Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug-or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and downregulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin b4-subunit). Importantly, gene expression profiling analyses reveal that vimentin expression correlates positively/ negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two-and three-dimensional matrices by vimentin is Axldependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process.

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDPS)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca2+ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDPS in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS to promote IP3R-mediated Ca2+ release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDPS-induced Ca2+ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDPS-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca2+ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca2+ signaling and cell death.

Systemic chemotherapy generally has been considered immunosuppressive, but it has become evident that certain chemotherapeutic drugs elicit immunogenic danger signals in dying cancer cells that can incite protective antitumor immunity. In this study, we investigated whether locoregionally applied therapies, such as melphalan, used in limb perfusion for melanoma (Mel-ILP) produce related immunogenic effects. In human melanoma biopsies, Mel-ILP treatment upregulated IL1B, IL8, and IL6 associated with their release in patients' locoregional sera. Although induction of apoptosis in melanoma cells by melphalan in vitro did not elicit threshold levels of endoplasmic reticulum and reactive oxygen species stress associated with danger signals, such as induction of cell-surface calreticulin, prophylactic immunization and T-cell depletion experiments showed that melphalan administration in vivo could stimulate a CD8 þ T cell-dependent protective antitumor response. Interestingly, the vaccination effect was potentiated in combination with exogenous calreticulin, but not tumor necrosis factor, a cytokine often combined with Mel-ILP. Our results illustrate how melphalan triggers inflammatory cell death that can be leveraged by immunomodulators such as the danger signal calreticulin. Cancer Res; 75(8);

Background: Evolutionary conserved Bax inhibitor-1 (BI-1) protects against ER stress-mediated apoptosis. Results: We identified a Ca 2ϩ -permeable channel pore in the C terminus of BI-1. Critical pore properties are an ␣-helical structure and two aspartate residues conserved among animals, but not among plants and yeast. Conclusion: C-terminal domain of BI-1 harbors a Ca 2ϩ -permeable channel pore. Significance: BI-1 has Ca 2ϩ channel properties likely relevant for its function in ER stress and apoptosis. Bax inhibitor-1 (BI-1) is a multitransmembrane domainspanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca 2؉homeostasis by acting as a Ca 2؉ -leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca 2؉ pore responsible for its Ca 2؉ -leak properties. We utilized a set of C-terminal peptides to screen for Ca 2؉ leak activity in unidirectional 45 Ca 2؉ -flux experiments and identified an ␣-helical 20-amino acid peptide causing Ca 2؉ leak from the ER. The Ca 2؉ leak was independent of endogenous ER Ca 2؉ -release channels or other Ca 2؉ -leak mechanisms, namely translocons and presenilins. The Ca 2؉ -permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca 2؉ -leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca 2؉ -leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca 2؉ -channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca 2؉ leak. Thus, we elucidated residues critically important for BI-1-mediated Ca 2؉ leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca2؉ -leak properties of BI-1 are an added function during evolution. leak from the ER (9, 10). BI-1 seems to be strongly evolutionarily conserved and BI-1 orthologs from plants can substitute for mammalian BI-1 in regard to its anti-apoptotic function (11). Besides this, other diverse functions of BI-1 have been described. BI-1 is a negative regulator of the ER-stress sensor (12), it interacts with G-actin and increases actin polymerization (13), enhances cancer metastasis by altering glucose metabolism and by activating a sodium-hydrogen exchanger (14), and it reduces production of reactive oxygen species through direct interaction with NADPH-P450 reductase (15), a member of the microsomal monooxygenase system.Recently, the role of BI-1 in Ca 2ϩ signaling has been further explored. The effect of BI-1 on cell death seems to involve changes in the amount of Ca 2ϩ that is releasable from intrace...

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane. Cell Death and Differentiation (2012) 19, 1013-1026 doi:10.1038/cdd.2011; published online 13 January 2012Apoptosis is a conserved cell death mechanism essential for normal development and tissue homeostasis in multicellular organisms. In mammals, the BCL-2 family of proteins is a group of crucial upstream regulators of the caspase cascade, comprising both pro-and anti-apoptotic components. 1Although apoptosis is observed in most multicellular organisms, the BCL-2 family of proteins as a whole is poorly conserved in invertebrates including worms, flies, and other species.1,2 In fact, only two BCL-2 homologues are present in flies with controversial roles in programmed cell death. 3,4 A pioneering screening to identify human genes that prevents BAX toxicity in a yeast assay identified transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX inhibitor-1 (BI-1), as a new mammalian gene that negatively regulates apoptosis (reviewed in Robinson KS et al. and Reimers K et al. 5,6 ). Further studies demonstrated that TMBIM6/BI-1 is a six transmembrane-spanning protein, located at the endoplasmic reticulum (ER) that has a relevant role in preventing apoptosis. 5 Remarkably, bioinformatic analysis defined a putative family of at least six highly conserved orthologs of TMBIM6/BI-1containing the consensus motif UPF0005 with unknown function, a domain encoding for six to seven transmembrane-spanning regions. 7TMBIM family of proteins includes the founder member TMBIM6/BI-1, TMBIM1/RECS1 (responsive to centrifugal force and shear stress gene 1 protein), TMBIM2/LFG (life guard), TMBIM3/GRINA (glutamate receptor ionotropic NMDA protein 1), TMBIM4/GAAP (Golgi anti-apoptoticassociated protein), and TMBIM5/GHTIM (growth hormoneinducible transmembrane protein). The TMBIM family of proteins is highly conserved in mammals, zebrafish, and flies, with homol...

Bax inhibitor-1 (BI-1) is an evolutionarily conserved pH-dependent Ca²⁺ leak channel in the endoplasmic reticulum and the founding member of a family of six highly hydrophobic mammalian proteins named transmembrane BAX inhibitor motif containing (TMBIM) 1-6 with BI-1 being TMBIM6. Here we compared the structure, subcellular localization, tissue expression and the effect on the cellular Ca²⁺ homeostasis of all family members side by side. We found that all TMBIM proteins possess the di-aspartyl pH sensor responsible for pH sensing identified in TMBIM6 and its bacterial homologue BsYetJ. TMBIM1-3 and TMBIM4-6 represent two phylogenetically distinct groups that are localized in the Golgi apparatus (TMBIM1-3), endoplasmic reticulum (TMBIM4-6) or mitochondria (TMBIM5) but share a common structure of at least seven transmembrane domains with the last domain being semi-hydrophobic. TMBIM1 is mainly expressed in muscle, TMBIM2 and 3 in the nervous system, TMBIM4 and 5 are ubiquitously expressed and TMBIM6 in skeletal muscle, kidney, liver and spleen. All TMBIM proteins reduce the Ca²⁺ content of the endoplasmic reticulum, and all but TMBIM5 also reduce the cytosolic resting Ca²⁺ concentration. These results suggest that the TMBIM family has comparable functions in the maintenance of intracellular Ca²⁺ homeostasis in a wide variety of tissues. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.