Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal–epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Abstract

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum (ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G 0 /G 1 arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor (EGFR) and plasminogen activator receptor (uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells.

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK

“…Activation of p38a downregulates cyclin D 2 and upregulates p15 and p21, which probably mediates the G 0 /G 1 arrest observed in our previous studies. 6 Furthermore, we show that p38a activation by ERp29 negatively regulates the expression of basal eIF2a, whereas upregulation of p58 IPK inhibits phosphorylation of eIF2a and downstream ATF4/CHOP/caspase-3 pro-apoptotic signaling. Thus, for the first time, our results demonstrate that concomitant p38a activation and upregulation of p58 IPK and the interplay between these molecules are central in sustaining ERp29-induced cell growth arrest and survival.…”

“…MDA-MB-231 cells transfected with pcDNA/ERp29 or pcDNA, and MCF-7 cells transfected with shRNA/ERp29 or scrambled control shRNA were generated as described previously. 6 All transfected cells were maintained in DMEM supplemented with 10% FBS and G418 (Invitrogen; 2 mg/ml for MDA-MB-231 transfectants, or 1 mg/ml for MCF-7 transfectants). Cells were cultured at 371C in a 5% CO 2 /95% air atmosphere in a humidified incubator.…”

“…6,9,26 In brief, cell lysates were extracted with radio-immunoprecipitation buffer (1% Igepal, 1% sodium deoxycholate, 0.15 M sodium chloride, 0.01 M SDS, pH 7.2 and 2 mM EDTA), supplemented with protease inhibitors (Roche Diagnostics) and phosphatase cocktail inhibitors I and II (1:100, Sigma-Aldrich). Cell lysates were centrifuged at 13 000 Â g for 20 min/41C and the protein-containing supernatant was collected.…”

“…5 Recently, we have demonstrated a significant role of ERp29 in tumorigenesis by exogenously overexpressing ERp29 in the highly proliferative and invasive MDA-MB-231 cancer cells. 6 Overexpression of ERp29 in MDA-MB-231 cells results in G 0 /G 1 arrest, thus leading to a marked delay in the onset of tumorigenesis in vivo. 6 Significantly, overexpression of ERp29 increases cell survival when cells are exposed to genotoxic stress induced by doxorubicin and radiation treatment.…”

“…6 Overexpression of ERp29 in MDA-MB-231 cells results in G 0 /G 1 arrest, thus leading to a marked delay in the onset of tumorigenesis in vivo. 6 Significantly, overexpression of ERp29 increases cell survival when cells are exposed to genotoxic stress induced by doxorubicin and radiation treatment. [7][8][9] These studies indicate a pivotal role of ERp29 in inducing cell growth arrest and cell survival.…”