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.2009 Nov 5;462(7269):104-7.
doi: 10.1038/nature08462. Epub 2009 Oct 21.

Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma

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Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma

Etienne Meylan et al. Nature..

Abstract

NF-kappaB transcription factors function as crucial regulators of inflammatory and immune responses as well as of cell survival. They have also been implicated in cellular transformation and tumorigenesis. However, despite extensive biochemical characterization of NF-kappaB signalling during the past twenty years, the requirement for NF-kappaB in tumour development in vivo, particularly in solid tumours, is not completely understood. Here we show that the NF-kappaB pathway is required for the development of tumours in a mouse model of lung adenocarcinoma. Concomitant loss of p53 (also known as Trp53) and expression of oncogenic Kras(G12D) resulted in NF-kappaB activation in primary mouse embryonic fibroblasts. Conversely, in lung tumour cell lines expressing Kras(G12D) and lacking p53, p53 restoration led to NF-kappaB inhibition. Furthermore, the inhibition of NF-kappaB signalling induced apoptosis in p53-null lung cancer cell lines. Inhibition of the pathway in lung tumours in vivo, from the time of tumour initiation or after tumour progression, resulted in significantly reduced tumour development. Together, these results indicate a critical function for NF-kappaB signalling in lung tumour development and, further, that this requirement depends on p53 status. These findings also provide support for the development of NF-kappaB inhibitory drugs as targeted therapies for the treatment of patients with defined mutations in Kras and p53.

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Figure 1
Figure 1
NF-κB activity depends on p53. (a) MEFs were infected with adenoviruses expressing Cre or FlpO recombinase. 6 days later, cytoplasmic (c) and nuclear (n) lysates were analyzed by western blot (WB). NEMO (cytoplasmic) and PARP (nuclear) were used to determine purity. (b,c)K-rasLA2/WT; p53LSL/LSL; Cre-ERT2–lung tumor derived cells were either untreated (ctl), cultured in 0.1% FBS or stimulated with 200 nM 4-hydroxytamoxifen (OHT). (b) 4 days later, cells were lysed and analyzed as in (a). (c) 4 days later, RNA was purified, reverse transcribed, and cDNA was amplified using probes forGlut3,Tnf orGapdh (internal control). Data show means +/− s.d. (n= 3 independent cell lines), presented as fold changes (treatment/control; see Methods) .
Figure 2
Figure 2
NF-κB p65 DNA-binding activity is increased in NSCLC cells with altered p53. (a,b) 5 µg of nuclear lysates of the indicated mouse (a) or human (b) cell lines were analyzed for p65 DNA-binding activity by an ELISA assay (see Methods). Cells harboring WT p53 are indicated. As a positive control of p65 activity, LKR13 cells (a) were stimulated with TNF for 30 minutes prior to lysis.
Figure 3
Figure 3
NF-κB inhibition results in apoptosis of p53-deficient lung adenocarcinoma cells. (a) Two WT p53 (LKR10 and LKR13) and twoK-rasLSL-G12D/WT; p53Flox/Flox (KP) mouse cell lines were uninfected (No Lenti), infected with control (ctl) or IκB-SR expressing lentiviruses. 72 hours later, cells were assayed for viability (see Methods). Data are means +/− s.d. (n=3) of cell viability relative to uninfected cells (set to 1). (b) Lung cell lines or fibroblasts (3TZ) were untreated (−) or infected with pSicoRev expressing aNemo (sh1, 2) or ctl hairpin. 72 hours later, cell extracts were analyzed by WB. *n.s., non-specific staining. (c,d) KP cells were infected with pSicoRev expressing the indicated hairpin, or were uninfected (−). 72 hours later, cells were assayed for viability (c), or cell extracts were analyzed by WB (d). (c) Data are means +/− s.d. (n=6) of cell viability relative to uninfected cells (set to 1). *, P<0.05 relative to ctl hairpin.
Figure 4
Figure 4
NF-κB inhibition impairs lung adenocarcinoma development. (a)K-rasLSL-G12D/WT; p53Flox/Flox mice were infected with Lenti-Pgk.Cre or Lenti-UbC.IκB-SR; Pgk.Cre viruses, sacrificed 17 (left) or 14 (right) weeks post-infection (w.p.i.) and tumor number was counted (see Methods). (b–d)K-rasLSL-G12D/WT; p53Flox/Flox; CCSP-rtTA+ (rtTA+) or -rtTA− mice (rtTA−) were infected with Lenti-TRE.IκB-SR; Pgk.Cre viruses (104 viral particles). (b) From the day of infection, mice were fed a doxycycline diet (Dox). At 15 w.p.i., µ-CT imaged tumors (magenta) and lungs were reconstructed and individual tumor volumes (mm3, white text) were measured. (c) From 15 to 19 w.p.i., lungs were µ-CT imaged, and individual tumor volumes were measured. Data points represent means of fold change +/− s.d. in tumor volume relative to 15 w.p.i. (set to 1). (d) After one week of Dox treatment, RNA was isolated from tumors of rtTA+ (n=4) or rtTA− (n=3) mice. cDNA was amplified using probes for the indicated genes orGapdh (internal control). Data show means +/− s.d. **, P=0.001; *, P=0.04 (Tnf) or 0.05 (Il6).
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