Background
CD74, better known as an invariant chain (Ii) of the major histocompatibility complex II (MHC II) [
1-
3], is indispensable for the proper development of B cells. CD74 is internalized into the endocytic compartment, where intramembrane cleavage releases the intracellular cytosolic domain (CD74-ICD). CD74-ICD then enters the nucleus, activates NF-kB p65/RelA, and controls the differentiation of B cells through the TAFII105 coactivator [
4],[
5]. CD74-ICD also induces expression of TAp63, which subsequently elevates expression of Bcl-2 and promotes survival of B cells [
6]. Macrophage migration inhibitory factor (MIF) is the assigned ligand for CD74 and its binding activates the extracellular signal-regulated kinase-1/2 (ERK 1/2) MAP kinase (MAPK) cascade and cell proliferation [
7], as well as NF-κB, through which it enhances the expression of Bcl-2 [
6].
CD74 expression is rather limited in normal human tissues, but it was found to be overexpressed in more than 85% of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) and a majority of multiple myeloma (MM) cells [
8-
12]. B cells from CLL patients have higher cell surface levels of CD74 than do normal B cells, and it was shown that the activation of CD74 by MIF in CLL cells activates NF-κB and induces secretion of IL-8, which promotes cell survival and tumor progression [
11],[
13]. CD74-mediated proliferative and pro-survival signaling can initiate or contribute to pro-carcinogenic events and enhance the survival of cancer cells.
The Fas receptor is widely expressed by many tissues, yet the extent of Fas-mediated apoptosis does not correlate with the extent of Fas expression. Hematological cancer cells are commonly resistant to Fas ligand (FasL)-induced apoptosis despite normal expression of the Fas receptor [
14]. This resistance is usually not caused by Fas/FasL mutations or overexpression of apoptosis inhibitors, such as cFLIP (cellular FLICE/caspase-8-inhibitory protein) [
15]. Identification of potential inhibitors of Fas-mediated apoptotic signaling in cancers and understanding of the mechanism involved are important steps necessary for the design and implementation of new targeted therapies. Reversing Fas resistance has become a primary interest in order to improve the efficacy of treatments for chemotherapy-resistant hematological cancers [
16-
19]. Several current treatments, like interferon gamma (IFN-γ), CD40L, and rituximab, are believed to improve responses to chemotherapy primarily through restoration of Fas apoptotic signaling [
16],[
17],[
20],[
21]. Our goal was to identify and to target inhibitors of the Fas receptor in order to reinstate Fas-mediated apoptotic signaling in cancer cells with limited off-target effects on normal cells.
In the light of well documented CD74-mediated pro-survival effects, we aimed to examine the effect of CD74 on Fas-mediated apoptosis, which is required for effective killing of cancer cells by most chemotherapies and radiation [
22-
26].
Methods
Cell lines and drug treatments
BJAB, Raji, Ramos, Daudi, and Jurkat cells were purchased from ATCC and grown in RPMI medium with 10% FBS (HyClone) in a 5% CO2 atmosphere at 37°C, and split 2-3 times per week. Cell lines were authenticated by STR analysis (MD Anderson Cancer Center Characterized Cell Line Core) and regularly tested for mycoplasma (Lonza).
For drug treatment, 0.5e6 cells/mL in RPMI + 5% FBS were seeded into 24-well plates and treated with indicated doses of FasL (Enzo) or edelfosine (Sigma-Aldrich) for 20 hours, doxorubicin (Sigma-Aldrich) for 48 h, or 10 ng/mL of super FasL (sFasL; Enzo) for 16 h.
BJAB cells (1e6 cells/mL) were incubated with 50 μg/mL of milatuzumab (humanized anti-CD74 antibody, hLL1; Immunomedics, Inc.) for 10 min prior to addition of 20 μg/mL of goat anti-mouse or goat anti-human IgG (both from Jackson ImmunoResearch). Cells were washed after 30 min and subsequently incubated with either 50 ng/mL of anti-Fas antibody CH-11 (Millipore) or recombinant FasL (Enzo). Cells were harvested 24 h later and analyzed for apoptosis by propidium iodide staining and flow cytometry, as described previously [
27].
Cloning and RNA interference
To knock-down CD74 expression, BJAB and Raji cells were transduced using MISSION shRNA lentiviral particles targeting CD74 or non-target (NT) controls, according to manufacturer’s protocol (Sigma-Aldrich), and kept under selection with 1.9 μg/mL of puromycin.
The shRNA sequences from MISSION shRNA lentiviral particles were inserted between BamHI and EcoRI restriction sites in the multiple cloning cassette of pSIREN-Shuttle vector (Clontech). The U6 promoter-shRNA cassettes were then recloned to KpnI and ApaI sites of the pEPI-1 vector kindly provided by Dr. A. C. Jenke (HELIOS Children's Hospital Wuppertal, Germany) [
28]. The correct sequences of inserted shRNAs were confirmed by sequencing (SeqWright).
