CD74 interferes with the expression of fas receptor on the surface of lymphoma cells

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 evaluated by flow cytometry and compared to 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.

Figure 1

Establishment of CD74 knock-down cells. (A) Indicated lymphoma cell lines were harvested, lysed and CD74 expression was detected by WB using anti CD74 antibody LN-2. β-actin staining was used as a loading control; (B) BJAB Lenti and Raji Lenti cell lines with CD74 expression knocked-down with lentiviral vectors encoding CD74 targeting shRNAs (Sigma-Aldrich, 35, 36, 37, 39) or no-target shRNA (NT) and BJAB Epi cells with shRNAs expressed from the episomal vector (CD74 knock down cells clone 5.7, no-target cells clone N.7 and parental cells - Par) were harvested, lysed and analyzed for the expression of CD74 as in (A).

Figure 2

Suppression of CD74 expression sensitizes lymphoma cell lines to Fas-mediated apoptosis and Fas-dependent chemotherapies. Cell death was evaluated by flow cytometry in (A) BJAB Epi (clones 5.7 and N.7), BJAB Lenti (clones 35, NT), Raji Lenti (clones 35, NT) cells treated with 20 ng/mL of superFasL for 20 hours, (B) BJAB Epi clones 5.7 (solid line) and N.7 (dashed line) treated with indicated concentration of FasL for 20 hours, (C) BJAB Epi clones 5.7 (solid line) and N.7 (dashed line) treated with indicated concentration of edelfosine for 20 hours; (D) BJAB Epi clones 5.7 (solid line) and N.7 (dashed line) were treated with indicated concentrations of doxorubicin for 48 hours. Values shown represent the average and standard deviation from 3 wells. Presented results are a representative of at least two independent experiments.

Figure 3

Over-expression of CD74 protects mice from a lethal dose of agonistic Fas antibody. Mice were transfected with an empty plasmid (vector) or plasmid encoding Myc-tagged CD74 using a hydrodynamic transfection method. Twenty-four hours post transfection, mice were challenged with a lethal dose of agonistic anti-Fas antibody Jo2 and monitored for survival up to 6 hours. (A) Survival of challenged mice at 6 hours; (B) gross appearance of livers from Jo2 challenged mice; (C) immunohistochemistry of liver tissues from control (vector) and Jo2-challenged (vector with Jo2 and CD74-Myc with Jo2) mice. H&E – hematoxylin and eosin staining, MYC – Myc-tag staining, Cleaved Caspase 3 – apoptosis staining.

Figure 4

Knock-down of CD74 sensitizes lymphoma cell lines to Fas-mediated apoptosis and increases Fas receptor levels on the cell surface. (A) BJAB Epi cells (clones 5.7 and N.7) were treated with 20 ng/mL of super FasL (sFL) or buffer (C) for 20 hours. Cells were harvested, lysed and processing of caspase-8 and caspase-3 was detected by Western blot using antibodies specific for cleaved forms of either caspase. β-actin staining was used as a loading control; (B) BJAB Lenti and Raji Lenti cell lines (clones 35, NT) and BJAB Epi cells (clones 5.7 and N.7) were harvested, lysed and analyzed for the expression of cFLIP_L and pro-caspase-8 using specific antibodies. β-actin staining was used as a loading control; (C) BJAB Epi cells (clones 5.7 and N.7) were stained with PE-conjugated anti-Fas antibody UB2 and analyzed by flow cytometry. Data shown are representative of 3 independent experiments; (D) graphic representation of flow cytometry analysis of BJAB Epi cells (clones 5.7 and N.7) stained with PE-conjugated anti-Fas antibody UB2 and positively selected for green fluorescent protein signal. Values shown represent means and standard deviations from 3 wells.

Figure 5

Pre-treatment with crosslinked anti-CD74 antibody sensitizes BJAB cells to Fas-mediated apoptosis. BJAB cells were treated with anti-CD74 antibody hLL1 crosslinked with goat anti-mouse (GAM) or anti-human (GAH) IgG followed by 50 ng/ml of either agonistic anti-Fas antibody CH-11 or FasL. Apoptosis was analyzed by flow cytometry after 24 hours. Presented values represent means and standard deviations from 3 different wells.