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Antiapoptotic Effects of Progastrin on Pancreatic Cancer Cells Are Mediated by Sustained Activation of Nuclear Factor-KB

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Antiapoptotic Effects of Progastrin on Pancreatic Cancer Cells Are Mediated by Sustained Activation of Nuclear Factor-KB Research Article Antiapoptotic Effects of Progastrin on Pancreatic Cancer Cells Are Mediated by Sustained Activation of Nuclear Factor-KB William Rengifo-Cam,1 Shahid Umar,2 Shubhashish Sarkar,1 and Pomila Singh1 Departments of 1Neuroscience and Cell Biology and 2Internal Medicine-Gastroenterology, University of Texas Medical Branch, Galveston, Texas Abstract pancreatic cancer (6), lung cancer (7), and ovarian cancer (8) also Progastrin (PG) exerts proliferative and antiapoptotic effects express the gastrin gene and PGs. It is possible that PG-like on intestinal epithelial and colon cancer cells via Annexin II peptides play an equally important role in the growth and survival (ANX-II). In here, we show that ANX-II similarly mediates of many of these PG-expressing cancers as reported for PG- proliferative and antiapoptotic effects of PG on a pancreatic dependent CRCs (2, 9). Therefore, understanding the mechanisms cancer cell line, AR42J. The role of several signaling molecules by which PG-like peptides mediate growth and survival effects on was examined in delineating the biological activity of PG. PG cancer cells will be extremely useful in developing a targeted (0.1–1.0 nmol/L) caused a significant increase (2- to 5-fold) in approach for treating PG-dependent cancer as previously discussed the phosphorylation of phosphatidylinositol 3-kinase (PI3K), (2, 9, 10). The high-affinity PG/gastrin-binding sites are distinct Akt (Thr308), p38 mitogen-activated protein kinase (MAPK; from CCK1R and CCK2R and bind PG-like peptides in the order of Thr180/Tyr182), extracellular signal-regulated kinases (ERK; PG>Gly gastrin>G17>CCK8 (3). Annexin II (ANX-II) was recently Thr202/Tyr204), IKB kinase A/B (IKKA/B; Ser176/180), IKBA discovered as one such molecule and is required for mediating (Ser32), and p65 nuclear factor-KB (NF-KB; Ser536). Inhibition growth factor effects of PG/gastrin peptides on intestinal epithelial of p44/42 ERKs (PD98059), p38 MAPK (SB203580), Akt, and and colon cancer cells (11). Although high-affinity binding sites for PI3K (LY294002), individually or combined, partially reversed PG-like peptides are present on normal IECs, and on gastrin- antiapoptotic effects of PG. The kinetics of phosphorylation of responsive CRCs, it is not known if high-affinity PG-binding sites IKKA/B in response to PG matched the kinetics of phosphor- are present on other cancer cell types as well. We used a rat ylation and degradation of IKBA andcorrelatedwith pancreatic cancer cell line, AR42J, as the in vitro pancreatic cancer phosphorylation, nuclear translocation, and activation of cell model. AR42J cells, unlike human pancreatic cancer cell lines, p65 NF-KB. NF-KB essential modulator–binding domain do not express autocrine growth factors and/or elevated levels of n n peptide (an inhibitor of IKKA/B) effectively blocked the growth-promoting factors [such as nuclear factor- B (NF- B) or activity of p65 NF-KB in response to PG. Activation of p65 mutant Ras] and are responsive to exogenous G17/Gly gastrin NF-KB, in response to PG, was 70% to 80% dependent on (reviewed in ref. 2). In initial studies, we confirmed if AR42J cells phosphorylation of MAPK/ERK and PI3K/Akt molecules. were positive for high-affinity PG-binding sites and if the cells Down-regulation of p65 NF-KB by specific small interfering expressed ANX-II. Next, we confirmed if AR42J cells respond to RNA resulted in the loss of antiapoptotic effects of PG on proliferative and antiapoptotic effects of PG and if ANX-II mediates AR42J cells. These studies show for the first time that the growth effects of PG on AR42J cells. canonical pathway of activation of p65 NF-KB mediates Activation of c-Src kinase is required for mediating proliferative antiapoptotic effects of PG. Therefore, targeting PG and/or effects of PG on IECs (12). In mice overexpressing PG (hGAS p65 NF-KB may be useful for treating cancers, which are mice), significant increases in the relative levels of phosphorylated dependent on autocrine or circulating PGs for their growth. c-Src, phosphatidylinositol 3-kinase (PI3K)/Akt, Janus-activated [Cancer Res 2007;67(15):7266–74] kinase (JAK) 2, signal transducers and activators of transcription (STAT) 3, extracellular signal-regulated kinases (ERK), and trans- Introduction forming growth factor-a in colonic mucosa were reported (13), suggesting that one or more of these signaling molecules may be The full-length progastrin peptide (PG) is the precursor 1-80 involved in mediating proliferative and/or antiapoptotic effects of peptide of gastrin (G1-G17). G17 plays an important role in gastric PG. In fact, several of these molecules, including p38 mitogen- acid secretion via CCK R (1). Additionally, PG