Matthew G. Annis

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c‐myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug‐treated c‐myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine‐specific modifying agent revealed that Bax inserted both the ‘pore domain’ (helices α5–α6), and the tail‐anchor (helix α9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc‐expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore‐forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.

Bcl‐2 changes conformation to inhibit Bax oligomerization

Bcl‐2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl‐2, but not the inactive point mutants Bcl‐2‐G145A and Bcl‐2‐V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl‐2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of α5–α6 helices (Bcl‐2‐S105C/E152C) was only active in a reducing environment. Thus, Bcl‐2 must change the conformation to inhibit tBid‐induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl‐2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl‐2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl‐2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane‐inserted Bax monomers and prevent productive oligomerization of Bax.

Myc Potentiates Apoptosis by Stimulating Bax Activity at the Mitochondria

ABSTRACT The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochromec release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event

The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Δψm) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Δψm, wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Δψm.

PDK1-Dependent Metabolic Reprogramming Dictates Metastatic Potential in Breast Cancer

Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer.

ADAM10 Releases a Soluble Form of the GPNMB/Osteoactivin Extracellular Domain with Angiogenic Properties

Background Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40–75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment. Methodology/Principal Findings We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro. Conclusions/Significance GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.

During Apoptosis Bcl-2 Changes Membrane Topology at Both the Endoplasmic Reticulum and Mitochondria

In healthy cells the antiapoptotic protein Bcl-2 adopts a topology typical of tail-anchored proteins with only the hydrophobic carboxyl terminus inserted into the membrane, as shown by labeling cell lysates with a membrane-impermeant sulfhydryl-specific reagent. Induction of apoptosis in cells triggered a change in the conformation of Bcl-2 such that cysteine 158 near the base of helix 5 inserted into the lipid bilayer of both endoplasmic reticulum and mitochondria where it was protected from labeling. Addition of a peptide corresponding to the BH3 domain of the proapoptotic protein Bim to cell lysates triggered a similar conformational change in Bcl-2, demonstrating that preexisting, membrane-bound Bcl-2 proteins change topology.

Claudin-2 is selectively enriched in and promotes the formation of breast cancer liver metastases through engagement of integrin complexes

The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α2β1- and α5β1-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α5β1 and α2β1 complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver.

Bcl-2 Homodimerization Involves Two Distinct Binding Surfaces, a Topographic Arrangement That Provides an Effective Mechanism for Bcl-2 to Capture Activated Bax

The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.

Signaling through ShcA Is Required for Transforming Growth Factor β- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion

ABSTRACT Cooperation between the Neu/ErbB-2 and transforming growth factor β (TGF-β) signaling pathways enhances the invasive and metastatic capabilities of breast cancer cells; however, the underlying mechanisms mediating this synergy have yet to be fully explained. We demonstrate that TGF-β induces the migration and invasion of mammary tumor explants expressing an activated Neu/ErbB-2 receptor, which requires signaling from autophosphorylation sites located in the C terminus. A systematic analysis of mammary tumor explants expressing Neu/ErbB-2 add-back receptors that couple to distinct signaling molecules has mapped the synergistic effect of TGF-β-induced motility and invasion to signals emanating from tyrosine residues 1226/1227 and 1253 of Neu/ErbB-2. Given that the ShcA adaptor protein is known to interact with Neu/ErbB-2 through these residues, we investigated the importance of this signaling molecule in TGF-β-induced cell motility and invasion. The reduction of ShcA expression rendered cells expressing activated Neu/ErbB-2, or add-back receptors signaling specifically through tyrosines 1226/1227 or 1253, unresponsive to TGF-β-induced motility and invasion. In addition, a dominant-negative form of ShcA, lacking its three known tyrosine phosphorylation sites, completely abrogates the TGF-β-induced migration and invasion of breast cancer cells expressing activated Neu/ErbB-2. Our results implicate signaling through the ShcA adaptor as a key component in the synergistic interaction between these pathways.