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5AZA-induced Gene Expression in Human Breast Cancer Cell Lines and Benign Primary Mammary Epithelial Cell Cultures

ABSTRACT: The goal of this experiment was to identify genes that were expressed at higher levels in benign human mammary epithelial cells than in breast cancer cell lines and that were induced by 5AZA treatment in breast cancer cell lines. Six breast cancer cell lines were selected for demethylation studies based on known tumor suppressor gene expression regulation by promoter region hypermethylation: HCC1569 (CCND2), HCC1954 (SCGB3A1, APC, RASSF1A), MCF-7 (RAR-beta2), MDA-MB-231 (ESR1), UACC3199 (BRCA1), and BT-549 (hypermethylator phenotype). Other than MCF10A we specifically avoided immortalized benign human mammary epithelial cell lines for this experiment as these cells frequently show tumor suppressor gene methylation (e.g. p16) and gene expression profiles that are intermediate between normal breast epithelial cells and breast cancer. Instead, we opted to test six first-passage benign human mammary epithelial cell cultures (HME) generated in serum-free media from small fragments of normal breast tissue obtained from young women undergoing fibroadenoma excision. The 5AZA dose (0.5 microM) was selected based on evaluation of growth curves and induction of BNC1, SERPINB, and TKTL1 gene expression measured by RT-PCR in benign and malignant cells. The breast cancer cell lines, HME cultures, and MC10A cells were treated with 0.5 microM 5AZA (Sigma-Aldrich, St. Louis, MO) in DMSO or DMSO alone for six days after which the cells were harvested, and RNA prepared using the Illumina TotalPrep kit (AMIL1791, Life Technologies, Grand Island, NY). Whole genome expression was assessed using the Illumina HumanWG-6-v3 chip Gene expression was evaluated in 6 breast cancer cell lines, 6 primary breast epithelial cell cultures, and MCF10A cells after 6 days in DMSO or DMSO plus 0.5 microM 5AZA.

ORGANISM(S): Homo sapiens

SUBMITTER: Euhus David

PROVIDER: S-ECPF-GEOD-41692 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundRecent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells.MethodsWe used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A), as a model of benign mammary epithelial cells (MECs), and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS), as a model of breast cancer cells.ResultsThe in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells +/- PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells.ConclusionsTaken together, these data shift our understanding of adjacent benign epithelial cells in the cancer process, from passive, noncontributory cells to an active and tumor-promoting vicinal cell population that may have significant effects early, when benign cells outnumber malignant cells.

Project description:BackgroundOverexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression.Methods17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the "Warburg effect".ResultsConsiderable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively.ConclusionUsing RT-qPCR and three different antibodies we observed only exceptional occurrence of TKTL1 in a panel of malignant human cell lines in vitro. The presence of TKTL1 was unrelated to either the rate of glucose consumption/lactic acid production or resistance against chemo- and radiotherapy.

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5AZA-induced Gene Expression in Human Breast Cancer Cell Lines and Benign Primary Mammary Epithelial Cell Cultures

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