Obtained plasmids were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s recommendations. Transfected cells were sorted 48 h post transfection based on green fluorescent protein (GFP) expression, using a flow-activated cell sorter BD FACSAria II (BD Biosciences) followed by limited dilutions and selection with geneticin (1350 μg/mL; Invitrogen) for 2 weeks.
Western Blotting (WB)
WB was performed according to standard protocols, as described previously [
29],[
30]. Protein expression in total cell lysates was analyzed with primary antibodies recognizing Fas (B-10), CD74 (LN-2), cFLIP, caspase-8 and caspase-3 [all at 1:1,000 dilution in 5% blotting-grade blocker (BioRad) in phosphate-buffered saline (PBS) with Tween 20], followed by anti-mouse-HRP or anti-rabbit-HRP antibodies when an unconjugated primary antibody was used. Equal loading was verified by β-actin-HRP antibody (1:10,000; Sigma-Aldrich). Visualization was achieved by Supersignal West Pico chemiluminescent substrate (Thermo Scientific). Intensity of bands was compared by densitometry using ImageJ software (NIH).
Flow cytometry
For detection of surface Fas or CD74, cells were washed with 2% FBS/PBS and blocked with 0.025 mg/mL of mouse IgG blocking reagent (Invitrogen) at 4°C for 15 min in the dark and washed once with 2% FBS/PBS. Cells were then incubated with 3 μL of PE-conjugated anti-Fas antibody UB2 or with FITC-conjugated anti-CD74 antibody M-B741 (both from BD Biosciences) in 50 μL of 2% FBS/PBS at 4°C for 20 min in the dark. After washing cells 2× with 2% FBS/PBS, flow cytometry was performed on an BD LSRFortessa flow cytometer with Diva software (BD Bioscience).
For evaluation of apoptosis and cell death, cells were collected and resuspended in 1 mL of cold 1% FBS/PBS and stained with Vybrant® Apoptosis Assay Kit #5 according to protocol provided by the manufacturer (Molecular Probes). Flow cytometry was performed on BD FACSCanto II flow cytometer with Diva software (BD Bioscience). Data were analyzed using FlowJo software (Tree Star Inc.).
Animal experiments and immunohistochemistry
All animal experiments were performed in accordance with the guidelines of MD Anderson Cancer Center’s Institutional Animal Care and Use Committee. C57BL/6 mice (6- to 8-weeks old; from the National Cancer Institute) were hydrodynamically transfected [
31] with 100 μg of plasmid (pEF4-myc or pEF4-CD74-myc [
4]; kindly provided by Dr. Idit Shachar, The Weisman Institute of Science, Rehovot, Israel). Mice were challenged with agonistic anti-Fas antibody Jo2 (0.4 μg/g; BD Biosciences) 24 h post transfection and monitored for survival up to 6 h post challenge. Livers were harvested at the time of death/sacrifice, paraffin embedded, and cut to 5 μm sections.
Formalin-fixed, paraffin-embedded liver tissue sections on microscope slides were treated with an unmasking kit (1:500; Vector Laboratories). Sections were then stained with hematoxylin and eosin (H&E; both from Fisher Scientific). Immunohistochemical staining was performed using a mouse monoclonal anti-myc antibody (1:500; Sigma-Aldrich) and a rabbit polyclonal anti-cleaved caspase-3 antibody (1:500; Cell Signaling), followed by staining and developing with an ABC staining kit (Vector Laboratories), according to manufacturer’s protocol. Stained samples were viewed using a Zeiss Axioskop 2 plus microscope (Carl Zeiss) with 4× and 20× magnification objectives.
Statistical analysis
Differences between groups were calculated using the two-tailed Student t test. P-values less than 0.05 were considered statistically significant.
Discussion
In the presented study we evaluated the effect of CD74 on the Fas-mediated apoptotic signaling in lymphoma cells by knocking down the expression of CD74 in BJAB and Raji human NHL cells. We successfully generated the first cell lines with stable knock-down of CD74 expression by lentiviral and episomal vectors expressing CD74-targeting shRNA (Figure
1B). Using these cell lines, we showed that removal of CD74 sensitizes cells to Fas-mediated apoptosis (Figure
2A-B) and subsequently also to Fas-dependent chemotherapies, doxorubicin and edelfosine (Figure
2C-D). On the other hand, the overexpression of CD74 in mouse livers protected mice from the lethality of agonistic Fas antibody Jo2 (Figure
3A) by interfering with apoptotic signaling (Figure
3C). Increased sensitivity to Fas-mediated apoptosis in cells lacking CD74 was due to increased activation/cleavage of the initiator caspase-8 and correspondingly increased activation of effector caspase-3 (Figure
4A). These results suggested that the enhancement of Fas-mediated apoptosis occurs at an immediate early step of Fas signaling at the plasma membrane - the activation of death-inducing signaling complex (DISC). MIF signaling through CD74 mediates activation of NF-κB, which is known to regulate expression of cFLIP, a well-known inhibitor of a DISC component caspase-8. However, the WB analysis of the steady-state levels of cFLIP and caspase-8 in control and CD74 knock-down cells did not reveal significant alterations of either cFLIP or pro-caspase-8 levels (Figure
4B), suggesting that the Fas signaling component responsible for the sensitization event due to CD74 removal being upstream of DISC assembly. We next tested and confirmed that removal of CD74 significantly increased the levels of Fas receptor at the cell surface (Figure
4C-D) and thus the amount of the Fas receptor available for activation. Moreover, pre-treatment of cells with crosslinked anti-CD74 antibody sensitized cells to Fas-mediated apoptosis (Figure
5).
