Opposing roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investigations

Abstract

In malignancies, enhanced nuclear factor-κB (NF-κB) activity is largely viewed as an oncogenic property that also confers resistance to chemotherapy. Recently, NF-κB has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-κB. Using a mouse lymphoma model and analyzing transcriptome and clinical data from lymphoma patients, we show here that therapy-induced senescence presents with and depends on active NF-κB signaling, whereas NF-κB simultaneously promotes resistance to apoptosis. Further characterization and genetic engineering of primary mouse lymphomas according to distinct NF-κB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes guided us to identify Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL as a clinically relevant subgroup with significantly superior outcome when NF-κB is hyperactive. Our data illustrate the power of cross-species investigations to functionally test genetic mechanisms in transgenic mouse tumors that recapitulate distinct features of the corresponding human entity, and to ultimately use the mouse model-derived genetic information to redefine novel, clinically relevant patient subcohorts.

Figure 1.

Enhanced NF-κB activity in therapy-induced senescent primary lymphoma cells. (A) GSEAs of NF-κB targets (left) and the subset of cytokines and their modulators therein (right) in the GEP of ADR senescent versus untreated (ut) Eμ-myc control;bcl2 lymphomas (n = 12 matched pairs). (B) DNA-binding activity of the indicated NF-κB subunits in lymphomas as in A (n ≥ 3 matched pairs per subunit). (C) Immunofluorescence of the NF-κB subunit p65 in cytospin preparations of lymphoma cells as in A (representative example of three cases analyzed; bars, 50 μm). (D) Immunoblot analyses of the indicated proteins in cell lysates from four matched lymphoma pairs as in A with H3K9me3 as a senescence marker (Reimann et al. 2010) and α-Tubulin as a loading control. (E) RQ-PCR analyses of the indicated nonsecreted (left) and secreted (right) transcripts in lymphomas as in A (n = 5 samples each). Shown are relative expression levels, normalized to an internal control, that are comparable throughout all data sets. (Note the log-scaled presentation in the cytokine panel. All secreted NF-κB targets presented here reflect SASP, while Igfbp7 is a SASP factor, but not a bona fide NF-κB target.) All histogram bars indicate mean values ± standard deviation (SDEV).

Figure 2.

TIS is an NF-κB-dependent condition in primary lymphomas. (A, top) RQ-PCR analyses of the indicated transcripts in lymphomas presented as the ratio of the relative values for ADR-exposed versus untreated control;bcl2 lymphomas stably expressing the NF-κB SR or being empty vector-infected as a control (at least five samples each). (Bottom) Matched ADR-exposed control;bcl2 lymphoma pairs ± SR are shown for the relative expression levels of two representative SASP factors. (B) Frequencies of SA-β-gal-positive cells from matched lymphoma pairs ± SR, exposed to ADR in vitro (n = 7 pairs). (C) In situ frequencies of SA-β-gal-positive as well as Ki67-positive cells in matched-pair (±SR) lymphoma sections, exposed to CTX in vivo (n = 3 pairs; representative photomicrographs shown; bars, 100 μm). All histogram bars or numbers indicate mean values ± SDEV.

Figure 3.

Eμ-myc lymphomas driven by high endogenous NF-κB levels display NF-κB/Bcl2-mediated chemoresistance. (A) Stratification of 12 primary control lymphomas in an “NF-κB low” (NL; black bars), an intermediate (gray bars), and an “NF-κB high” (NH; white bars) group according to their NF-κB p65 DNA-binding activities at diagnosis (measured in triplicate; horizontal lines indicate mean activity within the respective groups). (B) Viability analysis by trypan blue dye exclusion of the NL versus NH lymphoma cell populations exposed in vitro for 24 h to 5 ng/mL ADR relative to untreated cells of the same group (n = 4 each group). (C) RQ-PCR analyses of the transcript levels of the NF-κB target bcl2 in the lymphoma groups as in B. (D) Viability analysis as in B and with the same lymphoma samples, now overexpressing Bcl2 from retroviral alleles in both groups. (E) GFP enrichment analysis of MSCV-SR-IRES-GFP-expressing cells treated as in B, but for 48 h; selective drop of GFP-positive cells in the NH group indicating their increased chemosensitivity upon NF-κB inhibition (n = 3 each group). All histogram bars indicate mean values ± SDEV.

Figure 4.

Differential oncogenic wiring determines opposing roles of NF-κB in treatment outcome. (A) Grouping of 13 primary control lymphomas according to the long-term responses to CTX therapy following transplant tumor formation in vivo as either “never relapse” (NR) or “relapse-prone” (RP) lymphomas, and RQ-PCR analyses of their IκBα transcript levels at diagnosis (i.e., prior to any therapy) as a representative bona fide NF-κB target (left) and of Bcl2 levels of the same samples (right; presented in the same order); horizontal lines indicate mean activity within the respective groups. (B) Frequencies of SA-β-gal-positive cells in Bcl2-overexpressing NL lymphomas ± CARD11-L251P, exposed for 3 d to 50 ng/mL ADR in vitro (n = 4 matched pairs; representative photomicrographs of cytospin preparations are shown below the corresponding bars). (C) Progression-free survival of the 49 R-CHOP-like-treated GCB DLBCL patients from a total of 233 patients (as reported by Lenz et al. 2008b) with an above-median Bcl2 expression by microarray-based transcriptome analysis of their lymphomas at diagnosis, and further stratified by a 63-gene signature as either “NF-κB high” (NH, n = 25; green) or “NF-κB low” (NL, n = 24; blue). All histogram bars indicate mean values ± SDEV.