Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.

Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors.

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Background: Aldehyde dehydrogenase 1 (ALDH1) has been identified as a marker of breast cancer stem cells, and ALDH1 expression in primary breast tumors has been associated with resistance to chemotherapy and poor prognosis. ALDH1 is an enzyme that can detoxify reactive aldehyde compounds; however the mechanism of ALDH1 chemotherapy resistance and its promotion of the breast cancer stem cell phenotype is unclear. Materials and Methods: Illumina cDNA array was performed on primary breast tumors that were snap-frozen, and RNA was isolated from dense, viable tumor regions. Samples were normalized by rank-invariant method and gene expression was analyzed. Immunohistochemistry was performed for ALDH1A1 on initial and post-neoadjuvant tumor samples from patients who have undergone six cycles of chemotherapy. Clinical data including age, tumor pathology, lymph node status, and response to neoadjuvant therapy were available for analysis. Human breast cancer cell lines (MCF-7 and MDA-MB-468) were treated with increasing doses of retinoic acid (RA) with and without the retinoic acid receptor alpha antagonist, Ro 41-5253. Effects on cell proliferation and signal transduction pathways were assessed. Results: The expression of ALDH1A1 and/or ALDH1A3 isoform mRNA was found to be present in primary human breast tumors. The majority of these cases were noted as triple negative or Her-2 positive tumors. Evaluation of RA receptor expression revealed repressed RAR beta levels in all tumors, whereas RAR alpha expression was retained. Observations of patient tumor samples (pre and post neoadjuvant chemotherapy treatment) demonstrated high ALDH1A1 expression in tumor and stromal cell compartments. The expression was predominant in partial and non-responders to the neoadjuvant therapy. Interestingly, the tumor from a partial responder was converted to extremely high expresser of ALDH1A1 post-neoadjuvant therapy indicating a positive survival selection. Since ALDH1A1 can convert retinaldehyde to retinoic acid, breast cancer cell lines were treated with all-trans RA and responded with a significant decrease in cell proliferation. This effect was partially attenuated by pre-treatment with the RAR alpha antagonist, Ro 41-5253. Importantly, RA treatment of MCF-7 cells repressed MAPK signaling, which was also attenuated by pre-treatment with Ro 41-5253. Discussion: The observations from this study suggest that the expression of the breast cancer stem cell marker, ALDH1A1 on tumor cells or in tumor-associated stromal cells can provide a mechanism for survival of cancer during chemotherapy treatment. The conversion of therapeutic oxidant compounds to retinoic acid by ALDH1 may lead to increased RAR alpha signaling and suppression of proliferation. The suppression of proliferation correlates well with chemotherapy resistance in these neoadjuvant cases. This data demonstrates a possible mechanism for ALDH1 promotion of tumor cell survival under chemotherapy and suggests that treatment with RAR alpha antagonists may promote tumor cell susceptibility to current treatment modalities and improve the therapeutic window for primary, metastatic and chemo-refractory breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1135.

Background: Adenosine monophosphate-activated protein kinase (AMPK) is a major regulator of energy homeostasis at both the cellular and whole-body levels. AMPK is activated by elevated intracellular AMP level and by phosphorylation on threonine 172 (T172) in the activation loop of the α catalytic subunit by upstream kinases, LKB1 and Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Once activated, AMPK switches on ATP-producing pathways while switching off ATP-consuming processes. Recent studies suggest that AMPK also controls processes relevant to tumor development, including protein synthesis, cell cycle progression, cell growth, survival and stress-induced autophagy. The purpose of our study is to determine the role of CaMKK2 as an AMPK upstream kinase in breast cancer cells, as well as its function in regulating cell proliferation, migration and survival. We chose the prototypical ERα + MCF-7 breast cancer cell line as our research model since this cell line does not express CaMKK1, which has similar functions to CaMKK2 but cannot activate AMPK. Materials and Methods: Illumina cDNA expression array was preformed on MCF-7, T-47D and MDA-MB-231 breast cancer cell lines, and normalized by rank-invariant method. MCF-7 cells with suppressed CaMKK2 or LKB1 expression were established using lentiviral gene specific shRNA interference. Stable knockdown cells (shNon-Target, shCaMKK2, shLKB1) were subject to acute and chronic stress conditions (i.e. ionomycin, 2-deoxy-D-glucose, hypoxia) and signaling events were evaluated using immunoblot analysis. Proliferation, anchorage-independent growth and motility of the knockdown cells were determined by MTT, soft agar formation and in vitro migration assay. Xenograft tumor studies were performed by inoculating estradiol-supplemented athymic nude mice with shNon-Target, shCaMKK2 and shLKB1 MCF-7 cells into the mammary fat pad. Tumor growth was determined by external caliper measurements twice a week. Forty-five days post-injection, mice were euthanized, tumors removed, weighed and prepared for histological, protein and RNA analyses. Results: CaMKK2 and LKB1 mRNAs are expressed at a similar level in the ERα + and ERα — breast cancer cell lines tested in the Illumina cDNA expression array. CaMKK2 activated AMPK phosphorylation (T172) under increased intracellular Ca2+ level induced by ionomycin. Under acute stress conditions, CaMKK2 functioned as the predominant AMPK upstream kinase. However, when cells are subject to long-term metabolic and hypoxic stress, CaMKK2 and LKB1 were both required for AMPK activation. Loss of CaMKK2 in MCF-7 cells promoted a moderate growth advantage under metabolic stress, increased soft agar colony formation and elevated cell migration capacity. CaMKK2 knockdown xenografts demonstrated less ductal structure formation, decreased E-Cadherin expression and reduced angiogenesis, as well as reduced VEGF and Glut1 mRNA levels in vivo. Conclusions: We have demonstrated that Ca2+-CaMKK2 signaling plays an important role in regulating breast cancer cell proliferation, migration and survival pathways. These findings suggest that Ca2+ activation through CaMKK2 promotes AMPK signaling, survival and angiogenesis in response to stress conditions in breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-07-06.