Role of BI-1 Protein in Breast Cancer Apoptosis

Institution: The Burnham Institute for Medical Research
Investigator(s): Beatrice Bailly-Maitre, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10FB-0090 Award: $87,424
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2004)
Defects in apoptotic (programmed cell death) mechanisms play an important role in pathogenesis and progression of breast cancer. However, relatively little is known about which apoptosis-regulating genes are expressed in tumors of mammary epithelial origin. The Reed laboratory at The Burnham Institute has recently discovered a novel anti-apoptotic protein, called BI-1 (Bax Inhibitor-1), which appears to regulate a cell death pathway linked to stress in the endoplasmic reticulum. The endoplasmic reticulum is a network of tubules, vesicles and sacs that are interconnected inside of the cell's cytoplasm. One key function of the endoplasmic reticulum is to store calcium. The endoplasmic reticulum is known to trigger an apoptosis pathway that may be relevant to a variety of diseases, including breast cancer. BI-1 is an endoplasmic reticulum-resident protein that suppresses apoptosis induction by specific types of stimuli, predominantly linked to endoplasmic reticulum-stress situations. Indeed, endoplasmic reticulum stress results in caspase activation and apoptosis, both of which are blocked by BI-1 over-expression. Elevated BI-1 expression also inhibits calcium efflux from the endoplasmic reticulum. We have evidence indicating that BI-1 expression is elevated in a significant proportion of breast cancer cell lines, and we also observed that hypoxia (low tumor oxygen concentration) is a potent inducer of BI-1 expression.

We will test the hypothesis that BI-1 is an important regulator of cell death pathways linked to endoplasmic reticulum stress. We will: (i) dissect the point in apoptotic pathways where BI-1 blocks cell death induced by endoplasmic reticulum stress; (ii) determine the effects of BI-1 on endoplasmic reticulum -dependent functions such as calcium and protein sequestration; (iii) define the role of BI-1 in hypoxia; (iv) determine the role of BI-1 in breast cancer oncogenesis using mouse xenograft models; and (v) characterize the expression of BI-1 protein in human breast cancer clinical samples. Transmission electron microscopy will be used to evaluate the ultrastructure of the endoplasmic reticulum and Golgi in cells with varying amounts of BI-1. Caspase activation and activity we will be performed by immunoblotting and using selective flurogenic peptide substrates, respectively. Cell death will be monitored by propidium dye uptake/Annexin V assay. Intracellular calcium concentrations will be monitored by video time-lapse "deconvolution microscopy" using a calcium -sensitive fluorescent dye, such as Fura-2. Fluorescently-tagged endoplasmic reticulum luminal proteins will be employed for determining whether proteins are release into the cytosol. Hypoxia-induced increases in BI-1 will be determined by using nuclear "run-on" assays. Immunostaining will be used to analyze expression of BI-1 in breast cancer specimens.

By using BI-1 as a "window" into this poorly understood endoplasmic reticulum-apoptosis pathway, we eventually hope to devise strategies for bypassing roadblocks to cell death that commonly arise in cancers. From a therapy perspective, our research could assist in devising strategies to allow cancers to become more sensitive to chemotherapy. Finally, BI-1 expression could be useful a predictive "biomarker" for cancer progression or therapy options for patients.

Final Report (2006)
The goal of this CBCRP grant was to elucidate the potential role in breast cancer of a novel anti-apoptotic protein, recently characterized in our laboratory, called BI-1 (Bax Inhibitor-1). During our studies of BI-1, we discovered that its role in apoptosis appears to be predominantly linked to endoplasmic stress (ER) stress situations. The ER is known to trigger an apoptosis (cell death) pathway that may be relevant to a variety of diseases, including breast cancer, but details of how this pathway is controlled remain unclear. BI-1 is an ER-resident protein that suppresses apoptosis induction by specific types of stimuli, predominantly linked to ER-stress situations. Indeed, ER stress results in caspase (i.e., enzymes involved in the destruction of cells) activation and apoptosis, both of which are blocked by increased amounts of BI-1. Elevated BI-1 expression also inhibits calcium efflux from the ER. The ER, in its crucial capacity to regulate intracellular calcium in response to chemotherapeutic agents, has been implicated in breast cancer, but the mechanism is relatively unexplored.

Our findings included the following: BI-1-mediated protection from apoptosis induced by ER stress correlates with inhibition of Bax activation and translocation to mitochondria, maintenance of mitochondria' membrane potential, and suppression of caspase activation. Calcium imaging studies revealed that BI-1 over-expression reduces releasable calcium from the ER without having any effect on the Bcl-2 (apoptosis-inhibiting) family. The role of the ER in a variety of physiological situations that involve apoptosis was reviewed by our laboratory in the J. Clin. Invest. 115:2565 (2006), and the specific functions of the BI-1 protein were described in PNAS, USA 103:2809.

These studies set the stage for more detailed investigations of BI-1 as a pivotal regulator of the apoptosis pathway linked to ER stress in the breast.

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress.
Periodical:Molecular Cell
Index Medicus: Mol Cell
Authors: Chae HJ, Kim HR, Xu C, Bailly-Maitre B, et al and Reed JC
Yr: 2004 Vol: 15 Nbr: 3 Abs: Pg:355-66

Endoplasmic reticulum stress: cell life and death decisions.
Periodical:Journal of Clinical Investigation
Index Medicus: J Clin Invest
Authors: Xu C, Bailly-Maitre B, Reed JC
Yr: 2005 Vol: 115 Nbr: 10 Abs: Pg:2656-64

Cytoprotective gene bi-1 is required for intrinsic protection from endoplasmic reticulum stress and ischemia-reperfusion injury.
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Bailly-Maitre B, Fondevila C, Kaldas F, et al and Reed JC
Yr: 2006 Vol: 103 Nbr: 8 Abs: Pg:2809-14