Analysis of stem cells markers in human breast cancer cell lines

Author(s)

Aline Ramos Maia Lobba

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2014-04-28

Examining board members

Mari Cleide Sogayar; Maria Mitzi Brentani; Nathalie Cella; Eduardo Moraes Rego Reis; Deborah Schechtman

• Advisor: Mari Cleide Sogayar

Abstract

Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols. (AU)