Research ArticleCancer

Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1

See allHide authors and affiliations

Science Signaling  06 Oct 2015:
Vol. 8, Issue 397, pp. ra98
DOI: 10.1126/scisignal.aac5418
  • Fig. 1 YAP1 signaling is activated in colon-derived cell lines and colon cancer.

    (A to C) Western blot analysis of total and phosphorylated YAP1 (pYAP1) (A) in human colon cancer cell lines and HEK293A cells, (B) in the colons of ApcF/F and ApcF/F:CDX2ERCre mice 9 days after treatment with tamoxifen (n = 3 mice per group), and (C) in tumor and adjacent normal colon tissues of treated AOM/DSS mice (n = 3 mice per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Western blot analysis of YAP1 in HCT-116 and SW480 cells transfected with either control siRNA (siScr) or an siRNA targeting YAP1 (siYAP1). (E) MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay performed in HCT-116 and SW480 cells after knockdown of YAP1. Data are means ± SEM. (F) Luciferase analysis using TOPFlash promoter after knockdown of YAP1 in HCT-116 or SW480 cells. Luciferase values were normalized to β-galactosidase. Data are means ± SD. *P < 0.05, **P < 0.01 versus siScr. All cell line experiments were done in triplicates and repeated at least three times. RLU, relative light units. (G) Kaplan-Meier survival plots with hazard rates (HR) and confidence intervals computed using the data of 1332 patients stratified by differential expressions of YAP1 target genes CYR61, CTGF, and TEAD1.

  • Fig. 2 VP treatment decreases colon tumors in a mouse model of colon cancer.

    (A) Schematic representation of AOM/DSS CAC mouse model that were treated with VP (100 mg/kg) twice a week for 3 weeks. (B to D) Representative images of tumors (B); colon length and the number, size, and volume of colon tumors (C); and hematoxylin and eosin (H&E) staining (D) of representative tumors from mice treated with vehicle (Veh) (n = 9 mice per group) or VP (n = 8). Scale bar, 50 μm. (E and F) Quantitative real-time polymerase chain reaction (qPCR) analysis of CTGF and CYR61 expression in normal (NT) and tumor (Tum) colon tissues from vehicle- or VP-treated mice, normalized to mRNA encoding β-actin. Data are means ± SEM (n = 4 mice per group). (G and H) Immunostaining for the Ki67 or cleaved caspase 3 (c-Caspase 3) in (G) tumor and (H) normal colon tissues from mice treated with vehicle or VP. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 VP decreases cell growth in patient-derived enteroid and xenograft models.

    (A and B) Representative images of (A) adenoma or (B) adenocarcinoma patient–derived enteroids treated with VP for the indicated doses and times. (C and D) Immunofluorescence staining and quantification (% positive staining ± SEM, n = 3 enteroids) of Ki67 and cleaved caspase 3 in patient-derived (C) adenoma or (D) adenocarcinoma enteroids in the presence of vehicle or VP for 6 days. (E to H) Size (E), weight (F), H&E staining (G), and proliferative and apoptotic analysis (H) of CRC patient–derived xenografts in mice treated with vehicle (n = 6) or VP (n = 8). Data are means ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle. Scale bars, 50 μm.

  • Fig. 4 VP inhibits STAT3 signaling in colon tumors and colon-derived cell lines.

    (A) Western blot analysis of phosphorylated and total STAT3 in the tumors of AOM/DSS-treated mice from the vehicle and VP groups (n = 4 mice per group). (B) qPCR analysis of Socs3, Bcl2, and Il-1β in normal or tumor tissue from vehicle- and VP-treated AOM/DSS mice. Expression was normalized to mRNA encoding β-actin. *P < 0.05 versus vehicle. (C and D) Western blot analysis of phosphorylated and total STAT3 in (C) HCT-116 or (D) RKO that were pretreated with vehicle or VP (1 μg/ml) for 2 hours and then treated with IL-6 (10 ng/ml) for 20 min. (E and F) Time- and dose-dependent effects of VP on IL-6–induced STAT3 phosphorylation in (E) HCT-116 or (F) RKO cells. All cell line experiments were done in triplicates and repeated at least three times.

