Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies

Abstract

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Keywords: Diffuse large B-cell lymphoma; alternative NFκB pathway; canine model; comparative pathology; targeting therapy.

Figure 1.

Constitutive activation of the NFκB pathway in canine DLBCL. (A) Expression and nuclear translocation of NFκB proteins were analyzed using nuclear lysates prepared from two canine primary DLBCL cells and human Burkitt lymphoma cell line Raji by immunoblotting. (B) Immunoblotting for c-Rel using CLBL1, OCI-Ly3, and IGROV cell lysates. (C) Activation of NFκB pathway (binding of NFκB proteins to the NFκB consensus sequence probe) was analyzed using nuclear lysates prepared from 5 primary canine DLBCL cells, CLBL1 cells, and OCI-Ly3 cells by the EMSA. Super-shifts by anti-NFκB antibodies indicate that the specific NFκB forms active NFκB complexes.

Figure 2.

Constitutive activation of the NFκB pathway in canine DLBCL cell line CLBL1. (A) Luciferase activity in CLBL1 NFκB reporter cells cultured for 4 h with or without CD40L. (B) Luciferase activity in CLBL1 NFκB reporter cells cultured for 4 h with 25 ng/ml CD40L in the presence of indicated NFκB pathway inhibitors. (C) Cytotoxicity assay for NFκB pathway inhibitors against CLBL1 cells. Where applicable, error bars represent the 95% confidence interval as determined by either Excel or GraphPad Prism.

Figure 3.

Inhibition of the alternative NFκB pathway induces apoptosis in CLBL1 cells. (A) CLBL1 cells were transfected with 1000 nM (=10 pmol siRNA per 1 × 10⁵ cells) RelB or UNC siRNA using a nucleofection method. After 24 h, the expression level of RelB gene was analyzed using quantitative RT-PCR. RelB gene expression levels were normalized by beta-actin gene expression among samples. (B) Expression of RelB protein in CLBL1 treated by no siRNA, UNC siRNA, or RelB siRNA was analyzed by immuno-blotting. (C) NFκB pathway in CLBL1-NFκB-luc cells was effectively inhibited by RelB siRNA treatment. (D) Inhibition effect of RelB siRNA was analyzed using nuclear lysates prepared from CLBL1 treated by no siRNA, UNC siRNA, and RelB siRNA using the super-shift EMSA. (E) Induction of apoptosis in CLBL1 cells treated by RelB siRNA was analyzed by Annexin V binding on flow cytometry. (F) CLBL1 cells were treated with UNC or RelB siRNA for 72 h and the cell viability was analyzed using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay. Where applicable, error bars represent the 95% confidence interval as determined by either Excel or GraphPad Prism.

Figure 4.

Canine DLBCL TMA. Canine lymphoma TMA slides were stained for p52, p65, and RelB and scanned for the analysis. Manual annotations were drawn to segment the desired TMA spots into individual regions per the TMA maps. Negative annotations were drawn to exclude regions containing poor tissue, staining artifacts, and non-representative tissue (e.g. benign areas). p50, p65, RelB, and p52 antigens were present in tumor areas encompassing nuclear and cytoplasmic staining. The Nuclear aμgorithm was calibrated to the DAB/Hematoxylin stains and nuclear DAB staining was measured in tumor areas. (A) Representative photos with low and high frequency of NFκB nuclear translocation are shown. Blue dots represent unstained nuclei and yellow-orange dots represent positive NFκB staining in nuclei. (B) Summary of the percentage of NFkB positive nuclei in total nuclei among samples. Red color represents the result of normal lymph node samples.