The inherent or acquired chemoresistance is a common hindrance to the successful treatment of cancer. Multiple chemotherapies, including doxorubicin, have been shown to upregulate Fas and/or FasL in order to achieve their full effectiveness [
24],[
25],[
35-
39]. However, cancer cells frequently downregulate the key players in the Fas signaling cascade or upregulate apoptosis inhibitors (cFLIP, IAPs, and Bcl-2 family members) to avoid apoptosis [
30],[
40],[
41]. Recently, a new category of Fas inhibitors has been recognized; cell surface receptors HGFR/c-MET, human herpesvirus-8 K1, CD44v6/v9, and nucleolin can bind and block the initiation of Fas signaling at the plasma membrane [
27],[
29],[
42-
44].
The increasing body of evidence points to CD74, known as the invariant chain of the major histocompatibility complex II (MHC II), functioning apart from that at MHC II. It was shown that CD74 serves as a receptor for the macrophage migration inhibitory factor, MIF. MIF-binding to CD74 activates the extracellular signal-regulated kinase-1/2 MAP kinase cascade and cell proliferation [
7]. CD74 is overexpressed in a wide variety of cancers (gastric, thymic, colorectal, breast, renal and lung carcinomas, invasive thymomas, bladder, prostate and pancreatic cancers) [
9],[
12],[
45-
50]. Additionally, the vast majority of NHL, CLL, and MM express CD74 while limited expression is observed in normal hematopoietic tissues [
8-
12]. In CLL cells, MIF was shown to activate NF-κB signaling, promoting the expression of IL-8 and consequently cell survival [
11],[
13]. The surface expression levels of CD74 in hematological cancer cells and some solid tumors correlate with poor prognosis [
45],[
51],[
52].
A rapid internalization of CD74 (10
6 to 10
7 molecules/cell/day) in a wide range of cancer cells [
53], which is not affected by the binding of the mouse monoclonal anti-CD74 antibody LL1 [
54], give rise to several therapeutic approaches. LL1 linked with different radioisotopes greatly delayed growth of disseminated Raji lymphoma cells in a xenograft model and cleared tumors in 50% of the mice [
55]. In the second approach, doxorubicin (DOX) conjugated to LL1 (LL1-DOX) cleared disseminated Raji lymphoma xenografts in 90% of mice. More importantly, the DOX dose used was 2.5% of the maximum tolerated dose of free DOX [
56]. Multiple myeloma is in general difficult to treat. A single dose of LL1-DOX (IMMU-110, Immunomedics, Inc.) cleared multiple myeloma xenografts in 70% of mice and significantly prolonged survival [
57].
The humanized form of LL1 (hLL1 or milatuzumab, Immunomedics, Inc.) alone showed promising antitumor effects in disseminated Raji and Daudi NHL xenograft models [
58], and in multiple myeloma xenograft models [
59]. Unlike the cell killing mechanism employed by rituximab, milatuzumab-mediated elimination of tumor cells does not involve antibody-dependent cell-mediated cytotoxicity (ADCC) or a complement-dependent cytotoxicity (CDC) [
58]. It was demonstrated that milatuzumab combined with a crosslinking antibody induces aggregation of CD74 on the cell surface of CLL cells [
60]. In another report, crosslinked milatuzumab induced significant apoptosis at 48 h, accompanied with activation of caspase-8 and caspase-3 [
58], suggesting the involvement of an extrinsic (receptor-mediated) apoptotic pathway. Our results suggest an explanation of those results. A rapidly internalized CD74 (turnover completed in 10 minutes) can decrease the levels of surface Fas (Figure
4C-D) through its internalization from the cell surface. Crosslinked milatuzumab-induced aggregation of CD74 prevents internalization of CD74 [
60] and indirectly also internalization of Fas, consequently increasing surface Fas levels and sensitizing cells to Fas-mediated apoptosis (Figure
5).
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Competing interests
Dr. Goldenberg is employed by and has stock in Immunomedics, Inc., which has patented and owns the milatuzumab antibody used in this study. There are no other potential conflict of interests relevant to this article.
Authors’ contributions
ZB designed the experiments, generated and initially characterized CD74-suppressed cell lines and wrote the manuscript; SW performed animal and milatuzumab experiments; XA performed experiments with chemotherapeutic agents; JFW analyzed surface expression of Fas; FKB and LS performed Western blot analyses; AHR performed immunohistochemistry; DMG, and FS designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.