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The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.

Aerotaxis or chemotaxis to oxygen was described in bacteria 130 years ago. In eukaryotes, the main adaptation to hypoxia currently described relies on HIF transcription factors. To investigate whether aerotaxis is conserved in higher eukaryotes, an approach based on the self-generation of hypoxia after cell confinement was developed. We show that epithelial cells from various tissues migrate with an extreme directionality towards oxygen to escape hypoxia, independently of the HIF pathway. We provide evidence that, concomitant to the oxygen gradient, a gradient of reactive oxygen species (ROS) develops under confinement and that antioxidants dampen aerotaxis. Finally, we establish that in mammary cells, EGF receptor, the activity of which is potentiated by ROS and inhibited by hypoxia, represents the molecular target that guides hypoxic cells to oxygen. Our results reveals that aerotaxis is a property of higher eukaryotic cells and proceeds from the conversion of oxygen into ROS. Aerotaxis, chemotaxis towards oxygen, occurs in bacteria and likely in cancer cells. Here the authors find that confined cells from different tissues escape hypoxia by aerotaxis, a process independent of mitochondria and the HIF pathway, and dependent on EGF receptor interpretation of a ROS gradient in mammary cells.

Background Aerotaxis, the chemotactism to oxygen, is well documented in prokaryotes. We previously reported for the first time that non-tumorigenic breast epithelial cells also display unequivocal directional migration towards oxygen. This process is independent of the hypoxia-inducible factor (HIF)/prolyl hydroxylase domain (PHD) pathway but controlled by the redox regulation of epidermal growth factor receptor (EGFR), with a reactive oxygen species (ROS) gradient overlapping the oxygen gradient at low oxygen concentration. Since hypoxia is an acknowledged hallmark of cancers, we addressed the putative contribution of aerotaxis to cancer metastasis by studying the directed migration of cancer cells from an hypoxic environment towards nearby oxygen sources, modelling the in vivo migration of cancer cells towards blood capillaries. Methods We subjected to the aerotactic test described in our previous papers cells isolated from fresh breast tumours analysed by the Pathology Department of the Saint-Etienne University Hospital (France) over a year. The main selection criterion, aside from patient consent, was the size of the tumour, which had to be large enough to perform the aerotactic tests without compromising routine diagnostic tests. Finally, we compared the aerotactic properties of these primary cells with those of commonly available breast cancer cell lines. Results We show that cells freshly isolated from sixteen human breast tumour biopsies, representative of various histological characteristics and grades, are endowed with strong aerotactic properties similar to normal mammary epithelial cell lines. Strikingly, aerotaxis of these primary cancerous cells is also strongly dependent on both EGFR activation and ROS. In addition, we demonstrate that aerotaxis can trigger directional invasion of tumour cells within the extracellular matrix contrary to normal mammary epithelial cells. This contrasts with results obtained with breast cancer cell lines, in which aerotactic properties were either retained or impaired, and in some cases, even lost during the establishment of these cell lines. Conclusions Altogether, our results support that aerotaxis may play an important role in breast tumour metastasis. In view of these findings, we discuss the prospects for combating metastatic spread. Trial registration IRBN1462021/CHUSTE. Graphical abstract: EGFR-dependent aerotaxis of primary breast cancer cells

Intracellular Ca 2+ signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca 2+ pool and Ca 2+ pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca 2+ pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca 2+ -dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca 2+ homeostasis in cytoskeleton dynamics and cell migration.

Errors during cell division lead to aneuploidy, which is associated with genomic instability and cell transformation. In response to aneuploidy, cells activate the tumour suppressor p53 to elicit a surveillance mechanism that halts proliferation and promotes senescence. The molecular sensors that trigger this checkpoint are unclear. Here, using a tunable system of chromosome mis-segregation, we show that mitotic errors trigger nuclear deformation, nuclear softening, and lamin and heterochromatin alterations, leading to rapid p53/p21 activation upon mitotic exit in response to changes in nuclear mechanics. We identify mTORC2 and ATR as nuclear deformation sensors upstream of p53/p21 activation. While triggering mitotic arrest, the chromosome mis-segregation-induced alterations of nuclear envelope mechanics provide a fitness advantage for aneuploid cells by promoting nuclear deformation resilience and enhancing pro-invasive capabilities. Collectively, this work identifies a nuclear mechanical checkpoint triggered by altered chromatin organization that probably plays a critical role in cellular transformation and cancer progression. Hervé, Scelfo et al. show that chromosome mis-segregation induces mTORC2- and ATR-mediated p53 activation through a mechanosensitive checkpoint at the nuclear envelope triggered by altered heterochromatin content and increased nuclear membrane tension.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.Microtubules respond to mechanical compression by deforming, becoming more stable, which results in CLASP2 recruitment to the distorted shaft—a process crucial for cell migration through confined spaces.

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5–9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins. A dual illumination method reduces photobleaching for green and yellow fluorescent proteins.

Background The Transforming growth factor β (TGFβ) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGFβ1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFβ/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGFβ1, SMAD4 and TIF1γ expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. Methods Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Léon Bérard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0–6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGFβ1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson’s chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. Results 223 cases were assessable for TIF1γ, 204 for TGFβ1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2 % and 45.2 % had axillary lymph node (LN) metastasis (N1a to N3). 19.4 % of the patients had SBR grade I tumors, 46.8 % grade II tumors and 33.9 % grade III tumors. ER was positive in 85.4 %, PR in 75.5 % and Her2-neu was over-expressed in 10 % of the cases. Nuclear TIF1γ, cytoplasmic TGFβ1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9 %, 30.4 %, 27.7 % and 52.6 % respectively. TIF1γ expression was associated with younger age (p = 0.006), higher SBR grade (p < 0.001), more ER negativity (p = 0.035), and tumors larger than 2 cm (p = 0.081), while TGFβ1 was not associated with any of the traditional prognostic factors. TGFβ1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR = 2.28; 95 % CI: 1.4 to 3.8; p = 0.002), DFS (HR = 2.00; 95 % CI: 1.25 to 3.5; p = 0.005) and OS (HR = 1.89; 95 % CI: 1.04 to 3.43; p = 0.037). TIF1γ expression carried a tendency towards poorer DMFS (p = 0.091), DFS (p = 0.143) and OS (p = 0.091). In the multivariate analysis TGFβ1 remained an independent predictor of shorter DMFS, DFS and OS. Moreover, the prognostic significance of TGFβ1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR = 3.2; 95 % CI: 1.7 to 5.9; p < 0.0001), DFS (HR = 3.02; 95 % CI: 1.6 to 5.6; p < 0.0001) and OS (HR = 2.7; 95 % CI: 1.4 to 5.4; p = 0.005). Conclusion There is a crosstalk between the TIF1γ and the TGFβ1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.

The Transforming growth factor [beta] (TGF[beta]) signaling has a paradoxical role in cancer development and outcome. Besides, the prognostic significance of the TGF[beta]1, SMAD4 in breast cancer patients is an area of many contradictions. The transcriptional intermediary factor 1[gamma] (TIF1[gamma]) is thought to interact with the TGF[beta]/SMAD signaling through different mechanisms. Our study aims to define the prognostic significance of TGF[beta]1, SMAD4 and TIF1[gamma] expression in breast cancer patients and to detect possible interactions among those markers that might affect the outcome. Immunohistochemistry was performed on tissue microarray (TMA) blocks prepared from samples of 248 operable breast cancer patients who presented at Centre Lñon Bñrard (CLB) between 1998 and 2001. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between TGF[beta]1, SMAD4 and TIF1[gamma] expression with the clinico-pathologic parameters using Pearson's chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. 223 cases were assessable for TIF1[gamma], 204 for TGF[beta]1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2 % and 45.2 % had axillary lymph node (LN) metastasis (N1a to N3). 19.4 % of the patients had SBR grade I tumors, 46.8 % grade II tumors and 33.9 % grade III tumors. ER was positive in 85.4 %, PR in 75.5 % and Her2-neu was over-expressed in 10 % of the cases. Nuclear TIF1[gamma], cytoplasmic TGF[beta]1, nuclear and cytoplasmic SMAD4 stainings were high in 35.9 %, 30.4 %, 27.7 % and 52.6 % respectively. TIF1[gamma] expression was associated with younger age (p = 0.006), higher SBR grade (p < 0.001), more ER negativity (p = 0.035), and tumors larger than 2 cm (p = 0.081), while TGF[beta]1 was not associated with any of the traditional prognostic factors. There is a crosstalk between the TIF1[gamma] and the TGF[beta]1/SMAD4 signaling that deteriorates the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients.

Intracellular Ca signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca pool and Ca pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca -dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca homeostasis in cytoskeleton dynamics and cell migration.