AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

KRAS-Driven Metabolic Rewiring Reveals Novel Actionable Targets in Cancer.

Tumors driven by mutant KRAS are among the most aggressive and refractory to treatment. Unfortunately, despite the efforts, targeting alterations of this GTPase, either directly or by acting on the downstream signaling cascades, has been, so far, largely unsuccessful. However, recently, novel therapeutic opportunities are emerging based on the effect that this oncogenic lesion exerts in rewiring the cancer cell metabolism. Cancer cells that become dependent on KRAS-driven metabolic adaptations are sensitive to the inhibition of these metabolic routes, revealing novel therapeutic windows of intervention. In general, mutant KRAS fosters tumor growth by shifting cancer cell metabolism toward anabolic pathways. Depending on the tumor, KRAS-driven metabolic rewiring occurs by up-regulating rate-limiting enzymes involved in amino acid, fatty acid, or nucleotide biosynthesis, and by stimulating scavenging pathways such as macropinocytosis and autophagy, which, in turn, provide building blocks to the anabolic routes, also maintaining the energy levels and the cell redox potential (1). This review will discuss the most recent findings on mutant KRAS metabolic reliance in tumor models of pancreatic and non-small-cell lung cancer, also highlighting the role that these metabolic adaptations play in resistance to target therapy. The effects of constitutive KRAS activation in glycolysis elevation, amino acids metabolism reprogramming, fatty acid turnover, and nucleotide biosynthesis will be discussed also in the context of different genetic landscapes.

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

Kinesin-2 Controls the Motility of RAB5 Endosomes and Their Association with the Spindle in Mitosis.

RAB5 is a small GTPase that belongs to the wide family of Rab proteins and localizes on early endosomes. In its active GTP-bound form, RAB5 recruits downstream effectors that, in turn, are responsible for distinct aspects of early endosome function, including their movement along microtubules. We previously reported that, at the onset of mitosis, RAB5positive vesicles cluster around the spindle poles and, during metaphase, move along spindle microtubules. RNAi-mediated depletion of the three RAB5 isoforms delays nuclear envelope breakdown at prophase and severely affects chromosome alignment and segregation. Here we show that depletion of the Kinesin-2 motor complex impairs long-range movement of RAB5 endosomes in interphase cells and prevents localization of these vesicles at the spindle during metaphase. Similarly to the effect caused by RAB5 depletion, functional ablation of Kinesin-2 delays nuclear envelope breakdown resulting in prolonged prophase. Altogether these findings suggest that endosomal transport at the onset of mitosis is required to control timing of nuclear envelope breakdown.

High USP6NL Levels in Breast Cancer Sustain Chronic AKT Phosphorylation and GLUT1 Stability Fueling Aerobic Glycolysis.

USP6NL, also named RN-tre, is a GTPase-activating protein involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer, mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis. High levels of USP6NL in breast cancer cells delayed endocytosis and degradation of the EGFR, causing chronic AKT (protein kinase B) activation. In turn, AKT stabilized the glucose transporter GLUT1 at the plasma membrane, increasing aerobic glycolysis. In agreement, elevated USP6NL sensitized breast cancer cells to glucose deprivation, indicating that their glycolytic capacity relies on this protein. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing cell proliferation exclusively in breast cancer cells that harbored increased levels of USP6NL. Overall, these findings argue that USP6NL overexpression generates a metabolic rewiring that is essential to foster the glycolytic demand of breast cancer cells and promote their proliferation.Significance: USP6NL overexpression leads to glycolysis addiction of breast cancer cells and presents a point of metabolic vulnerability for therapeutic targeting in a subset of aggressive basal-like breast tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3432/F1.large.jpg Cancer Res; 78(13); 3432-44. ©2018 AACR.

Sema4C/PlexinB2 signaling controls breast cancer cell growth, hormonal dependence and tumorigenic potential.

Semaphorin 4C (Sema4C) expression in human breast cancers correlates with poor disease outcome. Surprisingly, upon knock-down of Sema4C or its receptor PlexinB2 in diverse mammary carcinoma cells (but not their normal counterparts), we observed dramatic growth inhibition associated with impairment of G2/M phase transition, cytokinesis defects and the onset of cell senescence. Mechanistically, we demonstrated a Sema4C/PlexinB2/LARG-dependent signaling cascade that is required to maintain critical RhoA-GTP levels in cancer cells. Interestingly, we also found that Sema4C upregulation in luminal-type breast cancer cells drives a dramatic phenotypic change, with disassembly of polarity complexes, mitotic spindle misorientation, cell-cell dissociation and increased migration and invasiveness. We found that this signaling cascade is dependent on the PlexinB2 effectors ErbB2 and RhoA-dependent kinases. Moreover, Sema4C-overexpressing luminal breast cancer cells upregulated the transcription factors Snail, Slug and SOX-2, and formed estrogen-independent metastatic tumors in mice. In sum, our data indicate that Sema4C/PlexinB2 signaling is essential for the growth of breast carcinoma cells, featuring a novel potential therapeutic target. In addition, elevated Sema4C expression enables indolent luminal-type tumors to become resistant to estrogen deprivation, invasive and metastatic in vivo, which could account for its association with a subset of human breast cancers with poor prognosis.

Rebound Effects Caused by Withdrawal of MET Kinase Inhibitor Are Quenched by a MET Therapeutic Antibody.

MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest. However, this antiproliferative effect allows persistence of a pool of cancer cells that are quiescent but alive. Once the inhibition is removed, rebound activation of MET-driven cell proliferative pathways and tumor growth may occur, an adverse event observed frequently in clinical settings after drug discontinuation. Here we show that inhibitor withdrawal prompts receptor phosphorylation to levels higher than those displayed at steady-state and generates a rebound effect pushing quiescent cancer cells back into the cell cycle, both in vitro and in experimental tumor models in vivo Mechanistically, we found that inhibitor treatment blocks MET endocytosis, causing a local increase in the number of receptors at the plasma membrane. Upon inhibitor washout, the receptor is readily rephosphorylated. The initial phosphorylation is not only increased but also prolonged in duration due to downmodulation of a phosphatase-mediated MET-negative feedback loop, which accompanies receptor internalization. Notably, treatment with a MET therapeutic antibody that induces proteolytic cleavage of the receptor at the cell surface substantially prevents this rebound effect, providing a rationale to combine or alternate these mechanistically different types of MET-targeted therapy. Cancer Res; 76(17); 5019-29. ©2016 AACR.

Tankyrase inhibition impairs directional migration and invasion of lung cancer cells by affecting microtubule dynamics and polarity signals.

BACKGROUND: Tankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front. RESULTS: Here we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation. CONCLUSIONS: Collectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells.

Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells.

Hydrogen sulfide (H(2)S) is a gasotransmitter that plays several roles in various tissues, including the cardiovascular system. Because it has been recently proposed to act as a mediator of angiogenesis progression, here we investigate the effects of H(2)S in a well-established model of tumor angiogenesis: endothelial cells obtained from human breast carcinoma (B-TECs). Ca(2+) imaging and patch-clamp experiments reveal that acute perfusion with NaHS, a widely employed H(2)S donor, activates cytosolic calcium (Ca(c)) increase, as well as potassium and nonselective cationic currents, in B-TECs. Stimulation with NaHS in the same concentration range (1 nM-200 µM) evoked Ca(c) signals also in "normal" human microvascular endothelial cells (HMVECs), but the amplitude was significantly lower. Moreover, although NaHS failed to promote either migration or proliferation on HMVECs, B-TEC migration was enhanced at low-micromolar NaHS concentrations (1-10 µM). Remarkably H(2)S mediates tumor proangiogenic signaling triggered by vascular endothelial growth factor (VEGF). B-TECs pretreated with dl-propargylglycine (5mM, 30 min), an inhibitor of the H(2)S-producing enzyme cystathionine γ-lyase, showed drastically reduced migration and Ca(c) signals induced by VEGF (20 ng/ml). We conclude that H(2)S plays a role in proangiogenic signaling of tumor-derived but not normal human ECs. Furthermore the ability of this gasotransmitter to interfere with B-TEC responsiveness to VEGF suggests that it could be an interesting target for antiangiogenic strategies in tumor treatment.

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.

Multiple roles of protein kinase a in arachidonic acid-mediated Ca2+ entry and tumor-derived human endothelial cell migration.

We recently showed that arachidonic acid (AA) triggers calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). In particular, AA-dependent Ca(2+) entry is involved in the early steps of tumor angiogenesis in vitro. Here, we investigated the multiple roles of the nitric oxide (NO) and cyclic AMP/protein kinase A (PKA) pathways in AA-mediated Ca(2+) signaling in the same cells. B-TEC stimulation with 5 µmol/L AA resulted in endothelial NO synthase (NOS) phosphorylation at Ser(1177), and NO release was measured with the fluorescent NO-sensitive probe DAR4M-AM. PKA inhibition by the use of the membrane-permeable PKA inhibitory peptide myristoylated PKI(14-22) completely prevented both AA- and NO-induced calcium entry and abolished B-TEC migration promoted by AA. AA-dependent calcium entry and cell migration were significantly affected by both the NOS inhibitor N(G)-nitro-l-arginine methyl ester and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, suggesting that NO release is functionally involved in the signaling dependent on AA. Moreover, pretreatment with carboxyamidotriazole, an antiangiogenic compound that interferes with agonist-activated calcium entry, prevented AA-dependent B-TEC motility. Interestingly, even in the absence of AA, enhancement of the cyclic AMP/PKA pathway with the adenylyl cyclase activator forskolin evoked a calcium entry dependent on NOS recruitment and NO release. The functional relevance of AA-induced calcium entry could be restricted to tumor-derived endothelial cells (EC) because AA evoked a smaller calcium entry in normal human microvascular ECs compared with B-TECs, and even more importantly, it was unable to promote cell motility in wound healing assay. This evidence opens an intriguing opportunity for differential pharmacologic treatment between normal and tumor-derived human ECs.

Genome-wide discovery of functional transcription factor binding sites by comparative genomics: the case of Stat3.

The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. However, the use of high throughput experimental methods, such as ChIP-chip and ChIP-sequencing, is limited by their high cost and strong dependence on cellular type and context. We developed a computational method for the genome-wide identification of functional transcription factor binding sites based on positional weight matrices, comparative genomics, and gene expression profiling. The method was applied to Stat3, a transcription factor playing crucial roles in inflammation, immunity and oncogenesis, and able to induce distinct subsets of target genes in different cell types or conditions. A newly generated positional weight matrix enabled us to assign affinity scores of high specificity, as measured by EMSA competition assays. Phylogenetic conservation with 7 vertebrate species was used to select the binding sites most likely to be functional. Validation was carried out on predicted sites within genes identified as differentially expressed in the presence or absence of Stat3 by microarray analysis. Twelve of the fourteen sites tested were bound by Stat3 in vivo, as assessed by Chromatin Immunoprecipitation, allowing us to identify 9 Stat3 transcriptional targets. Given its high validation rate, and the availability of large transcription factor-dependent gene expression datasets obtained under diverse experimental conditions, our approach appears to be a valid alternative to high-throughput experimental assays for the discovery of novel direct targets of transcription factors.