  • Fig. 5 VP inhibits growth of CRC-derived cells independent of YAP1.

    (A and B) Western blot analysis of HCT-116 cells transfected with (A) siScr or siYAP1. (B) HCT-116 cells stably expressing doxycycline (Doxy)–inducible shRNA against YAP1 (clone 1 and 2). Cells were untreated or exposed to IL-6 (10 ng/ml, 20 min). (C and D) Western blotting of lysates from HCT-116 cells stably expressing (C) control (shScr) or shRNA against TAZ (shTAZ clones 1 to 3) or (D) shTAZ (clones 1 and 2) and either siScr or siYAP1 and treated with IL-6 (10 ng/ml, 20 min). (E) Western blot analysis of HCT-116 cells transfected with siScr or siYAP1 and treated with VP (1 μg/ml) and then IL-6 (10 ng/ml) for 20 min. (F) MTT assay in HCT-116 cells transfected with siScr or siYAP1 and treated with vehicle or VP (1 μg/ml). (G to I) MTT assay in VP (1 μg/ml)–treated HCT-116 cells stably expressing (G) doxycycline-inducible shYAP1, (H) shTAZ, or (I) shTAZ and siScr or siYAP1. Data are means ± SEM of at least three independent experiments, each done in triplicate. ***P < 0.001 versus vehicle.

  • Fig. 6 VP-induced protein oligomerization causes apoptotic cell death.

    (A) Abundance of monomers and high–molecular weight (MW) oligomers of phosphorylated and total STAT3 in response to IL-6 (10 ng/ml) in HCT-116 cells pretreated with VP (left, 1 μg/ml VP for the indicated times; right, treatment for 2 hours). (B) Abundance of monomers and high–molecular weight oligomers of p62, lamin A/C, and STAT3 in CRC cell lines treated with VP (1 μg/ml) for 2 hours. (C and D) HCT-116 and RKO cells were exposed for 2 hours to vehicle, VP (1 μg/ml), 5 μM PPIX, 5 μM Meso, or 30 μM hemin. Both cell lines were then assessed for high–molecular weight oligomers or monomers by Western blot (C), and HCT-116 cells were additionally assessed for proliferation (D). (E) As in (C), also in response to 2-hour exposure to 100 μM chloroquine (CQ). (F) Identification and classification of VP-induced oligomers in HCT-116 (HCT) and SW480 (SW) by mass spectrometry and Panther Gene Ontology. (G to I) Coimmunostaining for cleaved caspase 3 and p62 in (G) HCT-116 cells treated with VP (1 μg/ml) for 4 hours; (H) patient-derived colon tumors from Fig. 3E; (I) patient-derived enteroids exposed to VP (1 μg/ml) for 8 hours. Scale bar, 50 μm. Data are means ± SEM of at least three independent experiments, each performed in triplicate. Blots and images are representative of three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001 versus vehicle.

  • Fig. 7 Tumor-selective apoptosis by VP is associated with impaired clearance of protein oligomers.

    (A) Western blot analysis of p62 and STAT3 in normal colon tissue and colon tumor tissue from mice treated intraperitoneally with VP (100 mg/kg) for 4 hours (n = 3 mice per group). (B) Intracellular amount of VP in colon tumor tissues and adjacent normal colon tissues after 4 hours of VP treatment (n = 3 samples per group). *P < 0.05 versus normal. (C) Western blot analysis of p62 in normal and tumor colon tissues treated ex vivo with VP (1 μg/ml), and then transferred to fresh Dulbecco’s modified Eagle’s medium (DMEM) (→ Med) for the indicated time. (D) Western blot analysis of HIF1α and HIF2α in normal and tumor tissues either freshly isolated from mice (in vivo) or after culturing for 4 hours (ex vivo). (E and F) Abundance of p62 and STAT3 high–molecular weight oligomers in HCT-116 cells exposed to VP (1 μg/ml) followed by a washout period under (E) normoxic (21% O2) or hypoxic (1% O2) culture conditions or (F) in the presence or absence of glucose. (G) MTT assays in HCT-116 and RKO cells exposed to VP under normoxic or hypoxic culture conditions. Data are means ± SEM; ###P < 0.001 versus hypoxic cells. All cell line experiments were done in triplicates and repeated at least three times.

  • Fig. 8 Schematic representations summarizing the tumor-specific cytotoxicity induced by VP.

    (A) Western blot analysis of p62, lamin A/C, and STAT3 in HCT-116 cells that were pretreated with 1 μM rapamycin (right) or 20 μM betulinic acid (left) for 16 hours then VP for 90 min. (B) Western blot analysis of cleaved PARP (cPARP) in HCT-116 cells pretreated with 1 μM rapamycin (Rapa) for 16 hours and then VP for 6 hours. (C) Cell proliferation assessed by bromodeoxyuridine (BrdU) staining in HCT-116 cells pretreated with vehicle, 1 μM rapamycin, 20 μM betulinic acid (BA), 1 μM trichostatin A (TSA), or 1 μM geldanamycin (Geld) for 16 hours followed by treatment with VP for 90 min. Experiments were done in triplicates and repeated twice. *P < 0.05 versus vehicle; ^P < 0.05 versus VP alone. (D) In normal cells, VP-induced protein oligomers are efficiently cleared through autophagy and proteasomal degradation. However, hypoxic tumor cells, which highly rely on autophagy and proteasomal machinery for nutrients and biomolecules, fail to clear the protein oligomers, resulting in apoptotic cell death.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/397/ra98/DC1

    Fig. S1. YAP1 signaling is activated in human colon cancer cell lines and tissues.

    Fig. S2. VP treatment does not induce toxicity in normal tissues.

    Fig. S3. VP treatment decreases growth in CT-26 xenograft model and colon cancer cell lines.

    Fig. S4. VP treatment inhibits STAT3 signaling.

    Fig. S5. The growth inhibitory effect of VP is YAP1-independent.

    Fig. S6. The growth inhibitory effect of VP is associated with oligomerization of proteins.

    Fig. S7. Hypoxia and nutrient deprivation impair clearance of VP-induced protein oligomers.

    Fig. S8. Activating autophagic and proteasomal pathways partially clears protein oligomers.

    Table S1. Proteins that are oligomerized in response to VP.

    Table S2. List of primers.

  • Supplementary Materials for:

    Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1

    Huabing Zhang, Sadeesh K. Ramakrishnan, Daniel Triner, Brook Centofanti, Dhiman Maitra, Balázs Győrffy, Judith S. Sebolt-Leopold, Michael K. Dame, James Varani, Dean E. Brenner, Eric R. Fearon, M. Bishr Omary, Yatrik M. Shah*

    *Corresponding author. E-mail: shahy{at}umich.edu

    This PDF file includes:

    • Fig. S1. YAP1 signaling is activated in human colon cancer cell lines and tissues.
    • Fig. S2. VP treatment does not induce toxicity in normal tissues.
    • Fig. S3. VP treatment decreases growth in CT-26 xenograft model and colon cancer cell lines.
    • Fig. S4. VP treatment inhibits STAT3 signaling.
    • Fig. S5. The growth inhibitory effect of VP is YAP1-independent.
    • Fig. S6. The growth inhibitory effect of VP is associated with oligomerization of proteins.
    • Fig. S7. Hypoxia and nutrient deprivation impair clearance of VP-induced protein oligomers.
    • Fig. S8. Activating autophagic and proteasomal pathways partially clears protein oligomers.
    • Legend for table S1
    • Table S2. List of primers.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.12 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Proteins that are oligomerized in response to VP.

    [Download Table S1]


    Citation: H. Zhang, S. K. Ramakrishnan, D. Triner, B. Centofanti, D. Maitra, B. Győrffy, J. S. Sebolt-Leopold, M. K. Dame, J. Varani, D. E. Brenner, E. R. Fearon, M. B. Omary, Y. M. Shah, Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1. Sci. Signal. 8, ra98 (2015).

    © 2015 American Association for the Advancement of Science

Navigate This Article