Dr. Weeks’ Comment: Cancer STEM cells need to be doctors for cancer to go to remission and stay in remission. Cancer STEM cells are what kill us. Determine what your oncologist knows about cancer STEM cells… if nothing… flee!
“…The most extensively validated hit from their screen is salinomycin, a highly selective potassium ionophore, facilitating bidirectional ion flux…”
Breast Cancer Stem Cells: Eradication by Differentiation Therapy?
1The Institute of Molecular Pathology, Dr. Bohrgasse 7, A 1030 Vienna, Austria
During metastasis, migrating breast cancer stem cells undergo a loss of polarity leading to an epithelial-to-mesenchymal transition (EMT). Gupta et al. (2009) use this attribute of cancer stem cells to develop a high-throughput screen, which successfully identifies small molecules that specifically inhibit cancer stem cell proliferation through the induction of differentiation.
Although tumor metastases are the cause of death in more than 80% of human cancer patients, the molecular mechanisms underpinning metastasis are still poorly understood. However, one theme that has emerged from recent work is that metastasis involves defects in the molecularmachines responsible for epithelial polarity and hence for the epithelial-to-mesenchymal transition (EMT) (Kalluri and Weinberg, 2009). Epithelial cell polarity is established by multiple cellular processes, including polarized trafficking of cytoskeletal and plasma membrane proteins, the maintenance of a diffusion barrier (tight junctions) (Humbert et al., 2008), and a 3D organization machinery that integrates extracellular information from receptors, adhesion proteins, and neighboring cells (Tanos and Rodriguez-Boulan, 2008). Hence, the loss of epithelial polarity during EMT can result from altered regulation of many different signaling pathways, transcription factors, chromatin regulators, and proteins involved in cell adhesion and polarity (Kalluri and Weinberg, 2009; Tanos and Rodriguez-Boulan, 2008).
Indeed, the list of oncogenes and tumor suppressors that modulate cell polarity is long and growing (Humbert et al., 2008; Tanos and Rodriguez-Boulan, 2008). In recent work, it has been shown that epithelial stem cells may undergo EMT and that EMT induction endows epithelial cells with salient features of stem cells. These cells can also exhibit properties of cancer stem cells upon overexpression of oncogenic Ras (Mani et al., 2008). In their current work, published in this issue of Cell, Gupta et al. (2009) use these newly discovered attributes of mammary epithelial cells (that is, induction of EMT and stem cell features by defined genetic alterations) to establish a high-throughput screen for compounds that selectively target cancer stem cells. Gupta et al. use telomerase-immortalized human mammary epithelial (HMLE) cells, in which knockdown of E-cadherin by RNA interference promotes EMT and the acquisition of features typical of cancer stem cells, including high levels of CD44, low levels of CD24, and the capacity to form mammospheres (Figure 1).
Cells depleted of E-cadherin also show increased resistance to several established tumor chemotherapeutics. In this respect, they resemble human breast carcinoma stem cells that contribute to tumor relapse in vivo. Gupta et al. establish that these cells are ideally suited for a high-throughput, cellular screen for compounds that selectively eliminate cancer stem cells (Figure 1). The most extensively validated hit from their screen is salinomycin, a highly selective potassium ionophore, facilitating bidirectional ion flux (Mitani et al., 1975). Salinomycin selectively impairs the viability of cells with features of cancer stem cells. In artificial mixtures of Ras-transformed HMLE cells in which some cells exhibit EMT and cancer stem cell features and some cells do not, salinomycin selectively eradicates cells with cancer stem cell properties. In contrast, established cancer chemotherapeutics (such as paclitaxel) have the opposite effect, even leading to cancer stem cell enrichment. In addition, in an orthotopic mouse model of lung metastasis using 4T1 murine carcinoma cells, salinomycin fully reverses the partial EMT-phenotype of these cells in that they adopt an epithelial phenotype. Although the mechanism of action for salinomycin is not yet clear, it appears that it might induce terminal epithelial differentiation accompanied by cell-cycle arrest rather than trigger cytotoxicity. This is consistent with evidence showing that salinomycin does not block proliferation in several other human mammary carcinoma cell lines.
