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. 2009 Aug;8(16):1567-76.
doi: 10.4161/cbt.8.16.8961. Epub 2009 Aug 8.

Constitutive non-canonical NFkappaB signaling in pancreatic cancer cells

Catherine E Wharry  1 , Kathleen M Haines, Richard G Carroll, Michael J May
Affiliations

Constitutive non-canonical NFkappaB signaling in pancreatic cancer cells

Catherine E Wharry et al. Cancer Biol Ther. 2009 Aug.

Abstract

Constitutive classical NFkappaB activation has been implicated in the development of pancreatic cancer, and inhibition of classical NFkappaB signaling sensitizes pancreatic cancer cells to apoptosis. However, the role of the more recently described non-canonical NFkappaB pathway has not been specifically addressed in pancreatic cancer. The non-canonical pathway requires stabilization of NIK and IKKalpha-dependent phosphorylation and processing of NFkappaB2/p100 to p52. This leads to the activation of p52-RelB heterodimers that regulate genes encoding lymphoid-specific chemokines and cytokines. We performed qRT-PCR to detect gene expression in a panel of pancreatic ductal adenocarcinoma cell lines (BxPC-3, PCA-2, PANC-1, Capan-1, Hs-766T, AsPC-1, MiaPACA-2) and found only modest elevation of classical NFkappaB-dependent genes. In contrast, each of the tumor cell lines displayed dramatically elevated levels of subsets of the non-canonical NFkappaB target genes CCL19, CCL21, CXCL12, CXCL13 and BAFF. Consistent with activation of the non-canonical pathway, p52 and RelB co-localized in adenocarcinoma cells in sections of pancreatic tumor tissue, and each of the tumor cell lines displayed elevated p52 levels. Furthermore, p52 and RelB co-immunoprecipitated from pancreatic cancer cells and immunoblotting revealed that NIK was stabilized and p100 was constitutively phosphorylated in a subset of the cell lines. Finally, stable overexpression of dominant negative IKKalpha significantly inhibited non-canonical target gene expression in BxPC-3 cells. These findings therefore demonstrate that the non-canonical NFkappaB pathway is constitutively active and functional in pancreatic cancer cells.

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Figures

Figure 1
Figure 1
Non-canonical NFκB-dependent gene expression in pancreatic adenocarcinoma cells. Expression of the classical and NC NFκB-dependent genes in (A) BxPC-3, (B) PCA-2 and, (C) a panel of pancreatic adenocarcinoma cell lines was measured by qRT-PCR and normalized to levels in untreated HeLa cells Where indicated, HeLa cells were treated with TNF (10 ng/mL) for 4 h to activate the classical NFκB pathway. The human multiple myeloma cell line RPMI-8226 has been shown previously to exhibit constitutive activation of upstream components of the NC NFκB pathway. Gene expression data in (C) are depicted such that white represents little to no expression above untreated HeLa cells, and shades of gray to black indicate the increasing levels of gene upregulation relative to the untreated HeLa control. Statistical significance in (A and B) was calculated by one-tailed unpaired t-test; *p < 0.05.
Figure 2
Figure 2
RelB and p100/p52 expression in human pancreatic adenocarcinoma. (A) Paraffin-embedded tissue sections of human pancreatic adenocarcinoma specimens were stained using anti-p100/p52 or anti-RelB as indicated. Positive staining is visualized by a dark brown color and the hematoxylin counter stain is blue. Two distinct fields from two separate specimens (specimens I and II) are shown. (B) Individual sections were dual-stained using fluorescent-labeled anti-p100/p52 (green) and anti-RelB (red). Nuclei were visualized by DAPI counterstaining (blue). On the merged image, the arrows indicate areas of yellow demonstrating co-localization of p100/p52 and RelB; and white, where the NFκB proteins are co-localized in the nucleus.
Figure 3
Figure 3
Non-canonical NFκB in pancreatic adenocarcinoma cells. (A) HUVEC were either untreated or treated with 10 ng/ml LIGHT for 24 hrs then whole cell extracts were generated. Extracts from HUVEC, HeLa and RPMI-8226 were immunoblotted using anti-p100/p52. (B) Whole cell lysates from HeLa, RPMI-8226 and the pancreatic adenocarcinoma cell lines indicated were immunoblotted using anti-p100/p52 (upper) and anti-RelB (lower). (C) Nuclear extracts (NE) from HeLa, BxPC-3 and Hs766T cells were immunoblotted using anti-p100/p52 and anti-RelB as indicated. To verify the integrity of the nuclear extracts, samples were also immunoblotted using anti-PARP and anti-tubulin that are expressed in the nucleus and cytoplasm respectively. A whole cell extract (WCE) from HeLa cells was immunoblotted with anti-PARP and anti-tubulin to confirm the location of these proteins (lane 1). (D) Lysates of HeLa, BxPC-3 and Hs766T cells were immunoprecipitated (IP) with either anti-p52 or a non-specific isotype-matched antibody (IgG) then samples immunoblotted using anti-RelB and anti-p52 as indicated. A sample of each cell lysate was retained prior to immunoprecipitation for immunoblotting (pre-IP).
Figure 4
Figure 4
Upstream non-canonical NFκB signaling in pancreatic cancer cell lines. (A) Whole cell lysates from HeLa, RPMI-8226 and the pancreatic cancer cell lines indicated were immunoblotted using anti-NIK (upper) and anti-TRAF3 (lower). (B) NIK protein levels in the panel of seven PC cell lines were assessed by immunoblotting whole cell lysates using anti-NIK then performing densitometry as described in Material and Methods. Immunoblots from three separate experiments were analyzed and for each experiment, the pixel intensity of the NIK band was normalized to the pixel intensity in an identical area at the location of NIK in the HeLa control lane. (C) Whole cell lysates from HeLa, BxPC-3 and Hs766T cells were immunoblotted using anti-p100 (lower) and anti-phospho-p100 (upper).
Figure 5
Figure 5
Dominant-negative IKKα reduces non-canonical NFκB-dependent gene expression in BxPC-3 cells. (A) A vector containing green fluorescent protein (GFP) was retrovirally transduced into BxPC-3 cells in parallel with cells transduced with the dominant negative IKKαSSAA mutant. The efficiency of transduction was quantified by FACS to determine the percentage of GFP-positive cells compared to untreated BxPC-3 controls. (B) Whole cell extracts from GFP-transduced and IKKαSSAA-transduced BxPC-3 cells were immunoblotted using anti-IKKα (top) and anti-tubulin as a loading control (bottom). (C) The expression levels of NC NFκB target genes in IKKαSSAA-transduced BxPC-3 cells were determined by qRT-PCR and normalized to expression in control vector-transduced cells. We did not detect any CCL19 in the control vector- or IKKαSSAA-transduced cells suggesting that retroviral transduction alone affects expression of this gene. Statistical significance was calculated by one-tailed paired t-test; *p < 0.05, **p < 0.005.
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