Berkova et al. Journal of Experimental & Clinical Cancer Research 2014, 33:80 http://www.jeccr.com/content/33/1/80 RESEARCH Open Access CD74 interferes with the expression of fas receptor on the surface of lymphoma cells Zuzana Berkova1, Shu Wang1, Xue Ao1, Jillian F Wise1, Frank K Braun1, Abdol H Rezaeian1, Lalit Sehgal1, David M Goldenberg2,3 and Felipe Samaniego1* Abstract Background: Resistance to Fas-mediated apoptosis limits the efficacy of currently available chemotherapy regimens. We identified CD74, which is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis. Methods: CD74 expression was suppressed in human B-lymphoma cell lines, BJAB and Raji, by either transduction with lentivirus particles or transfection with episomal vector, both encoding CD74-specific shRNAs or non-target shRNA. Effect of CD74 expression on Fas signaling was evaluated by comparing survival of mice hydrodynamically transfected with vector encoding full-length CD74 or empty vector. Sensitivity of cells with suppressed CD74 expression to FasL, edelfosine, doxorubicin, and a humanized CD74-specific antibody, milatuzumab, was evaluatedbyflowcytometryandcomparedto control cells. Fas signaling in response to FasL stimulation and the expression of Fas signaling components were evaluated by Western blot. Surface expression of Fas was detected by flow cytometry. Results: We determined that cells with suppressed CD74 are more sensitive to FasL-induced apoptosis and Fas signaling-dependent chemotherapies, edelfosine and doxorubicin, than control CD74-expressing cells. On the other hand, expression of full-length CD74 in livers protected the mice from a lethal challenge with agonistic anti-Fas antibody Jo2. A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74, suggesting that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. Cells with suppressed CD74 expression showed increased staining of Fas receptor on their surface. Pre-treatment with milatuzumab sensitized BJAB cells to Fas-mediated apoptosis. Conclusion: We anticipate that specific targeting of the CD74 on the cell surface will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis, and thus will increase effectiveness of chemotherapy regimens for hematological malignancies. Background elevates expression of Bcl-2 and promotes survival of B CD74, better known as an invariant chain (Ii) of the major cells [6]. Macrophage migration inhibitory factor (MIF) is histocompatibility complex II (MHC II) [1-3], is indispens- the assigned ligand for CD74 and its binding activates the able for the proper development of B cells. CD74 is extracellular signal-regulated kinase-1/2 (ERK 1/2) MAP internalized into the endocytic compartment, where intra- kinase (MAPK) cascade and cell proliferation [7], as well membrane cleavage releases the intracellular cytosolic do- as NF-κB, through which it enhances the expression of main (CD74-ICD). CD74-ICD then enters the nucleus, Bcl-2 [6]. activates NF-kB p65/RelA, and controls the differentiation CD74 expression is rather limited in normal human of B cells through the TAFII105 coactivator [4,5]. CD74- tissues, but it was found to be overexpressed in more ICD also induces expression of TAp63, which subsequently than 85% of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) and a majority of multiple * Correspondence: [email protected] myeloma (MM) cells [8-12]. B cells from CLL patients 1 Department of Lymphoma and Myeloma, The University of Texas MD have higher cell surface levels of CD74 than do normal Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA Full list of author information is available at the end of the article B cells, and it was shown that the activation of CD74 © 2014 Berkova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Berkova et al. Journal of Experimental & Clinical Cancer Research 2014, 33:80 Page 2 of 10 http://www.jeccr.com/content/33/1/80 by MIF in CLL cells activates NF-κB and induces secre- Cells were harvested 24 h later and analyzed for apoptosis tion of IL-8, which promotes cell survival and tumor by propidium iodide staining and flow cytometry, as de- progression [11,13]. CD74-mediated proliferative and scribed previously [27]. pro-survival signaling can initiate or contribute to pro- carcinogenic events and enhance the survival of cancer Cloning and RNA interference cells. To knock-down CD74 expression, BJAB and Raji cells The Fas receptor is widely