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TRAIL promotes membrane blebbing, detachment and migration of cells displaying a dysfunctional intrinsic pathway of apoptosis

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Abstract

Recently, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to be a potential candidate for cancer therapy. TRAIL induces apoptosis in various cancer cells but not in normal tissues. Here we show that HCT116 and SW480 cells with a deficient mitochondrial apoptotic pathway were resistant to TRAIL-induced apoptosis, whereas HCT116 and SW480 cells with a functional mitochondrial apoptotic pathway underwent apoptosis upon exposure to TRAIL. Surprisingly, TRAIL induced phenotypic changes in cells with a dysfunctional mitochondrial apoptotic pathway, including membrane blebbing and a transient loss of adhesion properties to the substratum. Accordingly, TRAIL stimulated the ability of these cells to migrate. This behavior was the consequence of a transient TRAIL-induced ROCK1 cleavage. In addition, we report that Bax-deficient HCT116 cells exposed to TRAIL for a prolonged period lost their sensitivity to TRAIL as a result of downregulation of TRAIL receptor expression, and became resistant to combination of TRAIL and other drugs such as MG-132 and bortezomib. These findings may have important consequences for TRAIL anti-cancer therapy.

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Abbreviations

TRAIL:

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand

TNFR1:

Tumor necrosis factor receptor-1

DISC:

Death inducing signaling complex

TRAIL-R1:

TRAIL receptor-1

TRAIL-R2:

TRAIL receptor-2

MLC:

Myosin light chain

ROCK1:

Rho-associated, coiled-coil containing protein kinase 1

IAPs:

Inhibitors of apoptosis

XIAP:

X-linked inhibitor of apoptosis

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Acknowledgments

SPS was supported by a grant of the EC (FP6-ONCODEATH research consortium). We are very grateful to our colleagues from the consortium for helpful discussions, in particular to Dr. A. Pintzas who initiated this collaboration. We are also grateful to members of the laboratory for fruitful discussions and Dr. Sunil Thomas for critical reading of the manuscript. This work was supported by Oncosuisse (subsidy KLS-02370-02-2009 to JCM), the Swiss National Science Foundation (subsidy 31003A_141068/1 to JCM) and the state of Geneva.

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Authors and Affiliations

  1. Department of Cell Biology, University of Geneva, Sciences III, 30 quai Ernest Ansermet, 1211, Geneva 4, Switzerland

    Syam Prakash Somasekharan & Jean-Claude Martinou

  2. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada

    Syam Prakash Somasekharan & Poul H. B. Sorensen

  3. Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, V5Z 1L3, Canada

    Syam Prakash Somasekharan & Poul H. B. Sorensen

  4. Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic

    Michal Koc & Ladislav Andera

  5. INSERM, UMR866, 21079, Dijon, France

    Alexandre Morizot & Olivier Micheau

  6. Department of Dermatology-Venereology, Geneva University Hospital, Michel-Servet 1, Geneva, Switzerland

    Olivier Gaide

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  1. Syam Prakash Somasekharan
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Supplementary material Movie 1 HCT116 Bax−/+ cells treated with TRAIL (100 ng/ml) for 30 h as described in the Materials and methods. Movie was created using ImageJ software using time-lapse images captured every 7 min. (AVI 2565 kb)

Supplementary material Movie 2 HCT116 Bax−/− cells treated with TRAIL (100 ng/ml) for 30 h as described in the ‘Materials and methods’. Movie was created using ImageJ software using time-lapse images captured every 7 min. (AVI 2753 kb)

10495_2012_782_MOESM3_ESM.jpg

Supplementary material Fig. 1 HCT116 Bax−/+ cells overexpressing Bcl-2 or Bcl-xL behave like Bax−/−HCT116 cells in response to TRAIL treatment. a HCT116 Bax−/+ cells stably expressing a control or Bcl-2 or Bcl-xL expression vector were treated with TRAIL (100 ng/ml) and photographed at the indicated time points using a phase contrast microscope. b The number of blebbing cells in each condition was counted in three different fields. Mean values ± SD are shown for three independent experiments with *p < 0.05, **p < 0.01 (JPG 518 kb)

10495_2012_782_MOESM4_ESM.tiff

Supplementary material Fig. 2 Detachment and blebbing of SW480 cells overexpressing Bcl-xL upon TRAIL treatment. a SW480 cells transfected with a control or Bcl-xL expression DNA vector were treated with TRAIL (100 ng/ml), proteosome inhibitor MG-132 (10 μM) or TRAIL (100 ng/ml) and MG-132 (10 μM) for 5 h. Cells were harvested, stained with propidium iodide and analyzed by FACS. Note that Bcl-xL overexpressing cells were found to be resistant to TRAIL while they were sensitized by a co-treatment with TRAIL and MG-132. b Control or Bcl-xL overexpressing SW480 cells were treated with TRAIL (100 ng/ml) or TRAIL (100 ng/ml) and ROCK1 inhibitor Y-27632 (10 μM) for the indicated time points and imaged. c SW480 cells overexpressing Bcl-xL were treated with TRAIL (100 ng/ml) for 5 h and the detached cells were collected, counted and expressed as percentage of the total number of cells in the well. Mean values ± SD are shown for three independent experiments with *p < 0.05. d SW480 cells overexpressing Bcl-xL or control were treated with TRAIL (100 ng/ml), the detached cells were collected and seeded in a 3.5 cm petriplate at a concentration of 1 × 105 cells/ml. The reattached cells were counted after 24 h of incubation. Results are an average of three independent experiments. e Control or Bcl-xL overexpressing SW480 cells were cultured in the presence or absence of TRAIL (100 ng/ml) for 5 h. Cell lysates were prepared and analyzed by Western blotting using antibodies as indicated. Note that caspase-8, caspase-3, PARP and ROCK1 were efficiently cleaved in control cells. In Bcl-xL overexpressing cells, caspase-8 and ROCK1 were efficiently processed while caspase-3 was partially processed. (TIFF 11804 kb)

10495_2012_782_MOESM5_ESM.jpg

Supplementary material Fig. 3 Cleavage of ROCK1 is sufficient to induce membrane blebbing. a HCT116 Bax–/– cells were transiently transfected (Neon Transfection System, Invitrogen) with a plasmid encoding GFP alone (upper panel) or GFP and ROCK1 (G1114opa) (at a ratio of 5:1) (middle panels) and imaged after 24 h. Cells showing blebbing phenotype are indicated by arroheads. A single cell transfected with both GFP and ROCK1 (G1114opa) is enlarged and shown in the bottom panel. b Green cells showing blebbing morphology were counted and expressed as percentage of the total number of green cells. Results are an average of three independent experiments. Mean values ± SD are shown for three independent experiments with **p < 0.01. (JPG 2079 kb)

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Somasekharan, S.P., Koc, M., Morizot, A. et al. TRAIL promotes membrane blebbing, detachment and migration of cells displaying a dysfunctional intrinsic pathway of apoptosis. Apoptosis 18, 324–336 (2013). https://doi.org/10.1007/s10495-012-0782-6

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