NFATc1 Links EGFR Signaling to Induction of Sox9 Transcription and Acinar–Ductal Transdifferentiation in the Pancreas

Abstract

Background & Aims Oncogenic Kras mutation is a defining genetic alteration in pancreatic ductal adenocarcinoma (PDAC), but is not sufficient to promote cancer formation on ist own. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. In this study we sought to identify the underlying mechanisms which link EGFR signaling to Sox9 gene induction during acinar–ductal metaplasia (ADM), a transdifferentiation process that precedes pancreatic carcinogenesis. Methods We analyzed pancreatic tissues from KrasG12D;pdx1-Cre and KrasG12D;NFATc1Δ/Δ;pdx1-Cre mice after intraperitoneal administration of caerulein or dimethyl suloxide (controls). Pharmacological inhibition of NFATc1 activation was achieved by application of cyclosporin A. Induction of EGFR signaling and its effects on expression of NFATc1 or SOX9 were investigated by quantitative reverse transcription PCR, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue. Results EGFR activation induced NFATc1 expression in metaplastic tissues from patients with chronic pancreatitis and in pancreatic tissues from KrasG12D mice and promoted complex-formation with c-Jun in dedifferentiating acinar cells, thereby stimulating the transcription of ductal gene signatures to provoke ADM. This process involved NFATc1:c-Jun-mediated activation of Sox9 transcription in converting acinar cells. Pharmacological inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar–ductal transdifferentiation and pancreatic cancer initiation. Conclusion Our findings identify an EGFR-NFATc1-Sox9 signaling cascade as a critical mediator of inflammation-induced PDAC initiation and suggest that disruption of this pathway may offer a novel chemopreventive target for high-risk pancreatitis patients.

Inferring TF activation order in time series scRNA-Seq studies

“…ATF3 is a known co-factor of c-Fos and both are correctly assigned to the same time (Fig 4(i)). In addition, SOX9 is known to be the downstream target of NFATC1 [54], and CSHMM-TF identified both of them in the same path and assign them at the same time point (Fig 4(j)). Finally, SRF are known to form a physical complex with NKX3-1 [55], and both of them are assigned at the same path with same time (Fig 4(l)).…”

Acinar cell plasticity and development of pancreatic ductal adenocarcinoma

“…Similarly, in mice, the absence of SOX9 reduces EGFR signalling and pancreatic tumorigenesis 50 . However, EGFR signalling can also regulate expression of SOX9 through activation of NFATC1 and NFATC4 (REFS 51,52). …”

“…Consequently, MMP inhibition in mice completely blocked caerulein-induced ADM 4 . Other transcription factors activated in acinar cells after inflammation that contribute to ADM are NFATC1 and NFATC4 (REFS 51,52). …”

New insights in the development of pancreatic cancer

“…Grimont et al [13 ▪ ] showed that Sox9 contributes to acinar neoplasia by stimulating the ErbB pathway resulting in epidermal growth factor receptor (Egfr) and Erk phosphorylation in Kras-induced ADM in the setting of pancreatitis. There also appears to be a feed-forward loop in the activation of Sox9 in transformed acinar cells where Egfr activation leads to upregulation of Sox9 expression through the transcription factor, nuclear factor of activated T cells c1 (Nfatc1) that complexes with signal transducer and activator of transcription 3 (Stat3) in the nucleus to activate target genes [14]. Both Nfatc1 and Stat3 have been shown to be critical in early PanIN tumorigenesis [15–17].…”