Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner

Currently several combination treatments of mTor- and Ras-pathway inhibitors are being tested in cancer therapy. While multiple feedback loops render these central signaling pathways robust, they complicate drug targeting. Here, we describe a novel H-ras specific feedback, which leads to an inadvertent rapalog induced activation of tumorigenicity in Ras transformed cells. We find that rapalogs specifically increase nanoscale clustering (nanoclustering) of oncogenic H-ras but not K-ras on the plasma membrane. This increases H-ras signaling output, promotes mammosphere numbers in a H-ras-dependent manner and tumor growth in ovo. Surprisingly, also other FKBP12 binders, but not mTor- inhibitors, robustly decrease FKBP12 levels after prolonged (> 2 days) exposure. This leads to an upregulation of the nanocluster scaffold galectin-1 (Gal-1), which is responsible for the rapamycin-induced increase in H-ras nanoclustering and signaling output. We provide evidence that Gal-1 promotes stemness features in tumorigenic cells. Therefore, it may be necessary to block inadvertent induction of stemness traits in H-ras transformed cells by specific Gal-1 inhibitors that abrogate its effect on H-ras nanocluster. On a more general level, our findings may add an important mechanistic explanation to the pleiotropic physiological effects that are observed with rapalogs.Peer reviewe

A cholesterol recognition motif in human phospholipid scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.Fil: Posada, Itziar M. D.. Universidad del País Vasco; EspañaFil: Fantini, Jacques. Universidad de Aix-Marsella; FranciaFil: Contreras, F. Xabier. Universidad del País Vasco; España. Ikerbasque; EspañaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas; ArgentinaFil: Alonso, Alicia. Universidad del País Vasco; EspañaFil: Goñi, Félix M.. Universidad del País Vasco; Españ

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

<div><p>Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.</p></div

ASPP2 dominates over Gal-1 thus robustly inducing senescence and abrogating mammosphere formation.

<p>(<b>A</b>) SA-β-gal assay of MCF-7 cells transfected with plasmids encoding H-rasG12V, ASPP2, Gal-1 or the combination of the latter two, as indicated. Cells were stained 7 days after transfection. On the left, percentages of SA-β-gal positive cells are shown in the graph (mean ± SEM, n = 3). On the right, representative images from the assay. (<b>B</b>) Mammosphere formation assay with MCF-7, MDA-MB-231 or HS-578T breast cancer cell lines. Mammospheres were transfected with Gal-1, ASPP2, or both (1:1 ratio) and cells were then grown under non-adherent conditions for 9 days. On the right, representative images of mammospheres are shown as indicated. (<b>A, B</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM n≥3; ns, not significant; ****, p<0.0001).</p

N- and C-terminal truncation mutants of ASPP2 can still promote Ras nanoclustering.

<p>(<b>A</b>) Schematic of full-length ASPP2, as well as ASPP2(1–360) and ASPP2(123–1128) truncation mutants. ASPP2 domains from left to right: Ubl, ubiquitin-like domain; α-helical domain; Pro, proline-rich domain; Ank, Ankyrin repeats; SH3, SRC homology 3 domain. (<b>B</b>) Confocal microscopic images of HEK cells cotransfected with mGFP-H-rasG12V (green) and full-length or truncated ASPP2 (red). (<b>C-E</b>) Nanoclustering-FRET analysis of HEK cells coexpressing mGFP- and mCherry-tagged (<b>C</b>) H-rasG12V, (<b>D</b>) K-rasG12V or (<b>E</b>) N-rasG12V. Cells were analysed after overexpression of Gal-1, full-length ASPP2 or its truncation mutants. (<b>C-E</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; ****, p< 0.0001). (<b>F</b>) Western blot of anti-GFP immunoprecipitation samples probed with anti-ASPP2- (top) or anti-GFP- (bottom) antibodies. Samples were lysates prepared from mGFP-H-rasG12V transfected HEK cells that were cotransfected with full-length ASPP2 or its truncation mutants or an empty plasmid (control), as indicated. In, input; Ft, flow-through; W1, wash; E, elution. Red boxes indicate the immunoprecipitated ASPP2 fragments.</p

The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca2+-binding

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca2+-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291\u2013309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCR\u394) in which the last 28 amino acid residues were lacking, including the \u3b1-helix. SCR\u394 had lost the scramblase activity and its affinity for Ca2+ was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca2+ exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes