Abstract P5-07-11: The ectopic FOXA1 expression correlates to the luminal breast cancer stem cells
Authors: Tachi K , Shiraishi A , Bando H , Yamashita T , Tsuboi I , Kato T , Ichioka E , Tsushima Y , Ikeda T , Iguchi A , Hara H , Ohneda O 
Published: March 2017

Abstract
Background: It is known that ER-positive breast cancer constitutes approximately 70% of all breast cancer and the particular population of these, called as luminal A breast cancer can lead to late recurrence, possibly due to the presence of dormant tumor cells. It is also argued that there are many similar biological characteristics between dormant cells and cancer stem cells (CSCs). So we hypothesized that some part of CSCs would be involved in the late recurrence of luminal type breast cancer and looked for the factors correlateing late recurrence by comparing CSC-enriched population and other population. Materials and Methods: We used the MCF-7 and HCC1500 human breast cell lines. Moreover, we isolated the BC#1 breast cancer cell from metastatic pleural effusion of a breast cancer patient (79 years old, ER+, PgR+, HER2 score 0). These cells are cultured in the adhesion culture condition or the mammosphere culture condition. The immunohistochemical characteristics such as ER, PgR, HER2, Cytokeratin (CK) 8, CK5/6 status of these cells were confirmed. The analysis of the ALDH activity and the CD44+/24- expression as the CSC marker were carried out using flow cytometry. The expression of target genes was analyzed using quantitative RT-PCR. Protein expression was determined by western-blotting. To assess the self-renewal potential of CSCs and the proliferation of immature cancer cells, we used mammosphere formation assay and colony formation assay. To investigate the effects of 4-hydroxytamoxifen(4-OHT) on mammosphere formation assay, 1µm of 4-OHT added to the medium in the particular experiment. Results: BC#1 was confirmed as luminal type by immunohistochemistry and qRT-PCR. The expression tendency between two CSC markers are almost same and correlated to the mammosphere formation capacity. We also confirmed that mammosphere culture have an increased ALDH-positive population compared with adherent culture cells. Using flow cytometry, we divided the cell lines and isolated cells into two populations, ALDH-positive and negative. By qRT-PCR analysis, ALDH-positive population showed higher expression of FOXA1 (ER expression related gene) than ALDH-negative population. In addition, we found the significantly higher expression of FOXA1 gene, and RPRM gene (p53 induced G2/M arrest related gene) in mammosphere culture samples than in adhesion culture samples. In terms of the effects of 4-OHT, 4-OHT resistant mammosphere samples showed significantly increased FOXA1 gene expression level. Finally, we established shFOXA1 MCF-7 cells to investigate the relationship between self-renewal potential and FOXA1 expression. We found that colony formation in shFOXA1 MCF-7 cells decreased compared with control while there is no significant difference in the number of mammospheres. Conclusion: CSC-enriched population showed higher FOXA1 expression and our results suggested that the expression of FOXA1 correlate to the proliferation of immature breast cancer cell rather than the induction of self-renewal potency of CSCs. Moreover, there is no report argued about the correlation between CSCs and G2/M arrest related gene like RPRM so that the biological mechanism should be investigated in future.
Citation Format: Tachi K, Shiraishi A, Bando H, Yamashita T, Tsuboi I, Kato T, Ichioka E, Tsushima Y, Ikeda T, Iguchi A, Hara H, Ohneda O. The ectopic FOXA1 expression correlates to the luminal breast cancer stem cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-11.