Research ArticleCELL BIOLOGY

Regulated lysosomal exocytosis mediates cancer progression

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Science Advances  18 Dec 2015:
Vol. 1, no. 11, e1500603
DOI: 10.1126/sciadv.1500603
  • Fig. 1 Pleomorphic sarcomas are prevalent in Neu1+//Arf/ mice.

    (A and B) Numbers and types of pleomorphic (A) and nonpleomorphic (B) sarcomas that developed in WT/Arf−/− and Neu1+/−/Arf−/− mice. (C) Number of contained versus invasive pleomorphic sarcomas. (D) Quantification of NEU1 and LAMP1 expression levels in 20 sarcomas from Neu1+/−/Arf−/− mice. (E) Representative images of four pleomorphic sarcomas from Neu1+/−/Arf−/− mice with epithelioid/rhabdoid and spindle cells, as shown by hematoxylin and eosin (H&E) staining, and absent or low Neu1 and strong Lamp1 immunostaining. Examples of rhabdoid cells are marked with arrowheads. Scale bar, 25 μm. (F) Neu1 activity in cells isolated from Neu1+/−/Arf−/− sarcomas compared with that in differentiated myofibers (MF) from WT/Arf−/− mice (n = 5). (G) Lamp1 levels in cells isolated from Neu1+/−/Arf−/− sarcomas compared with that in WT/Arf−/− MF. α/β-Tubulin was used as the loading control. (H) β-Hex activity assayed in the culture medium of Neu1+/−/Arf−/− sarcomas cells compared with that of WT/Arf−/− MF, as a measure of lysosomal exocytosis (n = 5). OS, osteosarcoma; P-HS, pleomorphic hemangiosarcoma; P-LMS, pleomorphic leiomyosarcoma; P-MPNST, pleomorphic malignant peripheral nerve sheath tumor; P-SSMD, pleomorphic sarcoma with smooth muscle differentiation; RMS, rhabdomyosarcoma; SNOS, sarcoma non-otherwise specified; UPS, undifferentiated pleomorphic sarcoma. Data are means ± SD. ****P < 0.0001, Student’s t test for unpaired samples.

  • Fig. 2 NEU1 and LAMP1 expression levels vary inversely in LMS and RMS.

    (A) Quantification of NEU1 and LAMP1 expression levels in 32 LMS tissue arrays. (B) Representative images of LMS and healthy tissues immunostained for NEU1 and LAMP1. (C) Quantification of NEU1 and LAMP1 expression levels in 33 RMS tissue arrays. (D) Representative images of RMS and healthy tissues immunostained for NEU1 and LAMP1. (E) Quantification of NEU1 and LAMP1 expression levels in 14 patient-derived RMS xenografts. (F) Representative images of primary and relapse RMS xenografts from the same patient immunostained for NEU1 and LAMP1. Scale bars, 25 μm.

  • Fig. 3 Combined low NEU1 and high LAMP1 levels result in exacerbated lysosomal exocytosis in RMS cells.

    (A) Real-time quantitative polymerase chain reaction (RT-qPCR) of NEU1 mRNA expression (n = 4). (B) NEU1 enzyme activity measured in RH41 and RH30 cells (n = 4). (C) Immunoblot of LAMP1 and NEU1 in RH41 and RH30 cells. (D) Sialic acid content detected on LAMP1 from RH41 and RH30 cells. The sialic acid–containing glycoproteins were purified through Sambucus nigra (SNA) and Maackia amurensis (MAL) columns, followed by LAMP1 immunoblotting. (E) Number of lysosomes distributed in the peripheral and perinuclear region of RH41 compared with RH30 cells (n = 11). (F) Number of lysosomes tethered to or docked at the PM of RH41 compared with RH30 cells quantified by live TIRF imaging (n = 4). (G) β-Hex activity assayed in the medium of RH41 and RH30 cell as a measure of lysosomal exocytosis (n = 3). (H) Quantification of exosomes released by RH41, RH30, and RH30shLAMP1 cells (n = 4). (I) Immunoblot of exosomal markers flotillin-1, CD81, syndecan-1, and syntenin-1 in exosome preparations purified from RH41, RH30, and RH30shLAMP1 medium. (J) Immunoblot of LAMP1 in RH30shLAMP1 cells and its empty vector control. (K) Number of LysoTracker+ lysosomes present in the evanescent field close to the PM of RH30shLAMP1 cells and their control cells quantified by TIRF microscopy (n = 40). (L) β-Hex activity assayed in the medium of RH30shLAMP1 cells and its empty vector control (n = 5). α/β-Tubulin was used as the loading control. Data are means ± SD or SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test for unpaired samples.

  • Fig. 4 NEU1 gene expression in human sarcomas is inversely correlated with that of MYH11.

