Identification of cytoskeleton-associated proteins whose depletion induces non-apoptotic cancer cell death.

<p>(A, B) MCF7 (A), HeLa (B) and U-2-OS (B) cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or three independent siRNAs against the indicated targets individually (8 nM; A) or in pools (3×6.67 nM; B). Cell density was measured after 72 h by the MTT reduction assay. (C) MCF7-Bcl-2 or MCF7-pCEP (control) cells were transfected as in (B). <i>Left bottom</i>, After 96 h, cell death was determined by counting Hoechst 33342-stained cells with condensed nuclei (three random fields of 100 cells). TNF (20 ng/ml, 24 h) served as a positive control for Bcl-2 sensitive apoptotic cell death. <i>Left top</i>, Western blot confirming overexpression of Bcl-2 in untreated MCF7-Bcl-2 cells. <i>Right</i>, Examples of images of Hoechst 33342-stained nuclei of MCF7-pCEP and MCF7-Bcl-2 cells 96 h after transfection with indicated siRNAs. (D) MCF7 cells were treated as in (B). <i>Left</i>, After 60 h, DNA was stained with propidium iodide and cell cycle distribution analyzed by flow cytometry (FL-2A). <i>Right</i>, Examples of histograms showing cell cycle distribution of cells 60 h after transfection with indicated siRNAs. Values represent means + SD of three independent experiments (A, C, D) or triplicates in one representative experiment (B, n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells or as indicated (C).</p

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

<div><p>Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, <em>i.e.</em> increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, <em>i.e.</em> photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.</p> </div

Effect of the identified siRNAs on autophagy and dextran uptake.

<p>(A–D) tfLC3-MCF7 cells (A, B) or MCF7 cells (C, D) were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools (3×6.67 nM). (A, B) After 48 h, tfLC3-MCF7 cells were analyzed by confocal microscopy. Representative images (A; <i>Bars</i>, 10 µm) and quantification of puncta (B) are shown. Raptor siRNA (RPTOR) served as a control for increased autophagic flux. Closed arrows indicate AVd, open arrows indicate AVi. (C) After 60 h, the level of p62/SQSTM1 (p62), which is degraded by autophagy, was examined by Western blot. Rapamycin (20 nM, 4 h) was used to induce autophagy. Numbers represent p62 levels as percentage of the level in untreated control siRNA-transfected cells. (D) <i>Top</i>, After 60 h, MCF7 cells were treated with 100 µg/ml Alexa Fluor 488-dextran (dextran-488) for 1 h and analyzed by flow cytometry (FL1-H). The threshold for high intensity staining was defined so that 88% of control siRNA-transfected cells were below. <i>Bottom</i>, Example histograms showing dextran-488 content of cells transfected with control or KIF20A siRNA. M1 = gate for high intensity staining. Values represent means + SEM of 20 cells in one representative experiment (B, n = 3) or means + SD of three independent experiments (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells.</p

Supplementary Figures 1-4 from Sunitinib and SU11652 Inhibit Acid Sphingomyelinase, Destabilize Lysosomes, and Inhibit Multidrug Resistance

PDF - 207KB, Supplementary Figure S1: Screen for small molecules that exhibit non-apoptotic cytotoxicity. Supplementary Figure S2: Kinetics of SU11652- and sunitinib-induced cytotoxicity. Supplementary Figure S3: SU11652 and sunitinib accumulate rapidly in lysosomes of MCF7 cells. Supplementary Figure S4: SU11652 does not alter the ultrastructure of mitochondria or ER.</p

Summary of cellular changes induced by the depletion of survival-associated motor proteins.

1<p>CC, cell cycle.</p>2<p>VAC, volume of the acidic compartment (late endosomes and lysosomes).</p>3<p>photo-oxidation-induced lysosomal leakage.</p>4<p>−, decrease, <i>P</i><0.05; +, increase, <i>P</i><0.05; ±, no change; () 0.05<<i>P</i><0.10.</p>5<p>It should be noted that these data might be affected by cell death that starts already 50 hours after the transfection with KIF21A siRNAs.</p

Reduction of lysosomal stability by the identified siRNAs and monastrol.

<p>(A–C) MCF7 cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools (3×6.67 nM). (A and B) After 60 h, cells were treated with acridine orange and analyzed by live cell imaging to measure the loss of lysosomal integrity (increased green fluorescence) upon laser treatment. A miminum of 25 cells from pre-defined areas was examined for each experiment. Three independent experiments are shown in A and values in B represent means + SD of these experiments at the 60 sec time point. (C) After 72 h, cytosolic and total cysteine cathepsin activities were measured by analyzing the cleavage of zFR-AFC. The activities in cytosolic extracts are shown as percentages of the activities in the corresponding total extracts. HSPA1 and CTSB siRNAs served as controls for the induction of lysosomal leakage and transfection efficacy, respectively. (D) Cytosolic cysteine cathepsin activities in MCF7 cells left untreated or treated with vehicle (2% dimethyl sulfoxide, DMSO) or indicated concentrations of monastrol for 72 h were determined as in (C). Values represent means + SD of five (C) or three (D) independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected (B, C) or vehicle-treated cells (D).</p

Sensitization to lysosome-disrupting drugs by the identified siRNAs and monastrol.

<p>(A, B) MCF7 cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA or indicated siRNA pools (3×6.67 nM). After 48 h, cells were left untreated or treated with 2 µM siramesine (<i>top</i>), 50 µM etoposide (<i>middle</i>) or 10 µM cisplatin (<i>bottom</i>) for additional 48 h before light microscopy pictures were taken (representative images in B) and cell death was quantified by the LDH release assay (A). (C) MCF7 cells were left untreated or treated with 2 µM siramesine together with indicated concentrations of monastrol for 72 h and cell death was quantified by the LDH release assay. Values represent means + SD of a minimum of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells (A) or cells treated with 2 µM siramesine alone (C).</p

Effect of the identified siRNAs on lysosomes and cytoskeleton.

<p>(A–D) MCF7 cells were left untreated, treated with Oligofectamine (Oligo) or transfected with control siRNA (CT) or indicated siRNA pools. (A) After 60 h, cells with enlarged acidic compartment (late endosomes and lysosomes) were identified by flow cytometry (FL-2A) of LysoTracker Red-stained cells. The threshold for high intensity staining was defined so that 90% of control siRNA-transfected cells were below. (B) After 72 h, total cysteine cathepsin activity (zFR-AFC cleavage) was determined. HSPA1 and CTSB siRNAs served as internal controls. (C, D) After 60 h, cells were stained for Lamp-2 (C) or F-actin (D) and analyzed by confocal microscopy. Representative images are shown. <i>Arrows</i>, aggregation of lysosomal structures in cell protrusions/periphery. <i>Bars</i>, 20 µm. Values represent means + SD of a minimum of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, vs. control siRNA-transfected cells.</p

Video1_Annexin A7 mediates lysosome repair independently of ESCRT-III.avi

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.</p

Video7_Annexin A7 mediates lysosome repair independently of ESCRT-III.wmv

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.</p