BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation

Abstract

Although the multi-functional, prosurvival protein, Bcl-2-associated anthanogene 1 (BAG-1) is frequently overexpressed in breast cancers, its role in the development or maintenance of the malignant state remains unclear. Here, we have used the established MCF-10A 3-dimensional (3D) model of mammary morphogenesis as a biologically relevant system to determine how BAG-1 expression may influence the development of breast cancer. When cultured in 3D, MCF-10A cells undergo a highly regulated morphogenic program leading to the development of polarized acinar structures containing a central, hollow lumen formed, in part, through the induction of BIM-dependent apoptosis. BAG-1 overexpression resulted in an attenuation of this normal apoptotic program characterized by a significantly increased number of acini with filled lumens-a phenotype commonly observed in ductal carcinoma in situ. BAG-1's effects were associated with an activation of RAF-1-a known binding partner of BAG-1, enhanced signaling through the MAP kinase pathway and a decrease in BIM expression. Reversal of the BAG-1-associated survival phenotype by the mitogen-activated kinase/ERK kinase inhibitor, U0126, implicates the RAF-1-extracellular signal-regulated kinase signaling pathway as a major mediator of BAG-1's effects in this model. As BAG-1 expression is often elevated in preinvasive breast cancers, these findings support a possible role for BAG-1 as an early contributor to the malignant process in the breast.

A novel MCF-10A line allowing conditional oncogene expression in 3D culture

“…We provide evidence that the impaired lumen formation in B-Raf V600E expressing cells results most likely from the combination of a lack of proliferative suppression in late stage cultures and the failure to induce systematic luminal apoptosis. Our observations are in full agreement with previous studies showing that increased MEK/ERK signaling in 3D cultures of MCF-10A cells blocks luminal clearance by preventing the function and expression of the pro-apoptotic BIM protein, a critical regulator of acinar morphogenesis [43,44]. This notion is also in agreement with the observation that B-Raf V600E is a potent suppressor of BIM in colorectal cancer models [30].…”

“…Its subline, MCF-10AecoR (a kind gift of Drs. Danielle Carroll and Joan Brugge; Harvard Medical School), has been described by various assays and in detail before [14,25,44,46-48]. MCF-10Atet cells were generated by transfecting MCF-10AecoR cells with the Ahd I-linearized expression vector pWHE644, which uses the EF-1α promoter [49] to express a tri-cistronic transcript encoding the tetracycline-regulated transcriptional activator rtTA2 S -M2, the Tet-transsilencer tTS D -PP as well as a puromycin resistance gene [15].…”

G1P3, an interferon- and estrogen-induced survival protein contributes to hyperplasia, tamoxifen resistance and poor outcomes in breast cancer

“…In addition, MCF10A Vector and MCF10A G1P3 acini were harvested on days 5 and 10 and caspase 3 cleavage was confirmed by immunoblot analysis and apoptosis was quantified as described (Reginato et al, 2003;Anderson et al, 2010). In immunoblots, caspase 3 cleavage was absent on day 5; however, caspase 3 was cleaved in MCF10A Vector acini lysates on day 10, but not in MCF10A G1P3 acini, which is in agreement with the results of immunofluorescence studies (Figure 2g).…”

BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia

“…The observed impairment of the three key anti-apoptotic molecules conferred to BAG1 a crucial role in the control of apoptosis in AML. BAG1 silencing affected significantly the MAPK-pathway, specifically through down-regulation of ERK1/2 activity, upholding this proliferative pathway to be in part maintained by BAG1, as described in other cell types [41]. Despite these findings, only a slight decrease in cell vitality and insignificant apoptosis activation was observed, indicating that leukemic cells were able to successfully over-come the transient reduction of BAG1 expression.…”