumors. (This study indirectly supports the concept of a stem cell which under conditions that inhibit differentiation can result in malignant transformation.)

Researchers from Johns Hopkins and Stanford Universities, C. D. Peacock et al., published in the Feb. 28th online edition of PNAS their study on cancer stem cells in multiple myelomas and hedgehog (Hh) signaling in the the maintenance of the tumor stem cell compartment. The study was based upon the hypothesis that cancer stem cells give rise to malignant growth in tissues and that aberrant activation of the Hh signaling pathway may be involved in the maintenance of such stem cells in cancer. For example, Hh signaling normally regulates progenitor cell fate in development and homeostasis; however, mutational activation of the Hh pathway is associated with medulloblastoma and basal cell carcinoma. Studying multiple myeloma with a well defined stem cell compartment (CD138^negCD19+ progenitors reflecting a memory B cell phenotype), the investigators examined role of Hh activity in a subpopulation of these cancer stem cells. In the study, the investigators found that Hh activity was associated within a subset of the multiple myeloma cells in the tumor stem cell compartment. Additionally, it was found that the Hh ligand induces activation of smoothened (Smo) which results in clonal expansion of the cancer stem cell compartment. However, an Hh blockade by cyclopamine inhibits Smo and Hh signaling. Thus, inhibition of Hh activity resulted in terminal differentiation of these cancer stem cells into CD138+CD19^neg malignant plasma cells. The authors concluded that their experimental data both in vitro and in vivo suggest that multiple myeloma provides an ideal model to study the use of clinical drugs such as cyclopamine as an inhibitor of Hh signaling and as and anti-cancer agent through terminal differentiation of the cancer stem cells.