Biochemical and Biophysical Research Communications
Ell3 stimulates proliferation, drug resistance, and cancer stem cell properties of breast cancer cells via a MEK/ERK-dependent signaling pathway
Introduction
Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Similar to other tumors, breast cancer shows intratumoral heterogeneity. Although the mechanism(s) underlying this heterogeneity are not understood, a growing body of evidence suggests that a small population of cells, called cancer stem cells (CSCs), which show some phenotypic similarities with adult tissue stem cells, contribute to tumor heterogeneity [1]. CSCs are operationally defined by self-renewal, as well as their ability to differentiate into the non-self-renewing multiple lineages of the bulk tumor [2], [3]. Human breast CSCs are characterized according to their expression of specific cell surface markers (e.g., CD44+highCD24−low), aldehyde dehydrogenase (ALDH) enzyme activity (ALDH-positive cells), and mammosphere formation in suspension culture [2], [3]. CSCs mediate tumor metastasis and drug resistance, which contributes to relapse of cancer [4], [5]. Thus, the successful targeting of this cell population is critical to successful cancer treatment.
The RAS/RAF/MEK signaling transduction pathway critically regulates proliferation and apoptosis in various cell types. These proteins represent a group of serine/threonine kinases that mediate signal transduction from the cell surface towards both nuclear and cytosolic targets in response to a variety of extracellular stimuli. Deregulation of the RAS/RAF/MEK pathway is detected in more than 30% of human tumors, mainly resulting from mutations in RAS and/or B-RAF [6]. In addition, recent studies reveal that the RAS/RAF/MEK pathway is associated with CSCs and sensitivity to targeted therapy [7], [8]. Drug resistant breast cancer cells showing increased activation of the RAS/RAF/MEK pathway also show properties consistent with CSCs [9].
Ell3 is a Pol II transcription elongation factor enriched in testis. The C-terminal domain of Ell3 shows strong similarities with that of the Ell (eleven−nineteen lysine-rich leukemia) gene, which acts as a negative regulator of p53 and regulates cell proliferation and survival [10], [11].
Ell3 primes gene activation in embryonic stem cells (ESCs) by marking the enhancers of developmentally-regulated genes [12]. The binding of Ell3 to inactive or poised enhancers is essential for the recruitment of the super elongation complex (SEC) upon differentiation signaling. Recent studies in this laboratory demonstrated that Ell3 enhances the differentiation of mouse ESCs by protecting differentiating cells from apoptosis through the promotion of p53 degradation [13]. Given the central function of Ell3 in p53 stability, we investigated the role of Ell3 in breast cancer. We found that ectopic expression of Ell3 promotes cell proliferation and induces 5-FU drug resistance in both breast cancer cell lines and non-tumorigenic MCF-10A cells. Moreover, the cancer stem cell properties of breast cancer were increased upon Ell3 expression. ERK1/2 was phosphorylated upon Ell3 expression, and chemical inhibition of ERK activity compromised the effect of Ell3 on the breast cancer cell lines. Our data suggest that Ell3 functions to promote oncogenesis in breast cancer cell lines by regulating the ERK signaling pathway.
Section snippets
Cell culture and establishment of Ell3 overexpressing breast cancer cell lines
MCF-7, BT-20 and MCF-10A cell lines were purchased from American Type Culture Collection (ATCC, Teddington, UK). MCF-7 and BT-20 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Ell3-overexpressing (Ell3 OE) breast cancer cell lines were generated by the chromosomal integration of an Ell3 expression plasmid, which was constructed by cloning PCR-amplified Ell3 cDNA into pcDNA3.1 vectors (Invitrogen, Carlsbad, CA). Three independent Ell3 OE cell
Ell3. promotes cell proliferation in breast cancer cells
To understand the role of Ell3 in breast cancer cells, we first examined the expression of Ell3 in various breast cancer cell lines and in MCF-10A cells, a normal mammary epithelial cell line. Ell3 expression in the breast cancer cell lines was higher than that in MCF-10A cells (Fig. 1A). Thus, we first sought to determine whether stable Ell3 overexpression regulates breast cancer cell line characteristics. MCF-7 cells overexpressing Ell3 were generated by retroviral infection (Fig. 1B). As
Discussion
Here, we examined the oncogenic activity of Ell3 in breast cancer cells. Ell3 is an RNA polymerase II transcription elongation factor, which shows approximately 50% sequence identity to both Ell and Ell2 throughout its ORF proteins [10]. The Ell C-terminal domain of a MLL-ELL fusion protein found in patients with acute myeloid leukemia represses p53 transcriptional activity by binding to it, which is sufficient to immortalize myeloid progenitors [10]. Since Ell3 shows a great degree of homology
Acknowledgments
This work was supported by the Korea Science and Engineering Foundation (KOSEF) of the Korean government (MOST) (2012-050367 and 2012-0005261). This work was also supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20120006679).
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