Importantly, salinomycin also eradicates cells with cancer stem cell properties in mice. Tumors derived from 4T1 cells (or from Ras-transformed HMLE cells) form less efficiently in mice after treatment of the cells with salinomycin. Interestingly, in mice inoculated with SUM159 human breast cancer cells, treatment with salinomycin or paclitaxel delays tumor formation by 14 days. Furthermore, salinomycin induces expression of plasma membrane-E-cadherin in these tumors, providing further evidence that salinomycin might eliminate cancer stem cells by inducing their differentiation. Significantly, salinomycin also suppresses the metastasis of 4T1 cells to the lung. This differs from paclitaxel, which increases the prevalence of lung metastases in this model.
Thus, salinomycin might selectively suppress metastasis by inducing differentiation in the migrating cancer stem cells that have undergone EMT. The authors employ then global expression profiling to show that the fraction of Ras-transformed HMLE cells with properties of cancer stem cells have a gene expression signature found in natural mammary stem cells and in cancer stem cells from human tumors. They compared three cell states: (1) paclitaxel treatment versus salinomycin treatment of Ras-transformed HMLE cells, (2) mammospheres versus differentiation cultures of primary human mammary epithelial cells, and (3) cells expressing high levels of CD44 versus those expressing high levels of CD24 as sorted from normal human mammary glands and mammary carcinomas. They report 25 genes that are upregulated and 14 that are downregulated consistently in all of the above comparisons. This analysis clearly establishes that the Ras-transformed HMLE cells eliminated by salinomycin have a gene expression signature characteristic of mammary and cancer stem cells. Established breast cancer therapies often fail to achieve long-term patient survival, possibly because of tumor relapse as a result of chemotherapyresistant mammary cancer stem cells.
Thus, new therapeutic strategies to specifically target these cancer stem cells are urgently required. Gupta et al. (2009) significantly advance this field by presenting the first clear proof of principle that it is feasible to screen for drugs that specifically target breast cancer stem cells. The approach they chose evolved from the concept that normal mammary gland stem cells and cancer stem cells display a highly plastic epithelial phenotype, which allows them to undergo EMT in cell culture (Mani et al., 2008).
It remains unclear, however, by which mechanisms salinomycin selectively targets cells after EMT. Salinomycin isused as an antibiotic against eukaryotic parasites in animals and inhibits cartilage degradation during bone development (Peters et al., 2002). Given that it is a highly selective potassium ionophore, salinomycin may interfere with the function of potassium channels in cancer stem cells. It has been shown that tumor cells express elevated levels of various types of K+ channels. Their overexpression enhances proliferation, and drugs acting as channel blockers inhibit cell proliferation (Le Guennec et al., 2007; Zhang et al., 2009). Perhaps more importantly, certain K+ channels regulate migration. In a similar vein, certain G protein-coupled K+ channels are overexpressed in breast cancer lymph node metastases (Zhang et al., 2009). Given the importance of the cell polarity machinery to cell migration (Etienne-Manneville, 2008), it is tempting to speculate that K+ channels targeted by salinomycin have a critical function in epithelial polarity and metastasis, which can be deregulated by salinomycin.
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Figure 1. Salinomycin Targets Breast Cancer Stem Cells
Depicted is the screening approach used by Gupta et al. (2009). Control human mammary epithelial (HMLE) cells express the basolateral marker E-cadherin
and high levels of CD24. These cells do not form mammospheres in suspension culture. In contrast, HMLE cells treated with a short hairpin RNA targeting Ecadherin
undergo changes consistent with an epithelial to mesenchymal transition (EMT), including upregulation of the mesenchymal marker vimentin. These
cells exhibit features of cancer stem cells, including high levels of CD44 expression and the ability to form mammospheres in suspension culture. These two
cell types were then subjected to a high-throughput chemical screen measuring cell viability, which led to the identification of salinomycin as a compound that
targets breast cancer stem cells selectively.
SOURCE: Cell 138, August 21, 2009 ©2009 Elsevier Inc. p 623-625