    (A) Heat map for NEU1 positively or negatively correlated genes (Z score–normalized log2 expression) within different locations in LMS. Scale colors are graded from 2 (green), to 0 (black), to 2 (red). (B) Negative linear correlation (Pearson’s r = 0.488) plot of NEU1 log2 expression signals to MYH11 log2 expression. The sarcoma class is distinctly split into a high-MYH11– and low-MYH11–expressing, with an isolated high-expressing class primarily composed of LMS (green box). (C) Correlation of NEU1 log2 expression with MYH11 (probe set 201497_x_at) log2 expression in LMS metastasis. A green box highlights the distinct subgroup of metastatic LMS expressing low NEU1 and high MYH11. (D) Boxplot of NEU1 expression depending on the histological type (P = 1.13 × 107) of sarcomas. (E) Boxplot of NEU1 log2 expression within the LMS dependent on tumor location (P = 0.0053). Sarcoma gene set: GSE21050. (F and G) Representative image of a pleomorphic LMS (F) and a pleomorphic RMS (G) from human TMAs immunostained for Myosin-11 (MYO11). (H) Patient-derived RMS xenograft of a primary tumor and relapsed tumor from the same patient immunostained for Myosin-11. Scale bars, 25 μm.

  • Fig. 5 Myosin-11 in sarcomas from Neu1+//Arf/ mice interacts with Lamp1 and contributes to lysosomal exocytosis.

    (A to C) Representative images of pleomorphic sarcomas from Neu1+/−/Arf−/− mice with rhabdoid cells immunostained for Myosin-11. Scale bars, 25 μm. (D) Immunoblot of Myosin-11 and Lamp1 in primary cells isolated from different Neu1+/−/Arf−/− sarcomas, that is, OS, MPNST, LMS, RMS, UPS, P-SSMD, and P-LMS. The sarcoma cells used for the coimmunoprecipitation assays and for silencing of Myh11 and are indicated with “*.” (E) Lamp1 antibody coimmunoprecipitated Myosin-11 from sarcoma cell extract. (F) Myosin-11 antibody coimmunoprecipitated Lamp1 from a cytoskeleton-enriched subcellular fraction of sarcoma cells. (G) Levels of Myosin-11 in sarcoma cells with silencing Myh11 (shMyh11), and with silencing of both Lamp1 and Myh11 (shLamp1/shMyh11), compared with their empty vector controls. (H) Levels of Lamp1 in sarcoma cells with silenced Lamp1 (shLamp1), and shLamp1/shMyh11, compared to their empty vector control. (I) β-Hex activity measured in the medium of shLamp1, shMyh11, or shLamp1/shMyh11 sarcoma cells compared to that assayed in their empty vector controls (n = 4). α/β-Tubulin was used as the loading control. Data are means ± SD. **P < 0.01, ****P < 0.0001, Student’s t test for unpaired samples.

  • Fig. 6 NEU1 levels and the extent of lysosomal exocytosis in RMS cells determine their invasive potential and predict their responsiveness to chemotherapy.

    (A) Number of RH41 and RH30 cells that invaded ex vivo peritonea isolated from Neu1+/+ (WT) or Neu1−/− mice (n = 10). (B) Number of RH30shLAMP1 cells and empty vector controls that invaded peritoneal membranes from WT or Neu1−/− mice (n = 5). (C) Number of RH30 and RH41 cells that migrated through a Matrigel Transwell chamber; number of migratory RH41 cells that were cultured in the presence of exosomes purified from RH30 or RH30shLAMP1 medium (exo) (n = 4). (D) NEU1 activity measured in a crude lysosomal fraction (CLF) of RH30PPCA cells compared to that in the CLF of RH30empty cells (n = 3). (E) Cathepsin A (CA) activity measured in CLF of RH30PPCA cells compared to that in the CLF of RH30empty cells (n = 10). (F) β-Hex activity assayed in the medium of RH30PPCA cells was compared to that in the medium of RH30empty control cells (n = 2). (G) Bioluminescence measurement of tumor growth after injection of RH30PPCA and RH30empty cells into the flank of WT/NSG (blue) or Neu1+/−/NSG (red) mice. (H) Arbitrary fluorescence units (AFU) of doxorubicin (Doxo) effluxed into the medium by RH41 and RH30 cells after exposure to doxorubicin (4 μg/ml) alone or combined with 50 μM verapamil (n = 4). (I) AFU of doxorubicin effluxed into the medium of RH30shLAMP1 and RH30empty cells after exposure to doxorubicin (4 μg/ml) (n = 4). (J) Quantification of the fluorescence intensity in individual RH41, RH30shLAMP1, and RH30empty cells after uptake of doxorubicin (n = 10). (K) Viability curves of RH41 and RH30 cells exposed for 16 hours to the indicated doses of doxorubicin alone or combined with 50 μM verapamil (n = 6). (L) Viability curves of RH30shLAMP1 and RH30empty cells exposed for 16 hours to the indicated doses of doxorubicin (n = 6). Data are means ± SD or SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, Student’s t test for unpaired samples.

  • Fig. 7 The extent of lysosomal exocytosis in tumor cell lines derived from a Neu1+//Arf/ sarcoma determines their invasive potential and predicts their responsiveness to chemotherapy.

    (A) Number of sarcomas cells with silenced Myh11 (shMyh11) and Lamp1 (shLamp1) and with silencing of both Lamp1 and Myh11 (shLamp1/shMyh11) that migrated through a Matrigel Transwell chamber, compared with their empty vector control (n = 8). (B) Quantification of doxorubicin fluorescence (AFU) effluxed into the medium-modified sarcomas cells (shMyh11, shLamp1, and shLamp1/shMyh11) compared to their unmodified cells after exposure to doxorubicin (4 μg/ml) (n = 4). (C) Quantification of the fluorescence intensity in individual modified sarcoma cells (shMyh11, shLamp1, and shLamp1/shMyh11) compared to their unmodified cells after uptake of doxorubicin (n = 14). (D) Sensitivity to doxorubicin was assayed by cleaved poly(adenosine diphosphate–ribose) polymerase (cPARP) expression after exposing Neu1+/−/Arf−/− sarcoma cells to the indicated doses of doxorubicin. α/β-Tubulin was used as the loading control. Data are means ± SD or SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, Student’s t test for unpaired samples.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/11/e1500603/DC1

    Fig. S1. UPS that developed in a Neu1+/−/Arf−/− mouse expresses epithelial and mesenchymal markers and TGFβ.

    Fig. S2. Excessive lysosomal exocytosis in RMS cells is caused by low NEU1 and high LAMP1 levels.

    Fig. S3. Genes whose expression is negatively correlated with that of NEU1 expression in a sarcoma data set (GSE21050).

    Fig. S4. Showing of entire immunoblots corresponding to panels in Fig. 5 (E and F).

    Fig. S5. Immunoprecipitation of Myosin-11 and CD63.

    Fig. S6. NEU1 expression levels in tumor cells and the surrounding microenvironment determine their invasiveness.

    Fig. S7. Increased lysosomal exocytosis confers chemoresistance to RMS cells.

    Table S1. Type of tumors developed in Neu1+/+/Arf−/− and Neu1+/−/Arf−/− mice.

    Table S2. Immunohistochemical markers used for diagnosis of sarcomas.

    Table S3. Metastasis status in the sarcoma data set (GSE21050).

    Movie S1. In RH41 cells, lysosomes localize in the perinuclear region.

    Movie S2. In RH30 cells, lysosomes distribute to the cell periphery.

    Movie S3. Live TIRF imaging of LysoTracker-labeled lysosomes in RH41 as a measure of tethering to or docked at the PM.

    Movie S4. Live TIRF imaging of LysoTracker-labeled lysosomes in RH30 as a measure of tethering to or docked at the PM.

    Movie S5. Doxorubicin exposure induces lysosome aggregation and subsequent apoptosis of RH41 cells.

    Movie S6. RH30 cells maintain lysosomal trafficking and are resistant to doxorubicin exposure.

    Movie S7. In RH41 cells, doxorubicin concentrates primarily in the nucleus.

    Movie S8. In RH30 cells, a fraction of doxorubicin remains lysosomal.

    Movie S9. RH30 cells cotreated with doxorubicin and verapamil cluster lysosomes in the perinuclear region and undergo apoptosis.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. UPS that developed in a Neu1+/−/Arf−/− mouse expresses epithelial and mesenchymal markers and TGFβ.
    • Fig. S2. Excessive lysosomal exocytosis in RMS cells is caused by low NEU1 and high LAMP1 levels.
    • Fig. S3. Genes whose expression is negatively correlated with that of NEU1 expression in a sarcoma data set (GSE21050).
    • Fig. S4. Showing of entire immunoblots corresponding to panels in Fig. 5 (E and F).
    • Fig. S5. Immunoprecipitation of Myosin-11 and CD63.
    • Fig. S6. NEU1 expression levels in tumor cells and the surrounding microenvironment determine their invasiveness.
    • Fig. S7. Increased lysosomal exocytosis confers chemoresistance to RMS cells.
    • Table S1. Type of tumors developed in Neu1+/+/Arf−/− and Neu1+/−/Arf−/− mice.
    • Table S2. Immunohistochemical markers used for diagnosis of sarcomas.
    • Table S3. Metastasis status in the sarcoma data set (GSE21050).
    • Legends for movies S1 to S9

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). In RH41 cells, lysosomes localize in the perinuclear region.
    • Movie S2 (.mov format). In RH30 cells, lysosomes distribute to the cell periphery.
    • Movie S3 (.mov format). Live TIRF imaging of LysoTracker-labeled lysosomes in RH41 as a measure of tethering to or docked at the PM.
    • Movie S4 (.mov format). Live TIRF imaging of LysoTracker-labeled lysosomes in RH30 as a measure of tethering to or docked at the PM.
    • Movie S5 (.mov format). Doxorubicin exposure induces lysosome aggregation and subsequent apoptosis of RH41 cells.
    • Movie S6 (.mov format). RH30 cells maintain lysosomal trafficking and are resistant to doxorubicin exposure.
    • Movie S7 (.mov format). In RH41 cells, doxorubicin concentrates primarily in the nucleus.
    • Movie S8 (.mov format). In RH30 cells, a fraction of doxorubicin remains lysosomal.
    • Movie S9 (.mov format). RH30 cells cotreated with doxorubicin and verapamil cluster lysosomes in the perinuclear region and undergo apoptosis.

    Files in this Data Supplement:

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