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 \u3c 0.05), colony formation (P \u3c 0.05), adhesion (P \u3c 0.001), migration (P \u3c 0.001) and invasion (P \u3c 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2 gamma receptor null mice, ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/- cells (P \u3c 0.05). These novel results suggest that stem-like ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis

Differential Functional Roles of ALDH1A1 and ALDH1A3 in Mediating Metastatic Behavior and Therapy Resistance of Human Breast Cancer Cells

Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDHhiCD44+ ) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor® assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control (p \u3c 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control (p \u3c 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors

The Role of ALDHhiCD44+ Cells in Breast Cancer Metastasis and Therapy Resistance

Breast cancer is a leading cause of death in women, due primarily to ineffective treatment of metastatic disease. Despite being a lethal process, metastasis is surprisingly inefficient, with less than 0.5% of cancer cells able to successfully navigate the metastatic cascade, indicating that only a small proportion of cancer cells possess the necessary characteristics to establish metastases. Cancer stem cells (CSCs) have recently been identified in leukemia and solid tumors; however, the role of CSCs in breast cancer metastasis and therapy resistance remains poorly understood. Sub-populations of cells demonstrating stem-cell like characteristics (high expression of CSC markers and/or high ALDH activity, an enzyme involved in stem cell self-protection) were identified in MDA-MB-435, MDA-MB-231, MDA-MB-468 breast cancer cells, but not in MCF-7 cells. When isolated and compared to ALDHlowCD44- cells, ALDHhiCD44+ cells demonstrated increased metastatic behaviours in vitro. Furthermore, following injection into immunocompromised mice, ALDHhiCD44+ cells showed enhanced tumorigenicity and metastasis compared to ALDHlowCD44- cells, indicating that stem-like ALDHhiCD44+ cells may be important mediators of breast cancer metastasis. ALDHhiCD44+ cells from MDA-MB-231 and MDA-MB-468 cells that were exposed to chemotherapy or radiotherapy also demonstrated significantly increased cell survival relative to ALDHlowCD44- cells, potentially through an ALDH1-dependent manner. ALDH1 expression has previously been correlated with poor patient outcome and incidence of metastatic disease. To test a potential functional role for ALDH1 expression in metastasis and therapy resistance, the ALDH1 isozymes ALDH1A1 or ALDH1A3 were knocked down using siRNA in MDA-MB-468 and SUM159 breast cancer cell lines. ALDH1A1low cells demonstrated decreased metastatic abilities in vitro and in vivo; whereas ALDH1A3low cells demonstrated minimal changes in metastatic abilities in vitro but showed a significant reduction in metastatic capacity in vivo. Additionally, ALDH1A1low cells but not ALDH1A3low cells demonstrated increased sensitivity to both chemotherapy and radiation. Collectively, these data suggest that ALDH1 expression demonstrates a functional role in breast cancer metastasis and therapy resistance. Thus, drug development that targets ALDH1-expressing tumor cells may represent a novel therapeutic strategy to treat metastatic breast cancer patients in the future

Lung-Derived Factors Mediate Breast Cancer Cell Migration through CD44 Receptor-Ligand Interactions in a Novel Ex Vivo System for Analysis of Organ-Specific Soluble Proteins

Breast cancer preferentially metastasizes to lung, lymph node, liver, bone, and brain. However, it is unclear whether properties of cancer cells, properties of organmicroenvironments, or a combination of both is responsible for this observed organ tropism. We hypothesized that breast cancer cells exhibit distinctive migration/growth patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast cancer cells and soluble organ-derived factors mediate this behavior. Using an ex vivo model system composed of organ-conditionedmedia (CM), human breast cancer cells (MDA-MB-231,MDA-MB-468, SUM149, and SUM159) displayed cell line—specific and organ-specific patterns of migration/proliferation that corresponded to their in vivo metastatic behavior. Notably, exposure to lung-CM increased migration of all cell lines and increased proliferation in two of four lines (P < .05). Several cluster of differentiation (CD) 44 ligands including osteopontin (OPN) and L-selectin (SELL) were identified in lung-CM by protein arrays. Immunodepletion of SELL decreased migration of MDA-MB-231 cells, whereas depletion of OPN decreased both migration and proliferation. Pretreatment of cells with a CD44-blocking antibody abrogated migration effects (P < .05). “Stemlike” breast cancer cells with high aldehyde dehydrogenase and CD44 (ALDHhiCD44+) responded in a distinct chemotacticmanner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (P < .05). In contrast, organ-specific changes in migration were not observed for ALDHlowCD44- cells. Our data suggest that interactions between CD44+ breast cancer cells and soluble factors present in the lung microenvironment may play an important role in determining organotropic metastatic behavior