Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

Daniela Basso,Dania Bozzato,Matteo Fassan,Eliana Greco,Michele Scorzeto,Francesca Simonato,Ambrogio Fassina,Sirio Dupont,Sergio Pedrazzoli,Filippo Navaglia,Andrea Padoan,Carlo-Federico Zambon,Michela Pelloso,Paola Fogar,Stefania Moz,Mario Plebani

doi:10.1186/1478-811x-12-20

Abstract

BackgroundIn order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines.ResultsNT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation).ConclusionsThe effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Highlights

In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NTS100A8, a Pancreatic ductal adenocarcinoma (PDAC)-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT)
Metastatic PDAC cell lines have high S100A8/A9 expression A comparative quantification was made of S100A8 and S100A9 mRNA expression by real time PCR and referred to their respective expression levels by the PDAC metastatic SUIT2 cell line (Table 1)
S100A8 proteolysis is induced by pancreatic cancer cell conditioned media Figure 1 shows low molecular weight MALDI-TOF/Mass spectrometry (MS) spectra obtained after 24 hours’ incubation at 37°C of S100A8 in complete control culture medium, Capan1 conditioned medium (CM) and Capan1 CM obtained after treatment with the Matrix metalloproteinase (MMP) inhibitor Ukrain [41]

Summary

Introduction

In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NTS100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). When the widely known PDAC precursor, PanIN, progresses from stage I to stage III, it accumulates cellular atypia and a series of genetic alterations, including activation of oncogenic k-ras and inactivation of tumor suppressors p16Ink4a and p53 [4]. As clearly demonstrated in genetically engineered mouse models of PDAC progression, the pancreatic epithelium-specific expression of k-ras (KrasG12D) induces PanIN lesions that can progress to PDAC, the average latency being more than one year; K-rasG12D expression coupled with the homozygous or heterozygous deletion of the tumor suppressor genes p53 and/or p16Ink4a underlies short latency PDAC [5,6,7]. In most cases of PDAC, k-ras, p16Ink4a and p53 mutations are present [8], whereas in about 50% of cases, Smad inactivation due to gene deletion or mutations associated with loss of heterozygosity, is identified [9]. When Smad is inactivated, PDAC is associated with widespread metastases [10] and a poorer prognosis [11]

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