Alison L. Allan

High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability.

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre‐clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre‐clinical model systems for studying stem‐like cells in breast cancer metastasis, and to test the hypothesis that stem‐like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA‐MB‐435, MDA‐MB‐231, MDA‐MB‐468, MCF‐7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self‐protection. We then used fluorescence‐activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony‐forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub‐populations of cells demonstrating stem‐cell‐like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF‐7. When isolated and compared to ALDHlowCD44low/– cells, ALDHhiCD44+CD24− (MDA‐MB‐231) and ALDHhiCD44+CD133+ (MDA‐MB‐468) cells demonstrated increased growth (P < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2γ receptor null mice, ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/– cells (P < 0.05). These novel results suggest that stem‐like ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis.

The Role of Human Aldehyde Dehydrogenase in Normal and Cancer Stem Cells

Normal stem cells and cancer stem cells (CSCs) share similar properties, in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields, there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently, it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells, and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition, emerging evidence suggests that ALDH1 is not only a marker for stem cells, but may also play important functional roles related to self-protection, differentiation, and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs, with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed.

Cancer stem cells: implications for the progression and treatment of metastatic disease

•  Introduction •  The metastatic process ‐  Metastatic inefficiency ‐  Organ‐specific metastasis: seed and soil ‐  Epithelial‐mesenchymal transition (EMT) •  The cancer stem cell hypothesis ‐  Evidence supporting the cancer stem cell hypothesis ‐  Defining the cancer stem cell ‐  Identifying and isolating cancer stem cells •  Parallels between stem cell behaviour and metastatic behaviour ‐  The metastatic niche ‐  The SDF‐1/CXCR4 axis ‐  Resistance to apoptosis and protection from cellular insult ‐  Origin of the cancer stem cell: normal stem cell gone bad? •  Therapeutic implications ‐  Radiation therapy and cancer stem cells ‐  Cytotoxic/targeted chemotherapy and cancer stem cells ‐  Differentiation therapy and cancer stem cells ‐  Prognostic implications •  Conclusions and future directions

Tumor Dormancy and Cancer Stem Cells: Implications for the Biology and Treatment of Breast Cancer Metastasis

Breast cancer remains a leading cause of morbidity and mortality in women, mainly due to the propensity of primary breast tumors to metastasize to regional and distant sites. Metastatic spread after the removal of a primary tumor can be difficult to identify, creating uncertainty in patients with regards to possible cancer recurrence. This is a particular problem in breast cancer, exemplified by the fact that recurrence can take place after decades of apparent disease-free survival. The mechanisms underlying tumor dormancy in breast cancer remain poorly understood, and this presents significant challenges to both experimental investigation and clinical management of breast cancer. This review will discuss what is currently known about the metastatic process and tumor dormancy, consider the growing evidence that cancer stem cells may contribute to tumor progression and dormancy, and speculate about the clinical importance and implications of this research.

Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic

Solid cancers are a leading cause of death worldwide, primarily due to the failure of effective clinical detection and treatment of metastatic disease in distant sites. There is growing evidence that the presence of circulating tumor cells (CTCs) in the blood of cancer patients may be an important indicator of the potential for metastatic disease and poor prognosis. Technological advances have now facilitated the enumeration and characterization of CTCs using methods such as PCR, flow cytometry, image-based immunologic approaches, immunomagnetic techniques, and microchip technology. However, the rare nature of these cells requires that very sensitive and robust detection/enumeration methods be developed and validated in order to implement CTC analysis for widespread use in the clinic. This review will focus on the important technical and statistical considerations that must be taken into account when designing and implementing CTC assays, as well as the subsequent interpretation of these results for the purposes of clinical decision making.

Osteopontin overexpression in breast cancer: Knowledge gained and possible implications for clinical management

Osteopontin (OPN) is a secreted protein that is overexpressed in a number of human cancers, and has been associated with increased metastatic burden and poor prognosis in breast cancer patients. The OPN protein contains several conserved structural elements including heparin‐ and calcium‐binding domains, a thrombin‐cleavage site, a CD44 binding site, and two integrin‐binding sites. Experimental studies have shown that the ability of OPN to interact with a diverse range of factors, including cell surface receptors (integrins, CD44), secreted proteases (matrix metalloproteinases, urokinase plasminogen activator), and growth factor/receptor pathways (TGFα/EGFR, HGF/Met) is central to its role in malignancy. These complex signaling interactions can result in changes in gene expression, which ultimately lead to alterations in cell properties involved in malignancy such as adhesion, migration, invasion, enhanced tumor cell survival, tumor angiogenesis, and metastasis. Therefore, OPN is not merely associated with cancer, but rather it plays a multi‐faceted functional role via complex molecular cross‐talk with other factors. This review will focus on the role of OPN in breast cancer, in particular on the malignancy‐promoting aspects of OPN that may reveal opportunities for new approaches to the clinical management of breast cancer. J. Cell. Biochem. 102: 859–868, 2007.

Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer.

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44hi/CD24lo cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44hi/CD24lo cells (P < 0.05; Kruskal–Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44hi/CD24lo phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal–Wallis). Notch1 knockdown reduced the expression of CD44hi/CD24lo phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal–Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity. Mol Cancer Res; 9(7); 834–44. ©2011 AACR.

Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry

Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer.

Detection of circulating tumor cells in advanced head and neck cancer using the cellsearch system

Early detection of circulating tumor cells (CTCs) offers the possibility of improved outcome for patients with head and neck squamous cell cancer (HNSCC).

The Role of Erythropoietin and Erythropoiesis-Stimulating Agents in Tumor Progression

Over the past few decades, understanding of the physiologic function of erythropoietin (EPO) has evolved significantly. EPO binds to erythropoietin receptors (EPOR), initiating signaling that stimulates growth, inhibits apoptosis, and induces the differentiation of erythroid progenitors to increase red blood cell mass. EPO has additionally been shown to exert tissue-protective effects on multiple tissues, suggesting a pleiotropic mechanism of action. Erythropoiesis-stimulating agents (ESA) are used clinically for treating cancer-related anemia [chemotherapy-induced anemia (CIA)]. Recent clinical trials have reported increased adverse events and/or reduced survival in ESA-treated cancer patients receiving chemotherapy, potentially related to EPO-induced cancer progression. Signaling pathways downstream of EPO/EPOR have been shown to influence numerous cellular functions in both normal and tumor cells, including proliferation, apoptosis, and drug resistance. Some studies have reported effects on proliferation, reduced chemotherapy efficacy, reduction of apoptosis, and resistance to selective therapies on cancer cell lines, whereas others have shown null effects. In addition, newer targeted cancer therapies that are directed toward specific signaling pathways may be antagonized by ESAs. This molecular interplay between anticancer agents and potential survival signals triggered by ESAs may have been underestimated and may contribute toward decreased survival seen in certain trials. As more targeted anticancer therapies become available, these types of interactions may mitigate therapeutic efficacy by allowing tumor cells to acquire drug resistance. Therefore, a more complete understanding of the complex pathways involved will allow for the rational use of ESAs for the safe treatment of CIA in oncology patients. Clin Cancer Res; 17(20); 6373–80. ©2011 AACR.