The TRAIL Receptor Agonist Drozitumab Targets Basal B Triple Negative Breast Cancer Cells

The TRAIL Receptor Agonist Drozitumab Targets Basal B Triple Negative Breast Cancer Cells that Express Vimentin and Axl

Jennifer L. Dine*1, 2, Ciara C. O’Sullivan*1, Donna Voeller1, Yoshimi E. Greer1, Kathryn J. Chavez1, Catherine M. Conway3, Sarah Sinclair4, Brandon Stone1, Laleh Amiri-Kordestani1, Anand S. Merchant5, Stephen M. Hewitt3, Seth M. Steinberg6, Sandra M. Swain4, and Stanley Lipkowitz1

1Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 2 Intramural Research Program, National Institute of Nursing Research, National Institutes of Health, Bethesda, MD, 3Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 4Washington Cancer Institute, MedStar Washington Hospital Center, Washington, DC., 5Center for Cancer Research Bioinformatics Core, Advanced Biomedical Computing Center, SAIC-Frederick, Frederick, MD, and 6Biostatistics & Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

*indicates equal contribution

Corresponding Author:

Stanley Lipkowitz, M.D., Ph.D.

Women’s Malignancies Branch, Center for Cancer Research

National Cancer Institute, National Institutes of Health,

Building 10, Room 4B54

Phone: (301) 402-4276; Fax: (301) 402-4275;

Supplementary Figure Legends

Supplementary Fig. 1. Axl negatively regulated GST-TRAIL-induced caspase activation.

Cells were transfected with a nontargeting siRNA pool (siNEG), an siRNA pool targeting Axl (siAxl), or an siRNA pool targeting vimentin (siVimentin) were incubated as indicated. (a) GST-TRAIL induced caspase-3/7 activity was measured after 1 h. (b) DR5 was immunoprecipitated from cells treated with or without GST-TRAILas described in the Materials and Methods section and caspase-8 recruitment to DR5 was analyzed by immunoblotting. Lanes 1 and 2, and lanes 8 and 9 show the whole cell lysates (WCL) for siNEG and siAxl, respectively. Lane 3 (Mock IP) shows lysates from untreated cells incubated with the agarose-GSH beads in the absence of GST-TRIAL. The antibodies used are shown to the right of the blots and MW in kDa is listed to the left. The ratio of caspase-8/DR5 in the DR5 immunoprecipitates is shown below the blot (lanes 5 and 7) normalized to the ratio of the siNEG lane. (c) The ratio of caspase-8/DR5 was plotted for four individual experiments (shown in black) with the mean +/- SE shown in red. The ratio was determined as the sum of the intensity of the four bands representing the precursor and cleaved caspase-8 divided by the sum of the intensity of the two bands representing DR5. The values obtained for siNEG were compared to those from siAxl by a paired two tailed Student’s t-test.

Supplementary Fig. 2. Expression of vimentin and Axl in normal breast tissue. Representative IHC images of the three cases with normal TDLUS for patterns of vimentin and Axl staining at 450X magnification.