expressed by many tissues, yet were transduced using MISSION shRNA lentiviral parti- the extent of Fas-mediated apoptosis does not correlate cles targeting CD74 or non-target (NT) controls, accord- with the extent of Fas expression. Hematological cancer ing to manufacturer’s protocol (Sigma-Aldrich), and kept cells are commonly resistant to Fas ligand (FasL)-induced under selection with 1.9 μg/mL of puromycin. apoptosis despite normal expression of the Fas receptor The shRNA sequences from MISSION shRNA lentiviral [14]. This resistance is usually not caused by Fas/FasL mu- particles were inserted between BamHI and EcoRI restric- tations or overexpression of apoptosis inhibitors, such as tion sites in the multiple cloning cassette of pSIREN- cFLIP (cellular FLICE/caspase-8-inhibitory protein) [15]. Shuttle vector (Clontech). The U6 promoter-shRNA Identification of potential inhibitors of Fas-mediated apop- cassettes were then recloned to KpnI and ApaI sites of totic signaling in cancers and understanding of the mech- the pEPI-1 vector kindly provided by Dr. A. C. Jenke anism involved are important steps necessary for the design (HELIOS Children's Hospital Wuppertal, Germany) and implementation of new targeted therapies. Reversing [28]. The correct sequences of inserted shRNAs were Fasresistancehasbecomeaprimaryinterestinorderto confirmed by sequencing (SeqWright). improve the efficacy of treatments for chemotherapy- Obtained plasmids were transfected into cells using resistant hematological cancers [16-19]. Several current Lipofectamine 2000 transfection reagent (Invitrogen) ac- treatments, like interferon gamma (IFN-γ), CD40L, and ri- cording to the manufacturer’s recommendations. Trans- tuximab, are believed to improve responses to chemother- fected cells were sorted 48 h post transfection based on apy primarily through restoration of Fas apoptotic signaling green fluorescent protein (GFP) expression, using a flow- [16,17,20,21]. Our goal was to identify and to target inhibi- activated cell sorter BD FACSAria II (BD Biosciences) tors of the Fas receptor in order to reinstate Fas-mediated followed by limited dilutions and selection with geneticin apoptotic signaling in cancer cells with limited off-target ef- (1350 μg/mL; Invitrogen) for 2 weeks. fects on normal cells. In the light of well documented CD74-mediated pro- Western Blotting (WB) survival effects, we aimed to examine the effect of CD74 WB was performed according to standard protocols, as on Fas-mediated apoptosis, which is required for effect- described previously [29,30]. Protein expression in total ive killing of cancer cells by most chemotherapies and cell lysates was analyzed with primary antibodies recog- radiation [22-26]. nizing Fas (B-10), CD74 (LN-2), cFLIP, caspase-8 and caspase-3 [all at 1:1,000 dilution in 5% blotting-grade Methods blocker (BioRad) in phosphate-buffered saline (PBS) Cell lines and drug treatments with Tween 20], followed by anti-mouse-HRP or anti- BJAB, Raji, Ramos, Daudi, and Jurkat cells were purchased rabbit-HRP antibodies when an unconjugated primary from ATCC and grown in RPMI medium with 10% FBS antibody was used. Equal loading was verified by β-actin- (HyClone) in a 5% CO2 atmosphere at 37°C, and split 2–3 HRP antibody (1:10,000; Sigma-Aldrich). Visualization was times per week. Cell lines were authenticated by STR ana- achieved by Supersignal West Pico chemiluminescent sub- lysis (MD Anderson Cancer Center Characterized Cell strate (Thermo Scientific). Intensity of bands was compared Line Core) and regularly tested for mycoplasma (Lonza). by densitometry using ImageJ software (NIH). For drug treatment, 0.5e6 cells/mL in RPMI + 5% FBS were seeded into 24-well plates and treated with indi- Flow cytometry cated doses of FasL (Enzo) or edelfosine (Sigma- For detection of surface Fas or CD74, cells were washed Aldrich) for 20 hours, doxorubicin (Sigma-Aldrich) for with 2% FBS/PBS and blocked with 0.025 mg/mL of mouse 48 h, or 10 ng/mL of super FasL (sFasL; Enzo) for 16 h. IgG blocking reagent (Invitrogen) at 4°C for 15 min in the BJAB cells (1e6 cells/mL) were incubated with 50 μg/mL dark and washed once with 2% FBS/PBS. Cells were then of milatuzumab (humanized anti-CD74 antibody, hLL1; incubated with 3 μL of PE-conjugated anti-Fas antibody Immunomedics, Inc.) for 10 min prior to addition of 20 μg/ UB2 or with FITC-conjugated anti-CD74 antibody M-B741 mL of goat