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 < 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 < 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.

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 organ microenvironments, 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-conditioned media (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 (ALDH(hi)CD44(+)) responded in a distinct chemotactic manner 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 ALDH(low)CD44(-) 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.

Inhibition of aldehyde dehydrogenase (ALDH) activity reduces chemotherapy and radiation resistance of stem-like ALDHhiCD44⁺ human breast cancer cells.

The majority of breast cancer deaths are because of ineffective treatment of metastatic disease. We previously identified a subpopulation of cells in human breast cancer cell lines that demonstrate high activity of aldehyde dehydrogenase (ALDH) and high expression of CD44. These ALDH(hi)CD44(+) cells displayed enhanced metastatic behavior in vitro and in vivo relative to ALDH(low)CD44(-) cells. The goal of this study was to test the hypothesis that ALDH(hi)CD44(+) breast cancer cells are more resistant to standard cancer therapy, and that inhibiting ALDH activity through all-trans retinoic acid (ATRA) or the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) sensitizes these cells to treatment. ALDH(hi)CD44(+) and ALDH(low)CD44(-) populations were isolated from MDA-MB-231 and MDA-MB-468 cells lines and exposed to chemotherapy (doxorubicin/paclitaxel) or radiotherapy ± ATRA or DEAB. Cell populations were assessed for differences in survival, colony formation, and protein expression related to therapy resistance and differentiation. Significantly more ALDH(hi)CD44(+) cells survived chemotherapy/radiotherapy relative to ALDH(low)CD44(-) cells (P < 0.001). Glutathione-S-transferase pi, p-glycoprotein, and/or CHK1 were overexpressed in ALDH(hi)CD44(+) populations compared with ALDH(low)CD44(-) populations (P < 0.05). Pre-treatment of cell populations with DEAB or ATRA had no effect on ALDH(low)CD44(-) cells, but resulted in significant initial sensitization of ALDH(hi)CD44(+) cells to chemotherapy/radiotherapy. However, only DEAB had a long-term effect, resulting in reduced colony formation (P < 0.01). ATRA also significantly increased expression of CK8/18/19 in MDA-MB-468 ALDH(hi)CD44(+) cells compared with control (P < 0.05). Our novel findings indicate that ALDH(hi)CD44(+) breast cancer cells contribute to both chemotherapy and radiation resistance and suggest a much broader role for ALDH in treatment response than previously reported.

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 ALDH(low)CD44(low/-) cells, ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)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/IL2gamma receptor null mice, ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P < 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis.