ß-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, ß-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway.

Atypical function of a centrosomal module in WNT signalling drives contextual cancer cell motility.

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.

Methods to Investigate the ß-Arrestin-Mediated Control of ARF6 Activation to Regulate Trafficking and Actin Cytoskeleton Remodeling.

ADP-ribosylation factors (ARF) are GTPases that act to control the activation of numerous signaling events and cellular responses. The ARF6 isoform, present at the plasma membrane, can be activated by the angiotensin II type 1 receptor (AT1R), a process dependent upon ß-arrestin recruitment to the activated receptor. Here, we describe classical methods used to assess ß-arrestin-dependent activation of ARF6 following agonist stimulation of cells. In addition, because ARF6 and ß-arrestin can form a complex, we describe the procedures used to detect the interaction of ß-arrestin with this GTPase.

ARF6 protects sister chromatid cohesion to ensure the formation of stable kinetochore-microtubule attachments.

Sister chromatid cohesion, facilitated by the cohesin protein complex, is crucial for the establishment of stable bipolar attachments of chromosomes to the spindle microtubules and their faithful segregation. Here, we demonstrate that the GTPase ARF6 prevents the premature loss of sister chromatid cohesion. During mitosis, ARF6-depleted cells normally completed chromosome congression. However, at the metaphase plate, chromosomes failed to establish stable kinetochore-microtubule attachments because of the impaired cohesion at centromeres. As a result, the spindle assembly checkpoint (SAC) was active and cyclin B ubiquitylation and degradation were blocked. Chromosomes and/or chromatids in these cells scattered gradually from the metaphase plate to the two poles of the cell or remained blocked at the metaphase plate for hours. Our study demonstrates that the small GTP-binding protein ARF6 is essential for maintaining centromeric cohesion between sister chromatids, which is necessary for the establishment of stable k-fibres, SAC satisfaction and the onset of anaphase.

ARF GTPases control phenotypic switching of vascular smooth muscle cells through the regulation of actin function and actin dependent gene expression.

Vascular smooth muscle cells (VSMC) can exhibit a contractile or a synthetic phenotype depending on the extracellular stimuli present and the composition of the extracellular matrix. Uncontrolled activation of the synthetic VSMC phenotype is however associated with the development of cardiovascular diseases. Here, we aimed to elucidate the role of the ARF GTPases in the regulation of VSMC dedifferentiation. First, we observed that the inhibition of the activation of ARF proteins with SecinH3, a blocker of the cytohesin ARF GEF family, reduced the ability of the cells to migrate and proliferate. In addition, this inhibitor also blocked expression of sm22α and αSMA, two contractile markers, at the transcription level impairing cell contractility. Specific knockdown of ARF1 and ARF6 showed that both isoforms were required for migration and proliferation, but ARF1 only regulated contractility through sm22α and αSMA expression. Expression of these VSMC markers was correlated with the degree of actin polymerization. VSMC treatment with SecinH3 as well as ARF1 depletion was both able to block the formation of stress fibres and focal adhesions, demonstrating the role of this GTPase in actin filament formation. Consequently, we observed that both treatments increased the ratio of G-actin to F-actin in these cells. The elevated amounts of cytoplasmic G-actin, acting as a signaling intermediate, blocked the recruitment of the Mkl1 (MRTF-A) transcription factor in the nucleus, demonstrating its involvement in the regulation of contractile protein expression. Altogether, these findings show for the first time that ARF GTPases are actively involved in VSMC phenotypic switching through the regulation of actin function in migration and proliferation, and the control of actin dependent gene regulation.

Arterial stiffness induced by carotid calcification leads to cerebral gliosis mediated by oxidative stress.

BACKGROUND: Arterial stiffness is a risk factor for cognitive decline and dementia. However, its precise effects on the brain remain unexplored. Using a mouse model of carotid stiffness, we investigated its effect on glial activation and oxidative stress. METHODS: Arterial stiffness was induced by the application of calcium chloride to the adventitial region of the right carotid. Superoxide anion production, NADPH activity and levels, as well as glial activation were examined with immunohistochemical and biochemical approaches, 2-week postcalcification. Antioxidant treatment was done with Tempol (1 mmol/l) administered in the drinking water during 2 weeks. RESULTS: The current study revealed that arterial stiffness increases the levels of the microglial markers ionized calcium-binding adapter molecule 1 and cluster of differentiation 68 in hippocampus, and of the astrocyte marker, s100 calcium binding protein ß in hippocampus and frontal cortex. The cerebral inflammatory effects of arterial stiffness were specific to the brain and not due to systemic inflammation. Treatment with Tempol prevented the increase in superoxide anion in mice with carotid stiffness and attenuated the activation of microglia and astrocytes in the hippocampus. To determine whether the increased oxidative stress derives from NADPH oxidase, superoxide anion production was assessed by incubating brain tissue in the presence of gp91ds-tat, a selective NADPH oxidase 2 inhibitor. This peptide inhibited superoxide anion production to a greater extent in the brains of mice with carotid calcification compared with controls. CONCLUSION: Carotid calcification leads to cerebral gliosis mediated by oxidative stress. Correcting arterial stiffness could offer a novel paradigm to protect the brain in populations where stiffness is prominent.

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation.

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC.