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Proteasomal regulation of caspase-8 in cancer cell apoptosis

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Abstract

Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8, in response to proteasome inhibitor and GST-TRAIL. Moreover in the LNCaP human prostate cancer cells, caspase-8 polyubiquitination occurs after TRAIL stimulation and caspase-8 processing. Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells. The present results suggest that caspase-8 stabilization through proteasome inhibition leads to reactivation of the extrinsic pathway of apoptosis and identify E3 ligase mediating caspase-8 polyubiquitination, as a novel molecular target. Inhibition of this E3 ligase in combination with TRAIL towards restoring apoptosis signaling activation may have potential therapeutic significance in resistant tumors.

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Abbreviations

IP:

Immunoprecipitation

PI:

Propidium iodide

TRAIL:

TNF-alpha related apoptosis inducing ligand

HDAC7:

Histone deacetylase 7

DISC:

Death inducing signaling complex

FADD:

Fas associated death domain

NB7:

Neuroblastoma 7

GST:

Glutathione S-transferase

TNF-α:

Tumor necrosis factor-α

ATCC:

American type culture collection

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

CRPC:

Castration-recurrent prostate cancer

APC:

Allophycocyanin

SH3RF1:

Plenty of SH3, POSH

PBS:

Phosphate buffered saline

c-FLIP:

Cellular FLICE-inhibitory protein

NMS:

Normal mouse serum

Siah2:

Seven in absentia homolog

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Acknowledgments

The authors acknowledge Drs. Douglas Green and Andrew Oberst for generously providing the NB7 cell line and caspase-8 constructs. We also thank Drs. Greg Baumen and Jennifer Strange at the University of Kentucky Flow Cytometry Facility, Sarah Martin for help with the statistical analysis and Lorie Howard for her assistance with the submission process. This work was supported by the Department of Defense USAMRMC PC073314 Grant, National Institutes of Health: NIDDK, R01 DK083761, the Markey Cancer Center, and James F. Hardymon Endowment for Urology Research at the University of Kentucky.

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There are no potential conflicts of interests to disclose.

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Author notes

  1. Michael V. Fiandalo

    Present address: Department of Urologic Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA

Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry and the Markey Cancer Center, University of Kentucky College of Medicine, Combs Cancer Research Building, 306, 800 Rose Street, Lexington, KY, 40536, USA

    Michael V. Fiandalo, Steven R. Schwarze & Natasha Kyprianou

  2. Division of Urology, Department of Surgery, University of Kentucky College of Medicine, Combs Cancer Research Building, 306, 800 Rose Street, Lexington, KY, 40536, USA

    Natasha Kyprianou

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  1. Michael V. Fiandalo
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Correspondence to Natasha Kyprianou.

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Supplemental Fig. S1

A polyubiquitin pull-down was performed with the Ubiquitin Enrichment kit using Ubiquitin Extraction buffer (a stringent buffer containing 1 % NP40, 0.1 % SDS, 5 mM EDTA and a 150 mM NaCL, and 0.5 % sodium deoxycholate) followed by Western blotting probing for caspase-8 with the IC12 antibody. a Reveals the input indicating caspase-8 cleavage status of untreated and treated cells with combination epoxomicin and GST-TRAIL. b Ubiquitin IP/Caspase-8 Western revealing polyubiquitin smear only in the epoxomicin and GST-TRAIL treated samples; no signal is detected in the un-treated controls. (TIFF 589 kb)

Supplemental Fig. S2

Western blot showing caspase-8 expression status following treatment of LNCaP cells with Epoxomicin and GST-TRAIL separately and in combination. This datum serves as proof of principle that the same caspase-8 expression pattern (p18 stabilization) is observed with either Velcade or MG132 is (p18 stabilization) is maintained upon cell treatment with Epoxomicin. There is no change in expression levels of pro-caspase-8 between the non-treated and epoxomicin treated lanes. The only change in the p18 subunit as indicated when comparing the GST-TRAIL only to the combination of Epoxomicin and GST-TRAIL only. (TIFF 273 kb)

Supplemental Fig. S3

It shows a large field at a higher magnification of cells after treatment with TRAIL and Epoxomicin (4hrs)and subsequently exposed to fluorescence staining for caspase -8 and Na K ATPase co-localization. (JPG 802 kb)

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Fiandalo, M.V., Schwarze, S.R. & Kyprianou, N. Proteasomal regulation of caspase-8 in cancer cell apoptosis. Apoptosis 18, 766–776 (2013). https://doi.org/10.1007/s10495-013-0821-y

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