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

Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58(IPK). In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, ß(1)-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (P<0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D(2) is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58(IPK) expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58(IPK) by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58(IPK) expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58(IPK) prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58(IPK) upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.

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

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.

Cytokeratin 19 regulates endoplasmic reticulum stress and inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in breast cancer cells.

Cytokeratin 19 (CK19) is widely used as a biomarker for the detection of disseminated tumor cells in blood and bone marrow, and its positivity is considered as an independent prognostication indicator in cancer patients. However, its role in breast cancer progression remains unknown. We had established a stable CK19-expressing clone in the CK19-negative BT549 human breast cancer cell line and found that CK19 expression in the BT549 cells caused cell cycle arrest, reduced cell motility and increased drug resistance. Further study revealed that CK19 expression regulated endoplasmic reticulum (ER) stress signaling by up-regulating p38/RNA-dependent protein kinase-like ER kinase (PERK)/p-eIF2alpha and 78 kDa glucose-regulated protein (Bip/GRP78), and down-regulating focal adhesion kinase (FAK). The level of ER protein 29 (ERp29) was shown to be decreased in the CK19-expressing BT549 cells by proteomic analyses and verified by Western blotting and RT-PCR. Pharmacological inhibition of p38 signaling by its specific inhibitor SB203580 or knockdown of p38 and transcription factor XBP-1 by siRNA in BT549/CK19 and MDA-MB-231 cells revealed that p38/XBP-1 signaling negatively regulated ERp29 expression. Our results indicated that CK19 modulates ER stress signaling and contributes to cell survival and dormancy in breast cancer cells.