IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration.

The influence of aging on intestinal stem cells and their niche can explain underlying causes for perturbation in their function observed during aging. Molecular mechanisms for such a decrease in the functionality of intestinal stem cells during aging remain largely undetermined. Using transcriptome-wide approaches, our study demonstrates that aging intestinal stem cells strongly upregulate antigen presenting pathway genes and over-express secretory lineage marker genes resulting in lineage skewed differentiation into the secretory lineage and strong upregulation of MHC class II antigens in the aged intestinal epithelium. Mechanistically, we identified an increase in proinflammatory cells in the lamina propria as the main source of elevated interferon gamma (IFNγ) in the aged intestine, that leads to the induction of Stat1 activity in intestinal stem cells thus priming the aberrant differentiation and elevated antigen presentation in epithelial cells. Of note, systemic inhibition of IFNγ-signaling completely reverses these aging phenotypes and reinstalls regenerative capacity of the aged intestinal epithelium.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

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.

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.

Regulation of the microtubular cytoskeleton by Polycystin-1 favors focal adhesions turnover to modulate cell adhesion and migration.

BACKGROUND: Polycystin-1 (PC-1) is a large plasma membrane receptor, encoded by the PKD1 gene, which is mutated in most cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). The disease is characterized by renal cysts. The precise function of PC-1 remains elusive, although several studies suggest that it can regulate the cellular shape in response to external stimuli. We and others reported that PC-1 regulates the actin cytoskeleton and cell migration. RESULTS: Here we show that cells over-expressing PC-1 display enhanced adhesion rates to the substrate, while cells lacking PC-1 have a reduced capability to adhere. In search for the mechanism responsible for this new property of PC-1 we found that this receptor is able to regulate the stability of the microtubules, in addition to its capability to regulate the actin cytoskeleton. The two cytoskeletal components are acting in a coordinated fashion. Notably, we uncovered that PC-1 regulation of the microtubule cytoskeleton impacts on the turnover rates of focal adhesions in migrating cells and we link all these properties to the capability of PC-1 to regulate the activation state of Focal Adhesion Kinase (FAK). CONCLUSIONS: In this study we show several new features of the PC-1 receptor in modulating microtubules and adhesion dynamics, which are essential for its capability to regulate migration.

The CDC42-interacting protein 4 controls epithelial cell cohesion and tumor dissemination.

The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

The GTPase-activating protein RN-tre controls focal adhesion turnover and cell migration.

BACKGROUND: Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. RESULTS: Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of ß1, but not ß3, integrins and delays the turnover of FAs, ultimately impairing ß1-dependent, but not ß3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. CONCLUSIONS: Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.