and gastrin peptides 2 activated protein kinase (MAPK)/ERK p44/42 and PI3K/Akt, have exert growth effects on normal and cancerous intestinal epithelial been shown to play an important role in proliferation and cells (IEC) via PG-preferring receptors (2–5). We now know that survival of pancreatic cancer cells (14–16). Additionally, activation colorectal cancers (CRC) express autocrine PG (2). Besides CRCs, of NF-nB has been reported to be one of the most frequent molecular alterations in pancreatic cancer cells (14) and is required for the proliferation and survival of the cells (14, 17). Note: Supplementary data for this article are available at Cancer Research Online Importantly, down-regulation of NF-nB sensitizes the pancreatic (http://cancerres.aacrjournals.org/). cancer cells to apoptotic death via anticancer agents (14, 18, 19). Requests for reprints: Pomila Singh, Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 10.104 Medical Research Building, Route We therefore focused on examining the role of the above-listed 1043, Galveston, TX 77555-1043. Phone: 409-772-4842; Fax: 409-772-3222; E-mail: signaling molecules in mediating the observed survival effects of [email protected]. I2007 American Association for Cancer Research. PG on AR42J cells. As a result of our studies, we report for the doi:10.1158/0008-5472.CAN-07-1206 first time that PG exerts potent antiapoptotic effects on AR42J Cancer Res 2007; 67: (15). August 1, 2007 7266 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. NF-kB Mediates PG Effects on Pancreatic Cancer Cells cells, which are mediated by the phosphorylation of InB kinase (Ser536; 7F1), anti–phosphorylated InBa (Ser32/36; 5A5), polyclonal anti– (IKK) a/h, resulting in the degradation of InBa and activation of phosphorylated stress-activated protein kinase/c-Jun NH2-terminal kinase n n (JNK; Thr183/Tyr185), anti-MAPK p38, anti-ERK p44/42, anti-phosphotyr- p65 NF- B; activation of NF- B pathway seems to be 70% to 80% 180 182 dependent on the phosphorylation (activation) of p38 MAPK/ERK osine p85 PI3K, anti–phosphorylated MAPK p38 (Thr /Tyr ), anti– phosphorylated ERK p44/42 (Thr202/Tyr204), anti-IKKa/h, and anti– and PI3K/Akt. phosphorylated IKKa/h (Ser176/180) antibodies were from Cell Signaling Technology. Monospecific polyclonal anti-PG antibodies were generated in house and used as described previously (3, 20, 21). Kinase inhibitors, Materials and Methods LY294002, Akt inhibitor, SB203580, PD98059, and NF-nB essential Materials. Leupeptin, aprotinin, benzamidine, phenylmethylsulfonyl modulator (NEMO)-binding domain peptide were from Calbiochem. fluoride, sodium, orthovanadate, EDTA, NP40, octyl-D glucoside, h- Stock solutions (1 mmol/L) of all the inhibitors were prepared in 100% mercaptoethanol, Tris, HEPES, sodium chloride, sodium fluoride, glycerol, DMSO (Sigma) and stored at À70jC. Recombinant human PG (rhPG) was camptothecin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium generated in our laboratory as described previously (3) and radiolabeled bromide (MTT) were obtained from Sigma Chemical Co. Polyclonal with Na125I by our published methods (3). G17 and CCK8 were purchased anti-caspase-3 and anti-caspase-9 antibodies were from BD PharMingen. from Bachem. Polyclonal anti-InBa (C-21), anti-p50 NF-nB (H-119), anti–phosphorylated Cell culture. The rat pancreatic cancer cell line AR42J was propagated in Akt1/2/3 (Thr308)-R, anti-Akt1/2 (H-136), anti-PI3K p85a (Z-8), anti-actin culture as described previously (22). In vitro (I-19), anti-CCK2R (C-18), and anti-ANX-II (H-50) antibodies were from growth assays. The effect of increasing concentrations of Santa Cruz Biotechnology. Monoclonal anti-lamin B (101-B7) antibody rhPG, in the presence or absence of specific antibodies against ANX-II, PG, was from Calbiochem. Monoclonal anti–phosphorylated p65 NF-nB or CCK2R, on the growth response of AR42J cells was measured either in a Figure 1. A, dose-dependent (0.1 nmol/L to 1.0 Amol/L) growth effects of PG on AR42J cells. Growth in response to 1%FCS as a positive control ( C). Total number of cells/35-mm dish at the end of the assay is presented. Columns, mean of six to nine separate measurements from three experiments; bars, SE. The control dishes were treated with the vehicle alone. Bi and Bii, antiapoptotic effects of an optimal dose of PG (0.1 nmol/L) were measured on AR42J cells in terms of relative levels of activated caspase-3 (Bi) and caspase-9 (Bii) in control (nontreated) versus PG-treated cells. Cells grown in the presence or absence (control) of 0.1 nmol/L PG for 48 to 72 h were treated with a potent proapoptotic agent camptothecin (10 Amol/L) for 4 h. At the end of the assay, relative levels of activated caspase-3 (Bi) and caspase-9 (Bii) were measured by Western blot analysis. Top, Western blot data from a representative experiment of a total of three experiments for caspase-3 and caspase-9. The Western blot membranes were stripped and reprobed for h-actin levels as an internal control.
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