Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (DN-ErbB2). Methodology/Principal Findings: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm. Conclusions/Significance: Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formatio

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

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 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

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

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